X6493

HCT-116 cells (ATCC CCL-247) were cultured in RPMI with 10% FBS.
Normal colon cells (CoN ATCC CRL-1790) were cultured in EMEM with 10% FBS.
For dose response experiments, 2 × 10³ cells were seeded
in 96-well plates and incubated for 24 hours in a 5% CO₂ /
37°C incubator. Cells were then treated with compounds ranging from 0
– 10 μM and incubated for another 24 hours. The XTT
(2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)
cell viability assay was performed according to manufacturer’s
protocol (ThermoFisher, Cat#X6493). Absorbance (450 nm) was read using
Tecan-Magellan plate reader (Tecan Trading AG, Switzerland). Cells for
RT-qPCR, NanoString mRNA expression, and protein analyses were cultured in
100 mm cell culture plates until 50–60% confluence before treatment.
Cells were treated with 50 nM bPGN and collected for time point studies at
0, 1, 4, 8, 16, 24, 36, 48, and 72 hours post-treatment and subjected to
protein and total RNA extraction following manufacturer’s protocol
for mirVana PARIS (ThermoFisher). For siRNA targeting miR-21, cells were
treated with DMSO (control), 30 nM mimic (Ambion #4464066), or 30 nM
inhibitor (Ambion #4464084) using Lipofectamine 2000 as the lipid carrier
and incubated for 48 hours prior to mirVana PARIS extraction. All
experiments were performed in experimental triplicate.