Cell Proliferation Assay by MTT, BrdU and Crystal Violet.

For MTT assay, cells were plated at 2.5 x 10³ per well in 96-well plates in complete media (10% FBS) or media supplemented with 10% charcoal-stripped serum. Cells were either treated with DMSO or with indicated inhibitors. After indicated times, cells were incubated in 0.5 mg/mL MTT (Invitrogen) for 1 hour at 37° C. MTT crystals were dissolved in isopropanol and absorbance was measured in BioTek plate reader at 570 nM and represented graphically. The BrdU assay was performed by BRDU cell proliferation assay kit according to manufacturer’s instructions (BrdU cell proliferation assay kit, Cell Signalling # 6813). Cells were plated at 2.5 x 10³ per well in 96-well plates in complete media (10% FBS) or media supplemented with 10% charcoal-stripped serum. Cells were either treated with DMSO or with indicated inhibitors. BrdU incorporation in the proliferating cells was measured in BioTek plate reader at 450 nM and represented graphically. For the Crystal Violet cell proliferation assay, cells (in 96-well plate, treated with indicated drugs or cultured in FBS or CSS supplemented medium) were fixed in chilled 100% methanol for 10 minutes followed by staining with crystal violet (MilliporeSigma) for 2 hours and then washed with water. Crystal violet was dissolved in 1% SDS and absorbance was measured in BioTek plate reader at 595 nM and represented graphically.