Full Paper
doi.org/10.1002/chem.202100907
Chemistry—A European Journal
with 3% BSA in 1X PBS, immunofluorescence was carried out using
research fellowship. The authors thank Mrs. Debapriya Dutta,
IACS for Confocal Microscopy. This work was supported by the
Wellcome Trust/DBT India Alliance Fellowship [Grant Number,
IA/S/18/2/503986] and Department of Science and Technology
SwarnaJayanti Fellowship [DST/SJF/CSA-01/2015-16].
°
regular or standard methods, incubating at 37 C with BG4 antibody
(Mouse monoclonal, from Merck) diluted 1:200 in 1X PBS over-
night, and Alexafluor 647-conjugated secondary antibody (Invitro-
gen) for 2 h on next day. Finally, cover slips were mounted with
antified solution (Invitrogen). The BG4 emission (570–670 nm) was
assembled with the excitation at 559 nm, sequentially.[26] Digital
images were taken in a Confocal Laser Scanning Microscope (LSM-
800, Zeiss). For the quantification of BG4 foci, >50 cells were
counted using FIJI ImageJ software and the standard error of the
mean was calculated from three replicates, *P <0.05 (Student’s ‘t’
test) was considered as statistically significant.
Conflict of Interest
The authors declare no conflict of interest.
Quantitative real-time PCR (qRT PCR): K562 cells (~106 per well)
were placed in a 6-well plate and allowed to incubate overnight.
Next day, cells were treated with TPW (20 μM, 40 μM) and TPE
(20 μM, 40 μM) and harvested for 24 h. Untreated (control) cells and
those treated with DMSO control were used to evaluate primary c-
KIT expression levels. Total RNA was isolated using the TRIzol
reagent (Invitrogen) according to the manufacturer’s instructions.
RNA quantification was carried out by a Cary Win 300 UV-Vis
spectrophotometer and the total 500 ng of RNA was employed as a
template for cDNA synthesis using a Verso kit (Thermo Fisher
Scientific) as per the supplied protocol. Real-time PCR was carried
out on Roche LightCycler 480 by the use of SYBR Premix (Applied
Biosystems), according to the manufacturer’s instructions. The CT
values were normalized to 18 s rRNA and compared to the
untreated or control cells. The CT method (comparative cycle
threshold method) was used to calculate relative mRNA expression.
The mRNA level was expressed in terms of fold changes of target
gene with respect to control or untreated value of 0. Three
biological replicates were employed for the quantifications. The
Keywords: c-KIT · G-quadruplexes · leukemia · polyamide · TPW
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significance level was statistically analyzed by employing
a
Student’s t test, and results were statistically significant when *P<
0.05.
Transfection and luciferase assay: K562 cells were seeded (~106
cells each well) in 35 mm 6 well plates. After 16 h, cells were
transiently transfected with c-KIT, c-MYC and BCL-2 promoter
(Addgene, USA) luciferase reporter construct by the use of Lipofect-
amine 2000 (Invitrogen), as per manufacturer’s instructions. Basic
empty vector pGL4.72 was employed as negative control for c-KIT
wild promoter. pRL-TK, a HSV-thymidine kinase promoter, used as
Renilla luciferase control gene (Renilla Luciferase for normalization)
was employed as transfection control. After 6 h of incubation, 10%
FBS was supplemented to the cells and incubated for 2 h followed
by treatment with TPW and TPE at two different doses (20 μM and
40 μM). Subsequently, after 48 h of incubation, cells were lysed by
150 μl 1X cell lysis buffer (Promega) with continuous pipetting
followed by vortexing for 30 secs and kept at room temperature for
10 mins. The concentration of cell lysate protein was evaluated by
Lowry method.[27] The assay was performed in triplicate using
Luciferase Reporter Assay System (Promega) in a Multimode micro-
plate reader (Molecular Devices, USA). The normalization of
luciferase activity was accomplished with protein concentration
and the effect of ligands upon G4 constructs were normalized
against the untreated or control leukemia cells.
Statistical analysis: Data were presented as the mean�SEM and
analyzed with a Student’s t-tests using OriginPro 2016 software. *P
value of �0.05 indicates statistically significant.
Acknowledgements
R.P. thanks DST-India for INSPIRE Fellowship. D.D. thanks CSIR-
India for a research fellowship. T.D. thanks DBT-India for a
Chem. Eur. J. 2021, 27, 1–11
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