Simultaneous Discrimination of Cysteine, Homocysteine, Glutathione, and H2S in Living Cells through a Multisignal Combination Strategy

Article abstract of DOI:10.1021/acs.analchem.8b03869

Biothiols are essential reactive sulfur species (RSS), which play crucial roles in various critical physiological activities. To clarify their complex correlations in signal transduction and metabolism pathways, methods to distinguish H₂S, cyst

Full text of DOI:10.1021/acs.analchem.8b03869

Oncogene
https://doi.org/10.1038/s41388-018-0524-5
ARTICLE
p53-dependent autophagic degradation of TET2 modulates cancer
therapeutic resistance
Jixiang Zhang1,2 Peng Tan3 Lei Guo1,3 Jing Gong4 Jingjing Ma2 Jia Li1 Minjung Lee1 Shaohai Fang1 Ji Jing3
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Gavin Johnson1 Deqiang Sun1 Wen-ming Cao5 Roderick Dashwood1,6 Leng Han
Yubin Zhou
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Wei-Guo Dong2 Yun Huang
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Received: 7 December 2017 / Revised: 4 July 2018 / Accepted: 25 July 2018
© Springer Nature Limited 2018
Abstract
Tumor cells with p53 inactivation frequently exhibit chemotherapy resistance, which poses a long-standing challenge to
cancer treatment. Here we unveiled a previously unrecognized role of TET2 in mediating p53-loss induced chemotherapy
resistance in colon cancer. Deletion of TET2 in p53-null colon cancer cells enhanced DNA damage and restored
chemotherapy sensitivity. By taking a two-pronged approach that combined pharmacological inhibition with genetic
depletion, we discovered that p53 destabilized TET2 at the protein level by promoting its autophagic degradation. At the
molecular level, we further revealed a physical association between TET2 and p53 that facilitated the nucleoplasmic
shuttling of TET2, as well as its recruitment to the autophagosome for degradation. Our study has unveiled a functional
interplay between TET2 and p53 during anti-cancer therapy. Our findings establish the rationale for targeting TET2 to
overcome chemotherapy resistance associated with mutant p53 tumors.
Introduction
[1]. p53 is a well-known “genome guardian” that maintains
the proper cell cycle arrest, DNA repair, and programmed
cell death during DNA damage response [2, 3]. Drug
resistance is one of the reasons accounting for the poor
prognosis in patients with p53 inactivating mutations. The
correlation between the p53 mutational status and sensi-
tivity to cytotoxic drugs has been confirmed in multiple
cancer types [1, 4, 5]. Recent exome sequencing in a large
cohort of acute myeloid leukemia (AML) patients revealed
mutually exclusive mutations in TP53 and TET2 [6]
Chemotherapy resistance often arises owing to enhanced
DNA repair and failure to apoptose, which is closely
associated with mutations in tumor suppressors, such as p53
These authors contributed equally to the work: Jixiang Zhang, Peng
Tan, Lei Guo
Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41388-018-0524-5) contains supplementary
material, which is available to authorized users.
* Yubin Zhou
and Technology, Texas A&M University, Houston, TX 77030,
USA
yzhou@ibt.tamhsc.edu
4
* Wei-Guo Dong
Department of Biochemistry and Molecular Biology, McGovern
Medical School at The University of Texas Health Science Center
at Houston, Houston, TX 77030, USA
dwg@whu.edu.cn
* Yun Huang
5
yun.huang@ibt.tamhsc.edu
Department of Breast Medical Oncology, Zhejiang Cancer
Hospital, Hangzhou, China310022
1
6
Center for Epigenetics & Disease Prevention, Institute of
Biosciences and Technology, Texas A&M University,
Houston, TX 77030, USA
Department of Molecular & Cellular Medicine, College of
Medicine, Texas A&M University, College Station, TX 77843,
USA
2
7
Department of gastroenterology, Renmin Hospital of Wuhan
University, Wuhan 430060 Hubei Province, China
Department of Medical Physiology, College of Medicine, Texas
A&M University, Temple, TX 76504, USA
3
Center for Translational Cancer Research, Institute of Biosciences

J. Zhang et al.
(Supplementary Figure S1), suggesting that cells with
genetic lesions in both genes might not undergo malignant
transformation or have less survival capability. This finding
implies that defining the interplay between p53 and TET2 in
tumor cells might yield novel insights into the management
of p53 mutant tumors and cancer therapeutic resistance.
Ten–eleven Translocation 2 (TET2) belongs to the TET
family of Fe (II)- and α-ketoglutarate-dependent dioxy-
genases that successively oxidize 5-methylcytosine (5mC)
to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine
(5fC) and 5-carboxylcytosine (5caC) in the metazoan
nucleus [7–11]. TET-mediated serial oxidation on 5mC is
essential for both active and passive DNA demethylation
that paly pivotal roles in various biological process [12, 13].
TET2 loss-of-function (LOF) mutations are frequently
detected in a large spectrum of hematological malignancies
[11, 14–20]. Compromised TET enzymatic activities with
consequential reduction in its major catalytic product,
5hmC, has been noted in several types of solid tumors [11,
20–23]. TET protein is known to regulate genome stability
through maintaining DNA damage repair pathways [24],
but whether decrease in TET/5hmC levels in tumor cells
would affect the DNA damage response induced by anti-
cancer therapy remains unclear.
Several protein regulators of TET enzymes have been
identified in recent studies [25–27]. For example, TET
protein levels can be modulated by IDAX, a CXXC
domain-containing protein that is speculated to be originally
encoded within TET2 gene and then undergoes chromo-
some inversion during evolution. IDAX downregulated
TET2 protein levels through activation of caspase-related
pathways [25]. In addition, TET protein stability can be
modulated by a calcium-dependent protease calpain and
P300-mediated acetylation [26, 27]. In addition to protease-
dependent protein degradation pathways, macroautophagy
(autophagy hereafter) is a highly conserved cellular degra-
dation and recycling mechanism to eliminate proteins and
organelles to maintain proper cell function [28]. Autophagy
is considered as a double-edged sword in cancer depending
on the context-specific roles during cancer initiation and
progression [29].
Results
TET2 deletion restores sensitivity of anti-cancer
treatment in p53-null tumor cells
To find the best model system to interrogate the interplay
between TET2 and p53 in cancer cells, we examined the
protein expression levels of TET2 and p53 in three repre-
sentative colon cancer cell lines, HTC116, HT29 and
SW480. We detected a relatively higher expression of TET2
protein but lower expression of p53 protein in HCT116 cells
compared to the other two cell lines HT29 and SW480 (Fig.
1a). HCT116 cells are known to display highly aggressive
properties with cancer stem cell-like phenotypes [30]. We
therefore decided to use WT and p53 knockout out
(designated p53KO; generated by Dr. B. Vogelstein’s
laboratory) [31] HCT116 cells in this study to further study
the interplay between TET2 and p53 during anti-cancer
treatment.
We set out to delete TET2 in HCT116 cells (WT and
p53KO; Fig. 1b) using the CRISPR/Cas9 genome editing
method, and then treated these cells with doxorubicin
(DXR) in four experimental groups (WT, p53KO,
TET2KO, p53KO + TET2KO or DKO). DXR is a com-
monly used chemotherapy agent in the clinic for cancer
treatment [32]. DXR acts on cancer cells by interacting with
DNA and inhibiting topoisomerase II during DNA repli-
cation [33]. We used the WST-1 cell viability assay and
colony-forming assay to monitor cell survival and pro-
liferation (Fig. 1c, d) on these cells treated with various does
of DXR (0-3.2 µM). Consistent with earlier studies [34],
p53KO HCT116 cells exhibited strong resistance to DXR.
In the presence of 3.2 µM doxorubicin, approximately 80%
of WT HCT116 cells died whereas up to ~50% of p53 KO
cells remained viable. Interestingly, in p53KO HCT116
cells further depleted of TET2 (DKO), we observed a pro-
nounced decrease in cell viability and proliferation when
compared to p53KO cells. Furthermore, deletion of TET2 in
HCT116 cells showed the most striking decrease in cell
survival and proliferation upon DXR treatment (Fig. 1c, d).
Re-expressing TET2 in TET2KO or DKO HCT116 cells
restored the doxorubicin resistance phenotype (Fig. 1d). To
confirm this phenotype in vivo, we subcutaneously injected
four experimental groups of HCT116 cells (WT, p53KO,
TET2KO, and DKO) into the flank of NOD scid gamma
mice in the present of DXR treatment, and then monitored
tumor growth up to 4 weeks (Fig. 1e). Compared to the WT
group, mice injected with TET2KO colon cancer cells
showed a shrink in tumor size. By contrast, the p53KO
group exhibited a significantly larger tumor volume. How-
ever, the DKO group with deletion of both P53 and TET2
displayed an appreciable reduction in tumor size when
compared to the p53KO group (Fig. 1e). These findings
In the current study, we report that the tumor suppressor
p53 regulates TET2 stability through autophagic degrada-
tion pathways. In addition, we found that TET2 renders
p53-null cancer cells resistant to cancer therapy that targets
DNA damage response (e.g. doxorubicin and cisplatin).
TET2 inactivation provides a new approach to restore drug
sensitivity in p53-null tumors. Our study also calls for
cautions in the future application of TET activators in the
treatment of cancer. Furthermore, our findings establish a
previously unrecognized functional interplay between p53
and TET2, which is critical for drug resistance in tumor
cells bearing p53 LOF mutations.

p53-dependent autophagic degradation of TET2 modulates cancer therapeutic resistance
A
B
HCT116-
p53KO
FLAG-
TET2
HCT116
TET2
p53
TET2
FLAG
p53
p53
GAPDH
GAPDH
GAPDH
C
WT
p53KO
TET2KO
DKO
100
80
60
40
20
0
TET2KO + FLAG-TET2
DKO + FLAG-TET2
**
*
*
**
0
1
2
3
Doxorubicin (μM)
D
Doxorubicin (DXR)
Doxorubicin (0.8μM)
1.5
μM
WT
**
**
1.0
0.5
0.0
**
TET2KO
p53KO
*
TET2KO+p53KO (DKO)
TET2KO+FLAG-TET2
DKO+FLAG-TET2
E
500
WT
p53KO
TET2KO
DKO
400
300
200
100
0
**
**
Drug treatment
**
0
1
2
3
4
Weeks
Fig. 1 TET2 depletion overcomes anti-cancer treatment resistance in
p53-null HCT116 cells. a Endogenous levels of TET2 and p53 pro-
teins in colon cancer cell lines (HCT116, HT29 and SW480). GAPDH
was used as loading control. b (Left) TET2 and p53 protein expression
levels in WT and p53KO HCT116 cells treated with and without
sgRNA to deplete endogenous TET2. (Right) FLAG-TET2 and p53
protein expression levels in TET2KO and p53KO + TET2KO (DKO)
HCT116 cells with over-expression of FLAG-TET2. c Assessment of
cell viability. WST-1 was used to measure the percentage of survived
HCT116 cells (WT, p53KO, TET2KO, p53KO + TET2KO (DKO),
TET2KO + FLAG-TET2, and DKO + FLAG-TET2) after incubation
with increasing doses (0, 0.4, 0.8, 1.6, 3.2 µM) of doxorubicin for 48 h
(n = 3 independent experiments). Data were shown as mean S.D.;
*p < 0.01, **p < 0.001 (two-tailed Student’s t test compared with WT).
d Colony-forming assay to measure the viability of HCT116 cells
(WT, p53KO, TET2KO, p53KO + TET2KO (DKO), TET2KO +
FLAG-TET2, DKO + FLAG-TET2) treated with different amounts (0,
0.2, 0.4, 0.8, 1.2, 1.6, 3.4, 3.2 µM) of doxorubicin for 60 h. (Left)
Representative images showing the colony-forming assay result;
(Right) quantifications of colony formation (n = 3 independent
experiments). The data were shown as mean S.D.; *p < 0.01, **p <
0.001 (two-tailed Student’s t test compared with WT). e Tumor
volume (left) and representative images (right) at the indicated time
points in nude mice that were orthotopically implanted with corre-
sponding HCT116 tumor cells. n = 3 mice per group. **p < 0.001
(two-tailed Student’s t test compared with WT)

J. Zhang et al.
suggested that TET2 is indeed a positive contributor to the
chemotherapy resistant feature in p53-null tumor cells
in vivo.
We noticed a clear decline in the 5hmC levels following
both doxycycline-inducible or transient expression of p53
(Fig. 2g). We further confirmed this reciprocal expression
pattern of p53 and TET2 in other cancer cell lines (SW48
and MCF7, Supplementary Figure S2B), as well as in colon
cancer patients (Fig. 2h). Among the twelve patient sam-
ples, three showed detectable p53 mutations (Supplemen-
tary Table S1) and had relatively high TET2 protein levels
(Fig. 2h). Among the nine patients without p53 mutations,
we detected a strong negative correlation between p53 and
TET2 protein levels (Fig. 2h). Taken together, our data
suggest that p53 can modulate the TET2 protein expression
level, likely through a post-translational mechanism, to
affect DNA hydroxymethylation.
Since p53 expression can be rapidly upregulated in
tumor cells in response to anti-cancer therapy (e.g.,
doxorubicin-based chemotherapy or radiotherapy) [36], we
asked whether therapeutics-triggered p53 upregulation
could reduce TET2 protein levels in HCT116 cells. We
monitored TET2 and p53 protein levels in the presence of
increasing amounts of doxorubicin (0, 0.2, 0.4, 1, 2 µM) by
western blotting (WB). We observed a gradual increase in
p53 protein levels with concomitantly reduced TET2 pro-
tein levels without a significant alteration in the TET2
mRNA level (Fig. 3a, b). Meanwhile, we did not observe a
significant change in the TET1 protein levels in HCT116
cells treated with various doses of doxorubicin (Supplentary
Figure S3A). Similarly, decrease of TET2 protein levels
was also observed in HCT116 cells treated with gamma-
irradiation (3 or 6 Gy; Fig. 3c) and cisplatin (Supplentary
Figure S3B). As control, we did not observe changes in
TET2 protein levels in p53KO HCT116 cells following
incubation with doxorubicin (Fig. 3d). Similar results were
also observed in other cancer cell lines including SW48 and
MCF7 (Supplentary Figure S3C). These data clearly
demonstrate that p53 per se could promote TET2 protein
degradation during anti-cancer treatment.
Accumulating evidence has suggested that p53 inacti-
vation is closely associated with cancer initiation and pro-
gression, partially because those mutant cells are escaped
from DNA damage surveillance. Furthermore, it is well
known that p53 is significantly upregulated in response to
DNA damage response-based cancer treatment. p53-null
cancer cells are usually resistant to such treatment due to
reduced DNA damage response [34]. Recent reports have
shown that TET proteins play indispensable roles in the
maintenance of genome stability and that deletion of TET
proteins could promote DNA damage [24, 37]. These
findings led us to hypothesize that p53-mediated TET2
degradation is important for safeguarding cells to efficiently
respond to DNA damage stress. To test this hypothesis, we
first measured double strand DNA break marker, γ-H2AX,
in HCT116 cells treated by gamma-irradiation (Fig. 3c),
To further examine whether this phenotype applies to
other chemotherapeutic drugs, we treated HCT116 cells
with cisplatin, a potent anticancer agent that inhibits DNA
replication [35]. We observed similar phonotypes as seen in
cancer cells treated with DXR (Supplementary Figure S1B).
To extend our study to other types of cancer cells, we
performed the similar experiments in two additional cell
lines, SW48 (colon cancer) and MCF7 (breast cancer;
Supplementary Figures S1C-D). Consistent with the sce-
nario seen in HCT116 cells, we found that deletion of TET2
in these engineered p53-null tumor cells could likewise
restore their sensitivity to DXR treatment (Supplementary
Figure S1C-D). These data strongly indicate that TET2
renders p53-null tumor cells less sensitive toward che-
motherapy agent treatment.
p53-mediated degradation of TET2 enhanced DNA
damage in cancer cells
The reciprocal expression patterns of p53 and TET2 in
colon cancer cells prompted us to hypothesize that p53
might negatively regulate TET2 expression. To quickly test
this idea, we compared the endogenous mRNA and protein
levels of TET2 in both tumor (p53KO HCT116; Fig. 2a)
and non- tumor cell lines (HEK293T; Fig. 2b), with and
without re-expression of FLAG-tagged p53. We found that
TET2 protein levels were pronouncedly reduced in both cell
types following transient expression of FLAG-p53 without
significant alterations in TET2 mRNA levels (Fig. 2a–c). To
more rigorously validate this observation, we further uti-
lized a tetracycline-controlled transcriptional activation
system to enable inducible and tunable expression of p53 by
adding the tetracycline derivative doxycycline (DOX) to
both p53KO HCT116 and HEK293T cells. Upon the
addition of increasing doses of doxycycline, we observed a
gradual increase in p53 expression with concomitant
reduction in the protein levels but not mRNA levels of
TET2 (Fig. 2d, e). This result was further independently
confirmed by immunofluorescent staining of TET2 in
HEK293T cells before and after inducible expression of p53
(Fig. 2f). Meanwhile, we did not observe a significant
change of TET1 mRNA levels in HCT116 cells with and
without p53 expression (Sypplementary Figure S2A), thus
ruling out the possibility of functional compensation from
the major TET homolog in this cell line.
To further examine whether the resultant reduction in
TET2 expression would affect its major catalytic product
(5hmC), we performed
a dot-blot assay to semi-
quantitatively examine 5hmC levels in HEK293T cells
with inducible p53 expression or in p53KO HEK293T cells.

p53-dependent autophagic degradation of TET2 modulates cancer therapeutic resistance
A
B
p < 0.001
p = 0.21
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
TET2
p53
Tet2
p53
GAPDH
GAPDH
HCT116 p53KO
HEK293T
C
D
Doxycycline (DOX)
p = 0.18
2.0
1.5
1.0
0.5
0.0
(μM)
0 2 4 6 8
1.5
1.0
0.5
0.0
p = 0.80
P = 0.10
TET2
p53
GAPDH
p53
-
-
+
+
HCT116 p53KO with
inducible FLAG-p53
tet-on expression
HEK293THEK293T
p53KO
E
P < 0.001
1.5
1.0
0.5
0.0
F
DAPI
TET2
Merge
TET2
p53
GAPDH
HEK293T
Inducible p53
G
H
Anti-5hmC Methylene blue
HEK293T
Inducible
p53 cell
DMSO
DOX
Mock
p53
HEK293T
p53-KO
Cell line
0.6
0.4
0.2
r = -0.74
#12
#7
#10
1
2
3
4
5
6
7
8
9 10 11 12
Patient ID
p = 0.021
#2
p53
#6
#8
TET2
#3
#11
#1
0.0
GAPDH
0.0
0.2
0.4
0.6
p53 / GAPDH
doxorubicin (Fig. 3d, e) or cisplatin (Supplementary Figure
S3B). Indeed, we observed significantly increased γ-H2AX
protein levels upon irradiation, DXR or cisplatin treatment
in WT HCT116 cells (Fig. 3e, S3B). By contrast, in p53KO
HCT116 cells with intact TET2, we detected a pronounced
decrease in γ-H2AX levels compared to WT HCT116 cells
receiving the same treatment, suggesting less DNA dama-
ges in p53KO HCT116 cells (Fig. 3d, e and Supplementary
Figure S3B). A similar phenotype was also observed in
SW48 and MCF7 cells (Supplementary Figure S3C). Fur-
thermore, the additional deletion of TET2 in p53KO
HCT116 cells (DKO) substantially restored the γ-H2AX
levels after DXR treatment (Fig. 3e). These results con-
verged to support a protective role of TET2 against DNA
damage in p53KO HCT116 cells. Together, p53 mediated
TET2 degradation is essential for cells responding to DNA

J. Zhang et al.
Fig. 2 p53 downregulates TET2 at the protein level, but not at the
mRNA level. a (Left) Western-blotting analysis on TET2 and p53
protein levels in p53KO HCT116 cells with and without re-expressing
FLAG-tagged p53. (Right) Quantification of band intensities of
Western blotting results (n = 3 independent experiments). Data were
shown as mean S.D. b (Left) Western-blotting analysis on TET2 and
p53 protein levels in HEK293T cells without transfection, transfected
with empty vector (EV) or FLAG-tagged p53. (Right) Quantification
of band intensities of western blotting results (n = 3 independent
experiments). Data were shown as mean S.D. c Real-time quantita-
tive PCR analysis of TET2 mRNA. (n = 3 independent experiments).
The data were shown as mean S.D. (two-tailed Student’s t test). d
(Left) Western-blotting to monitor TET2 and p53 protein levels in
p53KO HCT116 cells with inducible expression of p53 following the
addition of increasing doses of doxycycline (0, 2, 4, 6, 8 µM). (Right)
Real-time quantitative PCR analysis of TET2 mRNA in corresponding
experimental groups. (n = 3 independent experiments). Data were
shown as mean S.D. (two-tailed Student’s t test). e (Left) Western-
blotting for monitoring TET2 and p53 protein levels in HEK293T cells
in the indicated experimental groups. (Right) Quantification of band
intensities of Western blotting results (n = 3 independent experi-
ments). Data were shown as mean S.D. f Representative confocal
images of HEK293T cells stained with DAPI (blue, nuclei) and an
anti-TET2 antibody (green) in the indicated experimental groups.
Doxycycline was added to induce the expression of p53. EV, empty
vector. g Representative dot-blot assay results showing the measure-
ment of the global 5hmC levels in HEK293T cells (with doxycycline
inducible expression of p53; top two panels) or in p53KO
HEK293T cells (lower two panels). Shown on the right is the
methylene blue staining results to ensure equal loading of total DNA.
h (Left) Western-blotting analysis on p53 and TET2 in tumor samples
collected from patients with colon cancer. Patients 4, 5 and 9 had p53
mis-sense mutations. (Right) Scatter plot of normalized p53 and TET2
(normalized to GAPDH) protein levels in individual patient with WT
p53. Pearson correlation coefficient was calculated (r = –0.74 and p =
0.021)
validate this with a genetic approach, we individually
depleted key components in the autophagic pathway,
including Atg16L1, p62, Beclin 1, and LC3 (Fig. 4b). We
then compared the TET2 protein levels in those engineered
cells in the absence or presence of inducible p53 expression.
Following the addition of doxycycline to induce p53
expression, we only observed a significant reduction of
TET2 protein levels in WT HEK293T cells, but not in cells
lacking autophagic components (Fig. 4b). Clearly, both
pharmacological and genetic studies converged to support
the conclusion that p53 downregulates TET2 protein levels
through autophagy-related signaling pathways.
Next, we asked whether p53 is required for the autop-
hagic degradation of TET2. To examine this, we induced
autophagy in WT and p53KO HEK293T cells using two
well-established protocols: serum-free starvation and rapa-
mycin (Rap) treatment [38]. In WT HEK293T cells, we
observed increased LC3 and decreased p62 expression upon
serum-free starvation (Fig. 4c) or rapamycin treatment (Fig.
4d), indicating the successful induction of autophagy. Fol-
lowing starvation, we observed a remarkable reduction in
TET2 protein levels in native HEK293T cells (with WT
p53). Furthermore, autophagy-mediated TET2 degradation
can be inhibited by the autophagy inhibitor CQ during
starvation (Fig. 4c). Interestingly, although we observed
autophagy induction following treatment with rapamycin or
by starvation in p53KO HEK293T cells, the protein levels
of TET2 remained largely unaltered, clearly suggesting that
p53 is required for autophagy-mediated degradation of
TET2 (Fig. 4c, d).
To further examine whether p53 mediated autophagic
degradation of TET2 also occurs in cancer cells, we per-
formed the similar experiment in p53KO HCT116 cells
before and after inducible re-expression of p53 (Fig. 4e), as
well as in HCT116 cells before and after DXR treatment
(Fig. 4f). In p53KO HCT116 cells, autophagic inhibition by
BafA1 exerted no effects on TET2 protein levels. However,
following inducible p53 expression in the same p53KO
HCT116 cells, BafA1-mediated inhibition of autophagy
substantially suppressed TET2 degradation (Fig. 4e), a
finding that dovetails with the data obtained from
HEK293T cells. Next, we asked whether doxorubicin
treatment induced decrease of TET2 proteins (Fig. 3a) is
also mediated by p53 through a similar autophagic
mechanism. We monitored the TET2 protein levels in
HCT116 cells in the present of doxorubicin and / or BafA1.
We found that Baf1A- mediated autophagic inhibition
indeed prevented TET2 protein degradation in HCT116
cells treated with DXR (Fig. 4f). In summary, our data
clearly suggest that p53 is required for mediating TET2
protein degradation through an autophagic mechanism in
both normal and cancer cells.
damage stress. p53 inactivation maintains relatively
higher levels of TET2 in cancer cells to counteract cancer
therapy-induced DNA damage, which might contribute to
cancer therapeutic resistance in p53 null / mutated cancer
cells.
p53 downregulates TET2 protein levels through
autophatic degradation
To further dissect how p53 regulates the protein levels of
TET2, we treated cells with inhibitors of proteasome and
autophagy pathways in HEK293T cells with inducible
expression of p53 (with DOX). We found that treatment of
cells with autophagy inhibitors, including 3-methyladenine
(3-MA), bafilomycin A1 (BafA1), NH4Cl and chloroquine
(CQ), led to prominent TET2 accumulation when p53
expression was induced by DOX (Fig. 4a). By contrast, no
significant TET2 accumulation was observed in cells treated
with a proteasome inhibitor, MG-132. These findings
strongly imply that p53 could regulate TET2 protein levels
through the autophagic degradation pathway. To further

p53-dependent autophagic degradation of TET2 modulates cancer therapeutic resistance
Fig. 3 A negative correlation
A
B
between p53 and TET2 protein
levels in response to anti-cancer
treatment with doxorubicin. a
Western-blotting analysis on
TET2 and p53 protein levels in
HCT116 cells treated with
Doxorubicin
(DXR, μM)
0 0.2 0.4
1
2
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
TET2
p53
increasing doses (0, 0.2, 0.4, 1,
2 µM) of doxorubicin for 24 h. b
mRNA expression levels of
TET2 and TP53 measured by
qPCR in HCT116 cells treated
with DMSO or doxorubicin (0.4
and 2.0 µM) for 24 h (n = 3
independent experiments). Data
were shown as mean S.D. c
Western-blotting analysis on
TET2, p53 and γH2AX protein
levels in HCT116 cells exposed
to 3 or 6 Gy gamma irradiation
for 6 or 24 h, respectively. d
Western-blotting analysis on
TET2, p53 and γH2AX protein
levels in WT and p53KO
GAPDH
μM
0.4 2.0
+ DXR
0.4 2.0 μM
+ DXR
C
D
24h
6h
p53KO
WT
IR
DXR (μM)
TET2
0
2.4 0 2.4
TET2
p53
p53
γH2AX
γH2AX
GAPDH
GAPDH
HCT116 cells treated with 0 or
2.4 µM doxorubicin for 24 h,
respectively. e Western-blotting
analysis on TET2, p53 and
γH2AX protein levels in WT,
TET2KO, p53KO, and
E
DMSO
+ DXR
TET2KO + p53KO (DKO) cells
treated with DMSO (control) or
2.4 µM doxorubicin for 24 h
TET2
p53
γH2AX
Short exposure
γH2AX
long exposure
GAPDH
p53 regulates the nucleocytoplasmic shuttling of
TET2
biochemically validate this, we separated the cytoplasmic
and nuclear fractions and examined the partition of TET2
between these two fractions by immunoblotting in both WT
and Beclin1KO HEK293T cells. We found that deletion of
Beclin 1 suppressed p53-mediated TET2 degradation in the
cytoplasm (Fig. 5b, left) but not for the nuclear fraction
(Fig. 5b, right). Furthermore, loss of Beclin 1 resulted in
cytoplasmic accumulation of TET2 in cells with or without
over- expression of p53, suggesting that p53-mediated
TET2 deletion mainly occurred in cytoplasm (Fig. 5b).
To further examine whether p53 is required for
autophagy-mediated cytoplasmic TET2 degradation, we
deleted p53 in Beclin1KO HEK293T cells (Fig. 5c) and
measured the cytoplasmic and nuclear TET2 protein levels
in Beclin1KO or Beclin1KO + p53KO (DKO) cells. We
found a strong TET2 nuclear accumulation in the DKO cells
compared with that in Beclin1 single KO HEK293T cells
(Fig. 5c). Re-expressing p53 in the DKO cells attenuated
Given that autophagy primarily occurs in the cytoplasm
whereas TET2 predominantly resides within the nucleus,
we asked whether p53 could impinge on the nucleocyto-
plasmic shutting of TET2 to promote its autophagic
degradation. To better study this process, we knocked out a
critical component in the autophagy pathway, Beclin1, in
HEK293T cells. This enables us to suppress autophagy for
better visualization of protein accumulation in the autop-
hagosome (marked by p62) prior to autophagic degradation
of TET2. We first performed immunofluorescence staining
of TET2 in WT and beclin1 knockout (Beclin1KO)
HEK293T cells. Upon genetic depletion of Beclin 1, we
detected a substantial accumulation of TET2 in the cyto-
plasm. By contrast, in native Hek293T cells, the majority of
TET2 was located in the nucleus (Fig. 5a). To further

J. Zhang et al.
A
B
Atg16L1
KO
p62 Beclin1 LC3
KO KO KO
+ Doxycycline
WT
+
-
-
+
-
+
-
+
-
+
Doxycycline
TET2
TET2
p53
Atg16L1
p62
p53
LC3
GAPDH
LC3
HEK293T p53-inducible
GAPDH
HEK293T p53-inducible
C Starvation
D
-
-
+
-
+
+
-
-
+
-
+
+
CQ
TET2
Rap (μg/ml)
TET2
0
0.2 0.4 0.8 1.2
p53
p62
p62
LC3
LC3
GAPDH
GAPDH
HEK 293T
HEK293T
p53KO
p53KO HEK293T
E
F
Doxycycline
BafA1
-
+
-
-
+
+
+
-
-
+
0
-
+
BafA1
Doxorubicin (μM)
TET2
0
2.4 2.4
TET2
p53
p53
p62
p62
LC3
GAPDH
GAPDH
HCT116
HCT116
p53-inducible
Fig. 4 p53 downregulates TET2 through autophagic degradation. a
Western-blotting analysis on TET2, p53 and LC3 in HEK293T cells
with doxycycline inducible expression of p53 in the presence of cor-
responding inhibitors. MG-132 were used to suppress proteosomal
degradation whereas 3-MA, BafA1, NH4Cl and CQ were added to
inhibit autophagy (MG-132: 10 µM; 3-MA: 10 mM; BafA1: 25 nM;
NH4Cl: 5 mM; CQ: 25 µM). b Western blotting to monitor TET2, p53,
Atg16L1 and LC3 levels in p53-inducible HEK293T cells with or
without depletion of the indicated autophagy-related genes. c Western-
blotting analysis on TET2, p53 and autophagy-related proteins (p62
and LC3 in WT and p53KO HEK293T cells under the indicated
culture conditions (i.e., serum-free starvation and/or autophagy
inhibitor treatment with 25 µM chloroquine, CQ). d Endogenous
TET2, p62 and LC3 protein levels in p53KO HEK293T cells treated
with increasing doses of rapamycin (0, 0.2, 0.4, 0.8, 12 µg/ml) for 24 h.
e Western-blotting analysis on TET2, p53, p62 and LC3 in p53-
inducible HCT116 cells with and without bafilomycin A1 (BafA1)
treatment (25 nM for 24 h). Doxycycline was added to induce p53
expression. f Western-blotting analysis on TET2, p53, p62 and LC3 in
WT HCT116 cells with and without bafilomycin A1 (BafA1) treat-
ment (25 nM for 24 h) together with doxorubicin treatment for 24 h.
Doxorubicin was added to induce cellular stress and p53 upregulation
(Fig. 3a)
nuclear TET2 accumulation, suggesting that p53 is essential
for the nuclear exit of TET2 to facilitate cytoplasmic
degradation (Fig. 5c). In parallel, we performed immunos-
taining experiments to examine whether cytosolic TET2
would co-localize with the autophagosome maker, p62 in
Beclin1KO HE293T cells, so as to demonstrate that the
cytosolically accumulated TET2 could be degraded via
autophagy after recruitment toward the autophagosome. We
used Beclin1KO HEK293T cells with inducible expression
of p53 to prevent TET2 degradation in the cytoplasm and
promote its accumulation in autophagosome to increase the
signal. We detected a strong co-localization of TET2 and
p62 (Fig. 5d) in the present of doxycycline (for inducible
p53 expression) when compared with cells without dox-
ycycline treatment. The interaction between TET2 and p53
in Beclin1KO HEK293T cells was further independently

p53-dependent autophagic degradation of TET2 modulates cancer therapeutic resistance
Fig. 5 p53 is required for the
autophagic degradation of
A
B
Beclin1
KO
Beclin1
KO
DAPI
TET2
TET2
WT
WT
TET2. a Confocal images of
native (upper) or Beclin 1
knockout (Beclin1KO; lower)
HEK293T cells stained for
visualization of TET2 (green)
and the nuclei (DAPI). b
Western blotting analysis on the
cytoplasmic and nuclear
fractions of TET2, p53, Beclin1
in WT and beclin1KO
HEK293T
TET2
p53
Beclin1KO
HEK293T
Beclin1
Tubulin
HEK293T cells with and
C
Beclin1KO +
p53KO
without expression of FLAG-
tagged p53. c Western-blotting
analysis on the cytoplasmic (C)
and nuclear (N) fractions of
TET2, p53 in Beclin1KO or
becklin1KO + p53KO (DKO)
HEK293T cells with or without
expression of FLAG-tagged
p53. d Confocal images showing
the distribution of TET2 (green)
and p62 (red) in beclin1KO
HEK293T cells before (upper)
and after (lower) inducible
expression of p53. DAPI was
used to visualize the nuclei.
Shown on the right is the
Beclin1KO
LaminB1
Mock FLAG-p53 Mock FLAG-p53
Cytoplasm
Nucleus
C
N
C
N
C
N
C
N
TET2
p53
Tubulin
Lamin B1
Beclin1KO HEK293T
D
Beclin1KO HEK293T – p53 inducible
DAPI TET2 p62 Merge
quantification of colocalization
of TET2 and p62 (n = 68).
Doxycycline as added to induce
p53 expression. e Co-
immunoprecipitation (co-IP)
confirmed the interaction
p < 0.001
20
15
10
5
between p53 and TET2 in
beclin1KO HEK293T cells
0
DMSO Doxycycline
E
3% Input IP: HA
HA-TET2
-
+
+
+
-
+
+
+
FLAG-p53
IB: FLAG
IgG
IB:HA
Beclin1KO HEK293T
confirmed by co-immunoprecipitation (Fig. 5e). Further-
more, the interaction between TET2 and p53 did not seem
to be affected by the post-translational modifications of p53
(e.g., acetylation and phosphorylation) or by cell cycle
blockers (Supplementary Figure S4A-B).
To determine whether the subcellular localization of p53
could influence autophagic degradation of TET2, we
resorted to several well-studied p53 mutants that have dis-
tinct distribution patterns: R175H (cancer-associated mutant
without affecting its nuclear and cytoplasmic location) [39],
K305A (cytoplasmic accumulation) [40], and L348A/
L350A (nuclear accumulation) [41] (Fig. 6a). In p53KO
cells, notable TET2 degradation was observed following the
re-expression of WT p53 or the R175H mutant. However,
in p53KO cells expressing the K305A or L348A/L350A
mutants, we did not detect a significant decrease in TET2
protein levels (Fig. 6a). Next, we separated the nuclear and
cytoplasmic fractions of cells expressing WT or mutant p53,

J. Zhang et al.
Fig. 6 p53 mutations impair
autophagy-mediated TET2
degradation. a Western-blotting
analysis on TET2 and p53 in
p53KO HEK293T cells
A
C
p53 KO cells + FLAG
tagged p53 variants
expressing WT and the indicated
p53 mutants. K305A and
TET2
L348A/L350A, but not R175H,
are known to cause either
cytosolic or nuclear retention of
p53 proteins. b Confocal images
showing the subcellular
p53
GAPDH
WT
C N C N C N C N C N C N
localization of FLAG-tagged
p53 mutants (red). Nuclei were
stained with DAPI and shown in
blue. c Western blotting analysis
on the cytoplasmic and nuclear
fractions of TET2 and p53 in
p53KO HEK293T cells
expressing WT or the indicated
FLAG-tagged p53 mutants.
Tubulin and lamin B1 were used
to indicate the separation of
cytosolic (C) and nuclear (N)
fractions, respectively
p53KO HEK293T
Anti-FLAG
B
DAPI
TET2
FLAG
(p53)
Tubulin
Lamin B1
expressing WT p53 or the R175H mutant. This was further
confirmed by immunofluorescent staining (Fig. 6c). These
results suggest that proper subcellular localization of p53 is
essential for autophagy-mediated TET2 degradation.
Together, our data established that p53 can interact with
TET2 in the cytoplasm to facilitate its recruitment to the
autophagosome, thereby promoting the autophagic degra-
dation of TET2.
Discussions
Fig. 7 A tentative model depicting how chemotherapy (doxorubicin)
disrupts the p53-associated autophagic degradation of TET2 in p53-
null tumor cells. (Left) In WT cells, p53 facilitates the autophagic
degradation of TET2 through enhancing the TET2 nuclear exit with
subsequent recruitment toward the autophagosome. A balance of p53
and TET2 is needed to maintain the normal cellular function. (Middle)
In p53 KO cells, p53 loss leads to less TET2 protein degradation in the
cytosol but more accumulation in the nucleus during doxorubicin
treatment. Nuclear TET2 protects the genome from DNA damage
imposed by doxorubicin. This ultimately promotes tumor cell growth
and survival, which contributes to the chemotherapy resistance.
(Right) In p53KO and TET2KO (DKO) cells, the protective effect of
TET2 is removed following chemotherapy induced DNA damage,
thereby overcoming therapeutic resistance to restore tumor cell killing
It is well known that tumors bearing genetic alterations in
p53 are commonly aggressive and resistant to anti-cancer
treatment. In this study, we identified a novel interplay
between p53 and TET2 during anti- cancer treatment. We
discovered a negative correlation between p53 and TET2
protein levels in colon cancer cell lines, as well as in pri-
mary colon tumor samples. We demonstrated that p53
facilitates the autophagic degradation of TET2 in both
tumor (HCT116, SW48 and MCF7) and non-tumor cell
lines (HEK293T). Furthermore, we showed that such
negative correlation between p53 and TET2 is essential for
balancing the DNA damage response when cells are chal-
lenged with extracellular stresses, such as anti-cancer ther-
apy (e.g., doxorubicin, cisplatin and gamma-irradiation
treatment). Mechanistically, we revealed that p53 is
required for autophatic degradation of TET2 by promoting
the shuttling of nuclear TET2 toward the cytoplasmic
and further monitored TET2 protein levels using western
blotting. We observed a strong nuclear accumulation of
TET2 in p53 KO cells expressing the K305A or P53-
L348A/L350A mutants (Fig. 6b), but not in those

p53-dependent autophagic degradation of TET2 modulates cancer therapeutic resistance
autophagosome. Deletion of p53 resulted in the dis-
connection between TET2 and autophagosome, which led
to the increase of cellular survival when responding to DNA
damage and therefore promoted anti-cancer therapy resis-
tance in p53-null tumor cells (Fig. 7). Our study establishes
the rationale for targeting TET2 as a potential strategy for
overcoming anti-cancer treatment resistance associated with
deregulated p53.
Previous studies have shown that TET2 is a tumor sup-
pressor gene. Genetic alteration of TET2 are frequently
observed in hematological malignancies. TET2 loss or
downregulation is implicated in tumor initiation and inva-
sion [20, 42]. Furthermore, protective roles of the TET
protein family have been reported by regulating DNA
damage response during cellular malignant transformation
[24, 37, 43].
Materials and methods
Cell culture
HEK293T, HCT116, SW480, HT29, SW48 and MCF-7 cell
lines were purchased from ATCC and cultured in Dulbec-
co’s modified Eagle’s medium (Corning, Manassas, VA,
USA) with 10% fetal bovine serum (Omega, Tarzana, CA,
USA), 1% antibiotics (100 IU penicillin and 100 μg/mL
streptomycin) and 2 mM L-glutamine with 1 mM 2-
mercaptoethanol in a humidified incubator at 37 °C with
5% CO2. The medium was changed every 2 days and cells
were split when reaching a confluence of 80–90%.
Chemicals
Surprisingly, in our study, TET2-mediated protective
effect on DNA damage seems to act as a “double-edged
sword” during anti-cancer treatment. When tumor cells
(e.g., HCT116, SW48 and MCF7) bearing WT p53 are
treated with chemotherapy reagents (e.g, doxorubicin or
cisplatin), p53-mediated TET2 degradation induces sub-
stantial DNA damage, leading to increased tumor cell death.
Conversely, in p53KO HCT116 cells, the high level of
TET2 protein protects the genome from chemotherapy
induced DNA damage and subsequently promotes cell
survival (Fig. 7). Thus, TET2 depletion could at least par-
tially overcome anti-cancer treatment resistance in p53KO
tumor cells.
To date, autophagy-mediated protein degradation is
mainly focused on cytoplasmic components. Recently,
Dou et al. illustrated LC3-mediated nuclear lamin B1
degradation in autophagosome [44], which is essential for
preventing cells from malignant transformation to safe-
guard against tumorigenesis. Our studies demonstrated
that TET2 as a nuclear protein can be translocated from
nucleus to cytoplasm via p53 through direct interaction,
thereby trapping TET2 in the cytosol to promote its
association with autophagosome for ultimate degrada-
tion. The protein stability of TET2 can be modulated by
several pathways that involve caspase [25], calpains [26],
and p300-mediated acetylation [27]. Our study provides
an additional regulatory mechanism with respect to the
post-translational regulation of TET2 in response to
extracellular stress imposed by cancer therapeutics in the
context of a specific genetic background (e.g., p53-null
tumor cells). Our study points to the exciting possibility
that TET2 inhibition might serve as a promising strategy
to reduce cancer therapeutic resistance in p53-null
tumors. Our findings also call for extra cautions during
the future development and application of TET activa-
tors/agonists in the treatment of cancer.
Doxycyclin (D9891), Doxorubicin (D1515), MG-132
(M7449),
3-Methyladenine
(M9281),
Chloroquine
(C6628), Bafilomycin A1 (B1793), Cisplatin (1134357) and
rapamycin (R8781) were purchased from Sigma-Aldrich (St
Louis, MO, USA). Puromycin and Zeocin were purchased
from Thermo Fisher Scientific (Grand Island, NY, USA).
Cell cycle blockers, including MK-1775 (21266), Flavo-
piridol (10009197), Palbociclib (16273), were purchased
from Cayman Chemical Company.
Plasmids
Human TET2, p53, Atg16L1, Beclin1, LC3A, LC3B, p62
were cloned from HEK293T cells using reverse transcription
PCR. p53 mutants (R175H, K305A and L348A + L350A)
were constructed by using the QuikChange site-directed
mutagenesis kit (Catalog #200523) from Agilent Inc. For
transient expression in mammalian cells, the cDNA encod-
ing human TET2 was cloned into a modified pcDNA3-Flag-
Dest-B vector. The CRISPR/Cas9 genome editing construct
PX459 (62988; Addgene) was used for sgRNA- directed
knockout of TET2, p53, Atg16L1, Beclin1, LC3, p62.
TET2 sgRNA-1: 5′-caccgTGGAGAAAGACGTAACT
TCG-3′ TET2 sgRNA-2: 5′-caccgCAGGACTCACACG
ACTATTC-3′ p53 sgRNA-1: 5′-caccgCCATTGTTCAA-
TATCGTCCG-3′ p53 sgRNA-2: 5′-caccgCCCCGGAC-
GATATTGAACAA-3′ Atg16L1 sgRNA: 5′-caccgAGATG
TGGCGCTTCCAGCGG-3′ Beclin1 sgRNA: 5′-caccgG-
GACACGAGTTTCAAGATCC-3′ p62 sgRNA: 5′-caccg-
CACCGTGAAGGCCTACCTTC-3′ LC3 sgRNA: 5′-caccg
ACCTCCTTACAGCGGTCGGC-3′.
The sgRNA-containing plasmids were transfected into
HEK293T cells and subjected to puromycin selection (2 µg
ml−1) for 2–4 days. Survival clones were then seeded into
96-well plates and later expanded to 24-well plates for
maintenance in the presence of 1 µg ml−1 puromycin. The

J. Zhang et al.
gene disruption was confirmed by Western blotting. All the
plasmid information was listed in Table S2.
p53-11-F: 5′-AAGTCAGCTGTATAGGTACTTGAAG
TGC-3′, p53-11-R: 5′-TCAGCTGCCTTTGACCATGAA-
3′;
p53-12-F: 5′-ACCATCTTGATTTGAATTCCCG-3′, p53-
12-R: 5′-CCCCAGCCCACACTCATTG-3′.
Xenograft models
1 × 106 HCT116 cells were suspended in 50 µl PBS mixed
with an equal volume of pathogen-free BD Matrigel.
Basement Membrane Matrix were implanted sub-
cutaneously into the flank of NOD scid gamma mice (6–
8 weeks old, Jackson laboratory, 005557). Doxorubicin was
injected at 4 mg kg−1 every two days starting at day 4 after
tumor implantation for 4 weeks via intraperitoneal injection.
The tumor volume was measured with a caliper every week
according to the formula: 0.52 × length × width × height. All
animal experiments in this study were approved by the
Texas A&M University IACUC Review Panel.
Exome sequencing was performed using the BigDye
Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher,
catalog #4337458) on an ABI 3730xl DNA analyzer
(Thermo Fisher Scientific, Waltham, MA, USA).
Nuclear and cytoplasmic proteins extraction
Cells (1 × 107) were resuspended with cytoplasmic and
nuclear protein lysis buffer A (10 mM HEPES, pH 7.9, 10
mM KCl, 1 mM DTT, 0.1 mM EDTA) supplemented with
protease inhibitor and phosphatase inhibitor cocktail (Gen-
deport, Barker, TX, USA) on ice for 25 min. Next, 5 μl of
10% NP-40 was added into the samples and incubated on
ice for another 2 min. After centrifugation at 5,000 rpm for
3 min, the supernatant was collected as cytoplasmic
extracts. The nuclear pellet was further cleared by cen-
trifugation at 12,000 r.p.m. for 3 min followed by removal
of supernatant. The pellet was further lysed by 50 μl of
cytoplasmic and nuclear protein lysis buffer C (10 mM
HEPES, pH 7.9, 400 nM NaCl, 1 mM EDTA, 1 mM DTT).
After incubation for 1 h at 4 °C, the samples were cen-
trifuged at 12,000 r.p.m. for 15 min and the supernatant
(nuclear fractions) was collected.
Human subjects
A total of 12 tumor samples were collected from patients
with colorectal cancer (CRC) at the Renmin Hospital of
Wuhan University. Detailed clinical and pathological data
were summarized in Table 1. All tissue samples were
obtained from patients with approval from Institutional
Review Board of Renmin hospital of Wuhan University
China (No. 2018K-C007). Informed consent was obtained
from the individual patient.
P53 exome sequencing
Western Blotting and Co-immunoprecipitation
p53 exome sequencing was performed by following the
manufacturer’s protocols from Tsingke Biological Tech-
nology Inc. Genomic DNA was isolated from freshly frozen
CRC tissues using the Trelief TM Animal Genomic DNA
Kit (catalog #TSP201-200) and the Maxwell® 16 MDx
Instrument (Promega Corp., Madison, WI, USA). Purified
genomic DNA was then quantified using the Qubit® dsDNA
HS Assay Kit (catalog #Q32851) and a Qubit 2.0 fluo-
rometer (Thermo Fisher Scientific, Waltham, MA, USA).
Seven pairs of primers were designed to capture the p53
exome:
p53-1-F: 5′-CCTCCTCCCCAACTCCATTTC-3′, p53-
1-R: 5′-CCGAGAGCCCGTGACTCAGA-3′;
p53-2-3-4-F: 5′ -GGAAGCCGAGCTGTCTCAGACA-
3′, p53-2-3-4-R: 5′-GGGGATACGGCCAGGCAT-3′;
p53-5-6-F: 5′-GGTGTAGACGCCAACTCTCTCTAG
C-3′, p53-5-6-R: 5′-GGCCACTGACAACCACCCTTAA-
3′;
Tumor samples collected from CRC patients were flash-
frozen in liquid nitrogen and then lysed in ice-cold lysis
buffer (20 mM Tris-HCl pH 7.8, 150 mM NaCl, 2 mM
EDTA pH 8.0, 1% NP40, 10% glycerol, supplemented with
10 mM NaF, 10 mg/mL Leupeptin, 200 mM Na2VO4, 5 mM
PMSF, and Aprotinin) (Sigma-Aldrich). Homogenization
was performed on ice with a POLYTRON® system PT 1200
E (Kinematica). Lysates were centrifuged at 13.000 r.p.m.
4℃ for 30 min and the supernatants were collected.
Cells were lysed on ice by using RIPA lysis buffer (50
mM Tris-HCl pH 7.4, 1% NP-40, 0.5% Na- deoxycholate,
0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 Mm NaF)
supplemented with protease inhibitor and phosphatase
inhibitor cocktail (Gendeport, Barker, TX, USA). The total
protein concentration was measured by the BCA protein
assay kit (Thermo, Rockford, IL, USA). The protein
extractions were denatured by SDS-sample buffer and
incubated at 95 °C for 10 min. The denatured protein mix-
ture was subjected to SDS-PAGE, subsequently transferred
to nitrocellulose membranes (Millipore, Billerica, MA,
USA), and blocked with 5% non-fat milk in Tris-buffered
saline pH 7.6 containing 0.1% Tween-20. Then the
p53-7-F: 5′-GAGGCTGAGGAAGGAGAATGG-3′,
p53-7-R: 5′-GCCCAGGGGTCAGAGGCA-3′;
p53-8-9-10-F: 5′-AAGGGTGGTTGGGAGTAGATGG-
3′, p53-8-9-10-R: 5′-GCAGGCTAGGCTAAGCTATGA
TG-3′;

p53-dependent autophagic degradation of TET2 modulates cancer therapeutic resistance
membranes were probed with the corresponding primary
antibodies overnight at 4 °C. After washing with TBS-T
buffer for three times (10 min for each wash at room tem-
perature), the membranes were incubated with an anti-rabbit
secondary antibody (1:10,000, sigma, Cat# 7074) or anti-
mouse secondary antibody (1:3000, Cell Signaling Tech-
nology, Cat# 7076) for 1 h at room temperature followed by
washing with TBST three times (10 mins for each wash at
room temperature). The antigen–antibody complexes were
detected using West-Q Pico Dura ECL Solution (Gende-
port, Barker, TX, USA). The intensities of protein bands on
the films were quantified by scanning densitometry using
the Image J software (NIH).
performed using amfiSure qGreen q- PCR Master Mix
without ROX (Gendeport, Barker, TX, USA).
The primer sequences were listed as follows:
p53 forward: 5′-CAGCACATGACGGAGGTTGT-3′,
p53 reverse: 5′-TCATCCAAATACTCCACACGC-3′;
TET2 forward: 5′-ATACCCTGTATGAAGGGAAGCC-3′,
TET2 reverse: 5′-CTTACCCCGAAGTTACGTCTTTC-
3′; GAPDH forward: 5′-GCACCGTCAAGGCTGAGAAC-
3′, GAPDH reverse: 5′-TGGTGAAGACGCCAGTGGA-
3′.
The Dot blot assay
For co-immunoprecipitation, cells were lysed on ice with
CHAPS lysis buffer (Gendeport, Barker, TX, USA) including
50 mM Tris-HCl (pH 7.4), 110 mM NaCl, 5 mM EDTA and
1% CHAPS, supplemented with protease inhibitors and
phosphatase inhibitors cocktail (Gendeport, Barker, TX,
USA) for 5 min, and then subjected to centrifugation at
12,000 rpm at 4 °C for 5 min. Subsequently, cell lysate was
incubated with indicated antibodies and magnetic A/G beads
(Thermo Fisher Scientific) overnight at 4 °C. The beads were
washed with the wash buffer (50 mM Tris-HCl, pH7.5, 150
mM NaCl, 1 mM EDTA and 0.5% Triton X-100, containing
10 mg/ml pepstatin A, 10 mg/ml leupeptin and 1 mM PMSF)
and heated in SDS loading buffer at 95 °C for 5 min. Then the
cell lysate was subjected to SDS-PAGE.
The primary antibodies used in the study include: mouse
anti-TET2 (1:500, Active motif, Cat#61389), rabbit anti-
TET1 (1:500, Active Motif, Cat#61443), mouse anti-p53
(1:2500, Santa Cruz, Cat# sc-126), mouse anti-Flag
(1:3000, Sigma, Cat#F1804), mouse anti-HA (1:1000,
Santa Cruz, Cat#sc-7392), rabbit anti- ATG16L1 (1:1000,
Cell Signaling Technology, Cat#8089), rabbit anti-Beclin1
(1:1000, Cell Signaling Technology, Cat#3495), rabbit anti-
p62 (1:1000, Santa Cruz, Cat#sc-28359), rabbit anti-LC3
(1:1000, Cell Signaling Technology, Cat#12741), rabbit
anti-γH2AX (1:1000, Cell Signaling Technology,
Cat#9718), mouse anti-Tubulin (1:2000, Sigma,
Cat#TG074), rabbit anti-LaminB1 (1:3000, Abcam,
Cat#ab16048), and rabbit anti-GAPDH (1:10000, Cell
Signaling Technology), rabbit anti Phospho-p53-Ser15
(1:1000, Cell Signaling Technology) and rabbit anti
Acetyl-p53-Lys382 (1:1000, Cell Signaling Technology).
Genomic DNA from cells was isolated using NucleoSPIN®
Tissue (Macherey-Nagel, Bethlehem, PA, USA). 5 μg of
DNA was denatured in 1 M NaOH, 25 mM EDTA at 95 °C
for 10 min. Then the samples were neutralized with ice-cold
2 M ammonium acetate (pH 7.0) followed by twofold serial
dilution of DNA samples, with each being spotted on a
nitrocellulose membrane in an assembled Bio-Dot apparatus
(Bio- Rad). DNA samples were filtered under vacuum
pressure and the membrane was washed with 2 × SSC
buffer, air-dried at room temperature for 20 min and
vacuum-baked at 80 °C for 2 h. Next, the membrane was
blocked in 5% milk at room temperature for 1 h and incu-
bated with a rabbit anti-5-hmC primary antibody (1:5000,
Active motif, Cat#39769) overnight at 4 °C. The membrane
was washed with TBST buffer for 10 min three times and
then incubated with an anti-rabbit secondary antibody
(1:10,000, sigma, Cat#7074) for 1 h at room temperature.
Next, the membrane was washed for 10 min three times and
visualized by chemiluminescence with West-Q Pico Dura
ECL Solution (Gendeport, Barker, TX, USA). Finally, the
membrane was washed with TBST and stained with 0.02%
methylene blue in 0.3 M sodium acetate (pH 5.2) to ensure
equal spotting of total DNA on the membrane.
Immunofluorescent staining
Cells (2 × 106/ml) were seeded and grown on sterile cover
slips in 12-well plates. Following treatment, cells were
washed with warm PBS and fixed in 4% paraformaldehyde
for 10 min. Fixed cells were then washed
three times with PBS, permeabilized with 0.2% Triton X-
100 for 15 min and blocked in 5% BSA for 30 min. Next,
cells were incubated with primary antibodies: mouse anti-
TET2 (1:100, Active motif, Cat#61389), mouse anti-p53
(1:500, Santa Cruz, Cat#sc-126) and rabbit anti-p62
(1:1000, Santa Cruz, Cat#sc-28359) followed by washing
three times with PBS, and incubation with Alexa Fluor®
488 goat anti-mouse (Invitrogen, 1:2000, Cat#A11001) or
an Alexa Fluor® 568 goat anti-rabbit (Invitrogen, 1:2000,
Cat#11011) secondary antibody for 1 h at room
RNA extraction, reverse transcription and
quantitative real-time PCR
Total RNA was extracted with the TRIzol RNA isolation
reagent (Thermo Fisher Scientific, Grand Island, NY, USA).
cDNA synthesis was carried out by using the amfiRivert
Platinum One cDNA Synthesis Master Mix (Gendeport,
Barker, TX, USA). Quantitative real-time PCR were

J. Zhang et al.
temperature. Cells were then washed 3 times with PBS and
mounted with Prolong Gold (Life Technologies). Cells were
imaged with ×40 oil lens on an inverted Nikon Eclipse Ti-E
microscope customized with A1R confocal imaging mod-
ules and the images were analyzed by NIS- Elements
software (Nikon) or Image J (NIH).
YH, R01GM112003 to YZ), the Welch Foundation (BE-1913 to YZ),
the American Cancer Society (RSG-18-043-01-LIB to YH, RSG-16-
215-01-TBE to YZ), and by an allocation from the Texas A&M
University start- up funds (YH).
Author contributions YH and YZ conceived, directed and oversaw the
project. JZ, JM and W-GD collected CRC samples, p53 mutation
analysis and western blotting analysis. JL and DS analyzed the CRC
western quantification data. JZ and LG performed the western blotting,
doxorubicin / cisplatin treatment, gamma irradiation experiments. JZ
and LG performed WST-1, colony-forming assays and quantitative
real-time PCR analysis. JZ and ML performed the dot-blot assay. JZ
and SF performed immunofluorescent staining. JZ generated majority
cell lines used in this study. TP provided cell lines lacking autopha-
gosome components and cell cycle blocking experiments. JG and LH
performed the mutational analysis in AML patients. GJ and RD pro-
vided colon cancer cell lines. JJ, W-MC, RD and W-GD provided
intellectual inputs. YH and YZ wrote the manuscript with all the other
authors participating in discussion, data interpretation and manuscript
editing.
Cell viability assay
WST-1 (Sigma-Aldrich, Cat# 05015944001) was used to
quantitatively assess cell viability. Cells (5 × 103) were
seeded into 96-well plates. On day 2, the medium was
replaced and cells were cultured in fresh medium containing
doxorubicin at different concentrations for 12, 24, 36, 48
and 60 h. Then 10 μl WST-1 solution with 90 μl DMEM
medium were added to each well, and cells were incubated
at 37 °C for 1 h. The absorbance at 450 nm was determined
by using a Cytation 5 microplate reader (BioTek, Winooski,
USA). DMEM containing 10% WST-1 was used as nega-
tive control.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Colony formation assay
References
Cells (3 × 104 cells) were seeded in 48-well plates. The next
day, corresponding treatments were applied. Cells were then
washed with PBS two times 5 days later. After fixation with
100% methanol at room temperature for 15 min and wash
with PBS two times, the colonies were stained using crystal
violet (0.25%, Sigma-aldrich, Cat#C3886) at room tem-
perature for 5 min and photographed.
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