Discovery of a new autophagy inducer for A549 lung cancer cells

Article abstract of DOI:10.1016/j.bmc.2019.05.015

Biological activities of a series of fluorescent compounds against human lung cancer cell line A549 were investigated. The results showed that (E)-1,3,3-trimethyl-2-(4-(piperidin-1-yl)styryl)-3H-indol-1-ium iodide (8) and (E)-2-(5,5-dimethyl-3-(4-(piperazin-1-yl)styryl)cyclohex-2-en-1-ylidene) malononitrile (11) could inhibit the growth of A549 cancer cells in a dose and time-dependent manner. Furthermore, compound 8 could trigger autophagy and apoptosis, but not obviously induce necrosis under the stimulatory condition. Therefore, 8 can be used as autophagy activator to investigate the regulatory mechanism of autophagy and may offer a new candidate for the treatment of lung cancer.

Full text of DOI:10.1016/j.bmc.2019.05.015

Accepted Manuscript
Discovery of a new autophagy inducer for A549 lung cancer cells
Na Li, GuoJing Qu, JingNa Xue, Xiao Li, Xuan Zhao, YeHao Yan, DongFang
Gao, Lu Zhang, Peng Wang, Ming Zhang, BaoXiang Zhao, JunYing Miao,
ZhaoMin Lin
PII:
S0968-0896(19)30091-4
https://doi.org/10.1016/j.bmc.2019.05.015
BMC 14902
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
17 January 2019
28 April 2019
9 May 2019
Please cite this article as: Li, N., Qu, G., Xue, J., Li, X., Zhao, X., Yan, Y., Gao, D., Zhang, L., Wang, P., Zhang,
M., Zhao, B., Miao, J., Lin, Z., Discovery of a new autophagy inducer for A549 lung cancer cells, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.05.015
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Discovery of a new autophagy inducer for A549 lung cancer cells
Na Li,1 GuoJing Qu,1 JingNa Xue,1 Xiao Li,1 Xuan Zhao,1 YeHao Yan,2 DongFang
Gao3, Lu Zhang3, Peng Wang3, Ming Zhang3, BaoXiang Zhao,2 JunYing Miao,1
ZhaoMin Lin3 *
1
Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology,
School of Life Science, Shandong University, Jinan 250100, P.R. China
2
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering,
Shandong University, Jinan 250100, P.R. China
3
Institute of Medical Science, the Second Hospital of Shandong University, Jinan
250033, P.R. China. E-mail: linzhaomin@sdu.edu.cn.
*Correspondence to: Dr. ZhaoMin Lin, Institute of Medical Science, the Second
Hospital of Shandong University, Jinan 250033, P.R. China.
Fax: + 86 531 88565610; Tel.: + 86 531 88364929.
E-mail address: linzhaomin@sdu.edu.cn.
Abstract
Biological activities of a series of fluorescent compounds against human lung cancer
cell line A549 were investigated. The results showed that (E)-1,3,3-trimethyl-2-(4-
(piperidin-1-yl)styryl)-3H-indol-1-ium iodide (8) and (E)-2-(5,5-dimethyl-3-(4-
(piperazin-1-yl)styryl)cyclohex-2-en-1-ylidene) malononitrile (11) could inhibit the
growth of A549 cancer cells in a dose and time-dependent manner. Furthermore,
compound 8 could trigger autophagy and apoptosis, but not obviously induce necrosis

under the stimulatory condition. Therefore, 8 can be used as autophagy activator to
investigate the regulatory mechanism of autophagy and may offer new candidate for
the treatment of lung cancer.
Keywords: Small molecules; Autophagy; Apoptosis; A549 lung cancer cells; mTOR
1. Introduction
With the increasingly serious environmental problems and the dramatic increase of
smoking rate, lung cancer is becoming primary cause of cancer-related mortality not
only in China but also around the world1-5. There was approximately 85% of the lung
cancers were non-small cell lung cancer (NSCLC)6, 7. Many approaches for cancer
treatment, including surgery, radiation therapy, hormonal therapy, chemotherapy
(either alone or in combination), and targeted therapy have emerged. Considering
resistance to drug, much attention has recently been paid to the discovery and
development of new, more selective anticancer agents 8, 9
.
Autophagy is an important metabolic regulation mechanism that could remove
dysfunctional organelles and extra macromolecules to protect against cellular stress10,
11. More and more evidence have shown that autophagy plays a crucial role in many
human physiological and pathological processes, including tumors12, infection13,
aging14 and cardiovascular diseases15. Many research revealed that autophagy
deficiency has a positive effect on malignant transformation16, indicating autophagy
may be a potential anti-tumor target.
Chemical small molecules have become progressively more important tools in recent

years to investigate fundamental biological mechanisms and processes of cancers17-
24. Organic small molecules have also achieved greater prominence alongside
complementary reagent for target validation in drug discovery25. As a continuation of
our study on the discovery of new anti-cancer small molecules9, 22-24, in the present
study, we screened a series of compounds prepared in our lab (Chart 1) and
investigated their biological properties. We found that (E)-1,3,3-trimethyl-2-(4-
(piperidin-1-yl)styryl)-3H-indol-1-ium iodide (8) and (E)-2-(5,5-dimethyl-3-(4-
(piperazin-1-yl)styryl)cyclohex-2-en-1-ylidene) malononitrile (11) could inhibit the
growth of A549 cells by activating apoptosis and autophagy.

Chart 1 Chemical structures of the compounds.
2. Results and Discussion
2.1. Compounds 8 and 11 reduced cell viability of A549 cell.
Firstly, we screened a series of small chemical molecules (Chart 1) from the
compound library of our lab to study their anti-cancer biological effects. SRB assay
was used to determine the effects of the molecules on the viability of human lung cell
line A549. The results showed that many of the compounds had obvious cytotoxicity

to A549 cells (Fig. 1–3, Table 1). 8 and 11 could inhibit A549 cell growth in a dose
and time-dependent manner and had lower IC50 values. Therefore, they were selected
to explore their biological functions and mechanisms in A549 cells.
Fig. 1 Effects of compounds 1–6 on A549 cells viability. A549 cells were treated with
the compounds and 5-FU at the concentration of 0.1, 1 and 10μM or DMSO (0.1%,
v/v, Ctr) for 24 and 48h, respectively. The cell viability was analyzed by SRB assay
and illustrated in column figures. Results are presented as mean ± SE; (n = 3. *p<
0.05; **p< 0.01 vs control).

Fig. 2 Effects of compounds 7–11 on A549 cells viability. A549 cells were treated
with compounds and 5-FU at the concentration of 0.1, 1, 5, 10 and 20μM or DMSO
(0.1%, v/v, Ctr) for 24 and 48h, respectively. The cell viability was analyzed by SRB
assay and illustrated in column figures. Results are presented as mean ± SE; (n = 3.
*p< 0.05; **p< 0.01 vs control).

Fig. 3 Effects of compounds 12–16 on A549 cells viability. A549 cells were treated
with compounds and 5-FU at the concentration of 0.1, 1 and 10μM or DMSO (0.1%
v/v, Ctr) for 24 and 48h, respectively. The cell viability was analyzed by SRB assay
and illustrated in column figures. Results are presented as mean ± SE; (n = 3. *p<
0.05; **p< 0.01 vs control).

Table. 1 Growth inhibitory properties (IC50, 48h) of compounds 8–11 and 5FU in
A549 cells. Results are mean ± SEM.
Compounds 5-FU
8
9
10
11
IC50(μM)
7.1±0.36
7.1±0.42
1.1±0.12
3.9±0.21
13.6±0.22
2.2. Compounds 8 and 11 induced the morphological change of A549 cells.
In order to find out how 8 and 11 impact the viability of A549 cells, we firstly
observed the morphological change of A549 cells after treated by 8 and 11 for 3, 6, 12
and 24h at different concentrations by using a phase contrast microscope (Fig.4).
Compared with the control group, the density of A549 cells decreased with increase
of compound concentration and treating time. Moreover, we observed that the
apoptosis body released after treated by 8 and 11.

Fig. 4 Compounds 8 and 11 induced morphological changes of A549 cells. A549 cells
were treated with 8 (A) and 11 (B) at the concentration of 0.1, 1, 5, 10 and 20 μM or
DMSO (0.1% v/v, Ctr) for different time, respectively. Microscopic photographs
(200×) were taken under an inverted phase contrast microscope (Nikon). Scale bar: 20
μm.

2.3. Effects of compounds 8 and 11 on LDH activity of A549 cell.
Necrosis is an alternative mode of programmed cell death and will induce an
inflammatory response in the surrounding tissue when cells are exposed to extreme
injury or other stimuli. Necrosis is usually avoided because of its detrimental effects26-
28. In order to find whether 8 and 11 will provoke necrosis, we performed the LDH
assay. There was no significant change of LDH activity between treated samples (0.1,
1, 5, 10μM) and the control group (Fig. 5), except the higher concentration of 11
(10μM). Therefore, 8 and 11 did not induced obviously necrosis in A549 cells at
lower concentrations (0.1, 1 and 5μM).
Fig. 5 Effects of the compounds 8 and 11 on the release of LDH from A549 cells. The
culture media from the cells treated with the compounds at the concentration of 0.1, 1,
5 and 10 μM for 24h, respectively. Results are mean ± SEM (*p < 0.05 vs control, n =
3).
2.4. Compounds 8 and 11 induced apoptosis in A549 cells.

To investigate whether 8 and 11 can induce apoptosis in A549 cells, we detected the
activation of poly (ADP-ribose) polymerase (PARP). Pervious investigation shows
that PARP is a key protein in DNA repair and death of cells29. In addition, up-
regulation of cleaved-PARP is a major characteristic of cell apoptosis. The results
showed a significant increase in the level of cleaved-PARP after A549 cells were
treated by 8 and 11 (Fig. 6). Therefore, 8 and 11 could induce apoptosis of A549 cells.
Fig. 6 The protein level of PARP in A549 cells treated by compounds 8 (A) and 11
(B) at the concentration of 1, 5 and 10 μM or DMSO (0.1%, v/v, Ctr) for 24h,
respectively. The relative levels of cleaved-PARP were normalized by the level of β-
actin, and represented as ratio of control. Results are mean ± SEM (*p < 0.05 vs
control, n = 3).
2.5. Compounds 8 and 11 induced autophagy in A549 cells.

Subsequently, AO (Acridine Orange) staining assay was performed to test whether
acidic vesicles accumulate in A549 cells. After A549 cells were treated by 8 and 11,
acidic vesicles were slightly increased (Fig. 7). Therefore, we could postulate that
autophagy might occur in A549 cells.
The microtubule-associated protein 1 light chain 3 (LC3) plays an important role in
autophagic vacuole formation30. An main characteristic of autophagosome is that
LC3-I conjugates with phosphatidylethanolamine (PE) and then change into LC3-II31.
It has been proved that p62 (sequestosome-1) will directly bind to LC3-I and LC3-II
through the LC3 interacting region (LIR)32. Afterwards, p62 and LC3-II positive
bodies will be degraded by autophagy. Western blot was used to determine the
changes in the level of LC3-I, LC3-II and p62 protein in A549 cells with treatment of
8 and 11. Our results showed that the level of LC3-II significantly increased
compared to control groups after A549 cells were treated by 8 and 11 (Fig.8A, 8B). In
addition, the level of p62 decreased compared to control groups after treated by 8
(Fig. 8A). However, the level of p62 increased compared to control groups after
treated by 11 (Fig. 8B). Accordingly, we speculated that 8 and 11 induced autophagy
but autophagic flux was blocked in the presence of 11. To further confirm that 8 could
induce autophagy, we observed the effect of 8 on the formation of punctiform pattern
of LC3-Ⅱ, using U87 cells with stable transfection of green fluorescent protein-
labeled LC3-Ⅱ. 8-treated cells appeared a punctiform pattern of GFP-LC3 compared
with control groups. Moreover, the level of punctiform pattern of LC3-Ⅱ increased in
a dose-dependent manner (Fig. 8C). To confirm whether autophagic flux was intact

after A549 cells were treated by 8, western blot were performed to observe the
changes in the level of LC3-I, LC3-II in A549 cells treated by 8 alone and 8 plus
Bafilomycin A1. Bafilomycin A1 can disrupt autophagic flux through inhibiting V-
ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-
lysosome fusion independently33. Disruption of autophagic flux could result in the
accumulation of LC3-Ⅱ. Our results showed that LC3-Ⅱ accumulated more in A549
cells treated by 8 plus Bafilomycin A1 than the controls and 8 (10μM) alone (Fig. 9).
So autophagic flux in A549 cells was intact after treatment by 8 (10μM).
Consequently, 8 could induce autophagy in A549 cells.
The signaling pathway of mTOR is a classic signaling pathway for the control of
autophagy. Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and
p70S6K are involved in the signaling pathway. To explore the mechanism by which 8
induces autophagy, the level of phosphorylation of p70S6K in A549 cells was
examined. p70S6K is one substrate of mTORC1 that is one of two complexes of
mTOR enzyme 34. The results showed that the phosphorylation of p70S6K (P-
p70S6K) decreased when A549 cells were exposed to 8 (10μM) for 3, 12 and 24h
(Fig. 10). Therefore, 8 could inhibit the phosphorylation of p70S6K, which might
result from the inhibition of mTORC1 signal pathway.

Fig. 7 AO stain assay was used to detect the level of acidic vesicle of A549 cells after
treated with compounds 8 (A) and 11 (B) at different concentrations for 12h and 24h.
Scale bar: 10 μm.

Fig. 8 Effects of compounds 8 and 11 on autophagy in A549 cells. A549 cells were
treated with compounds 8 (A) and 11 (B) at the concentration of 1, 5 and 10 μM or
DMSO (0.1%, v/v, Ctr) for 24h, respectively. The protein levels of LC3-II and p62
were detected by Western blot analysis. The relative levels of LC3-II and p62 were
normalized by the level of β-actin, and represented as a ratio of control. Results are
mean ± SEM (*p < 0.05, **p < 0.01 vs control, n = 3). (C) After treating EGFP-
MAP1LC3B U87 cells with compound 8 (1, 5, 10μM) or DMSO (0.1%, v/v, Ctr) for
24h, the punctuate patterns of LC3 were observed under a laser scanning confocal
microscope. Results are mean ± SEM (*p < 0.05, **p < 0.01 vs control. n = 3), Scale
bar: 10 μm.

Fig. 9 Compound 8 induced intact autophagic flux. The protein levels of LC3-II were
detected by Western blot analysis after treated by compound 8 (10μM) and
Bafilomycin A1 (10μM). The relative levels of LC3-II were normalized by the level
of β-actin, and represented as a ratio of control. Results are mean ± SEM (*p < 0.05
vs control, n = 3).

Fig. 10 Compound 8 inhibited mTORC1 activity. A549 cells were treated with
compound 8 (10 μM) for different time. The phosphorylation levels of p70S6K (P-
p70S6K) were detected by Western blot. Densitometry results were the ratio of P-
p70S6K to p70S6K, normalized by the level of β-actin. Results are mean ± SEM (*p
< 0.05 vs control, n = 3).
3. Conclusion
In conclusion, we screened a series of compounds and investigated their biological
activities against human lung cancer cell line A549 in vitro. We observed that
compounds 8 and 11 could inhibit growth of A549 cells in a dose-dependent and

time-dependent manner and induce apoptosis and autophagy. Furthermore, we found
that 8 could inhibit the phosphorylation of p70S6K, indicating that it might induce
autophagy by inhibiting mTORC1 pathway. Therefore, 8 could be used to investigate
the molecular mechanisms of apoptosis and autophagy regulation and might be a
potential candidate for a new anticancer drug.
4. Materials and methods
4.1. Synthesis of chemical small molecules
4.1.1. Phenothiazinederivatives 1 and 2
10-(3-Chloropropyl)-10H-phenothiazine-3-carboxylic acid 5,5-dioxide (1) and 1-(3-
(3-carboxy-5,5-dioxido-10H-phenothiazin-10-yl)propyl)-4-methylpyridin-1-ium
chloride (2) were synthesized from phenothiazine by three or four steps (Scheme 1).
To a mixture of 1-chloro-3-iodopropane (6.133 g, 0.03 mol) and NaH (3 g, 0.075
mol) in THF (25 ml) at 0℃, phenothiazine (5 g, 0.025 mol) in THF (25 ml) was
added. The mixture was stirred at room temperature for 9 h. After work-up, 10-(3-
chloropropyl)-10H-phenothiazine was obtained in37% yield.
A mixture of DMF (3ml, 7.13 mmol) and POCl3 (1.5 ml, 8.22 mmol) was stirred at
0oC for 1h to produce Vilsmeier reagent. Then a solution of 10-(3-chloropropyl)-
10H-phenothiazine (1.551 g, 5.48 mmol) in 1, 2-dichloroethane (20 ml) was added.
The mixture was refluxed for 11 h. After work-up, 10-(3-chloropropyl)-10H-
phenothiazine-3-carbaldehyde was obtained in 32% yield.
To a solution of 10-(3-chloropropyl)-10H-phenothiazine-3-carbaldehyde (1.200 g,
3.95 mmol) in a mixed solvent of THF and H2O (20 ml, 1:1, v/v), excess

KMnO4(6.344 g) was added in batches at 60 oC and stirred for 6 h. After work-up, 10-
(3-chloropropyl)-10H-phenothiazine-3-carboxylic acid 5, 5-dioxide (1) was afforded
in 66% yield. 1H NMR ((DMSO-d6，300 MHz), (ppm): 13.36 (s, 1H), 7.97-8.52 (m,
3H), 7.72-7.81 (m, 3H), 7.46 (S, 1H), 4.47 (t, J = 6.9 Hz, 2H), 3.75 (t, J = 6.6 Hz,
2H), 2.18-2.28 (m, 2H).
The mixture of compound 1(100 mg, 0.284 mmol), 4-methypyridine (132 mg, 1.418
mmol) and KI (catalytic amount) in acetonitrile (25 ml) was refluxed for 10 h. After
work-up,1-(3-(3-carboxy-5,5-dioxido-10H-phenothiazin-10-yl)propyl)-4-
methylpyridin-1-ium chloride (2) was obtained in 75% yield.1H NMR (DMSO-d6,
300 MHz),  (ppm): 8.94 (s, 1H), 8.47 (s, 2H), 8.23 (d, J = 9.0 Hz, 1H), 8.06 (d, J =
7.8 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H),7.80-7.97 (m, 3H), 7.47 (t, J = 7.5 Hz, 1H), 7.29
(s, 1H), 4.46-4.51 (m, 2H), 3.20-3.50(m, 2H), 2.35-2.25 (m, 2H), 2.34(s, 3H).
4.1.2. Carbazole derivatives 3 and 4
9-(3-Chloropropyl)-9H-carbazole-3-carbaldehyde (3) was synthesized according to a
literature procedure35.
Compound 4 was synthesized by the reaction of compound 3 with piperazine in the
presence of K2CO3 and KI in DMF.1H NMR (CDCl3, 300 MHz),  (ppm): 10.10 (s,
1H), 8.61 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.99 (dd, J = 1.2 and 7.8, 1H), 5.85 (d, J
= 8.7, 1H), 7.53-7.55 (m, 2H), 7.30-7.35 (m, 1H), 4.46 (t, J = 6.6 Hz, 2H), 2.88-2.95
(m, 4H), 2.25-2.53 (m, 7H), 2.04-2.10 (m, 2H).
4.1.3. 3-Hydroxyflavone derivatives
4-(3-Hydroxy-4-oxo-4H-chromen-2-yl) benzoic acid (5) and N,N-diethyl-4-(3-

hydroxy-4-oxo-4H-chromen-2-yl) benzamide (6) were synthesized according to our
previous paper36.
4.1.4. Indolium iodide and 2-styrylindolium derivatives
1,2,3,3-Tetramethyl-3H-indol-1-ium iodide (7) was synthesized according to literature
procedure37.
According to a literature procedure 38, the 2-styrylindolium derivatives (8-10) were
obtained by the condensation of compound 7 with the respective carbonyl
components in acetic acid as a reaction medium under reflux for 3-10 h in yields of
about 80%.
(E)-1,3,3-trimethyl-2-(4-(piperidin-1-yl)styryl)-3H-indol-1-ium iodide(8)
1H NMR (DMSO-d6, 300 MHz),  (ppm): 2.29 (d, J = 15.6 Hz, 1H), 8.06 (d, J =
9 Hz, 2H), 7.70-7.79 (m, 2H), 7.46-7.58 (m, 2H), 7.27 (d, J =15.6 Hz, 1H), 7.08 (d, J
= 9 Hz, 2H), 3.98 (s, 3H), 1.62 (s, 6H).
(E)-2-(4-aminostyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide (compound 9)
1H NMR (DMSO-d6, 300 MHz),  (ppm): 8.24 (d, J = 15.6 Hz, 1H), 7.96 (d, J =
8.7 Hz, 2H), 7.70-7.80 (m, 2H), 7.44-7.55 (m, 2H), 7.18 (d, J= 15.6 Hz, 1H), 7.03 (s,
2H), 6.70 (d, J = 9 Hz, 2H), 3.96 (s, 3H), 1.73 (s, 6H).
(E)-1,3,3-trimethyl-2-(4-(piperazin-1-yl)styryl)-3H-indol-1-ium iodide (10) was
reported in our previous paper39.
4.1.5.
(E)-2-(5,5-dimethyl-3-(4-(piperazin-1-yl)styryl)cyclohex-2-en-1-ylidene)
malononitrile (11) was synthesized according to a literature procedure40.
4.1.6. Coumarin derivatives 7-(diethylamino)-2H-chromen-2-one (12), 7-

(diethylamino)-3-(p-tolyl)-2H-chromen-2-one (13) and 3-(4-aminophenyl)-7-
(diethylamino)-2H-chromen-2-one (14) were synthesized according to a literature
procedure41.
4.1.7. 1, 8-Naphthalic Anhydrideand 1, 8-naphthalimidederivatives
6-(pyrrolidin-1-yl)-1H,3H-benzo[de]isochromene-1,3-dione (15)42 and 4-(1,3-
dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid (16)43 were synthesized
according to a literature procedure.
Scheme 1 Synthesis of phenothiazine derivatives
4.2. Cell culture
A549 lung cancer cells were cultured in RPMI 1640 medium at 37 ℃ with 5%
CO2, and 95% air, supplemented with 10% (v/v) bovine calf serum and 80 U/ml
gentamicin. The cells were seeded onto 96-well plates or other appropriate dishes
containing the medium at the density of 6250/cm2.
4.3. Cell viability assay
Cells were seeded onto 96-well plates and treated with compounds at different
concentrations for 24 and 48 h, respectively. Cell viability was determined by

sulforhodamine B (SRB) assay, according to the method of Skehan. Light absorption
was measured at the wavelength of 540 nm using a SpectraMAX190 microplate
spectrophotometer (GMI Co, USA).
4.4. Acridine orange (AO) staining
The cells were incubated with compounds 8 and 11 at 0.1, 1, 5, 10, 20μM for 12 and
24 h, respectively, and stained with 5μg/ml of acridine orange (AO) at room
temperature for 1 min. Then the cells were observed and photographed under a Nikon
fluorescence microscope.
4.5. LDH assay
Lactate dehydrogenase (LDH) assay was performed on A549 cells treated with
compounds 8 and 11 at different concentrations (0.1, 1, 5 and 10 μM) for 24h, using a
LDH kit (Nanjing Jiancheng, China) according to the manufacturers protocol. Light
absorption was measured at 340 nm using a model Cintra 5 UV–vis spectrometer
(GBC, Australia).
4.6. Western blot
Cells were washed twice with ice cold PBS and were then lysed in protein lysis buffer
(0.5% SDS in 25 mM Tris–HCl, pH 7.5, 4 mM EDTA, 100 mM NaCl, 1 mM PMSF,
10 μg mL−1 leupeptin and 10 μg mL−1 soybean trypsin inhibitor). The protein
concentration was determined by the Bradford method. After adding loading buffer
and boiling, equal amount of protein was loaded onto a 15% SDS polyacrylamide gel.
After electrophoresis, the resolved protein was electrophoretically transferred to a
Polyvinylidene Fluoride (PVDF) membrane (Millipore, MA, USA). The membrane

was blocked with 5% non-fat milk in PBST (PBS containing 0.05% Tween 20) for 1 h
at room temperature. Subsequently the membrane was probed with Rabbit anti-human
LC3b antibody (1:1000), or anti-β-actin mouse monoclonal antibody (1:1000)
overnight at 4 °C, and was then washed twice with PBST, each time for 5 min. The
membrane was subsequently incubated with HRP conjugated goat anti-rabbit IgG
(1:5000) or polyclonal goat anti-mouse immunoglobulins/HRP (1:5000) for 1 h at
room temperature and then washed three times with PBST. Then the membrane was
incubated with HRP substrate for 5 min and the fluorescence signals were detected
with X-ray films. Intensity of the protein bands was quantified using Quantity-One
software (Bio-Rad).
4.7. Punctiform pattern of LC3
Treated U87 cells with compound 8 for 24 h. U87 cells were washed three times with
0.1 M PBS. Following fixation (15 min in 4% paraformaldehyde), the cells were
observed by a laser scanning confocal microscope (Zeiss, Germany).
4.8. Statistical analyses
Data were presented as means ± SE and analyzed by SPSS software. Pictures were
processed with Photoshop software. Mean values were derived from at least three
independent experiments. Differences at *p < 0.05 and **p < 0.01were considered
statistically significant.
Conflict of interest
The authors declare no conflicts of interest.

Acknowledgments
This work was supported by the National Natural Science Foundation of China (No.
81502948, 31870831, 31871407, 31741083), Shandong Excellent Young Scientist
Award Fund (No. BS2015YY031, ZR2017BH108, ZR2017BH084, BS2013SW001)
and Shandong Natural Science Foundation (No. ZR2017PH033)
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