Clonogenic Assay of Cancer Cell Response to Small Molecule Inhibitors

The colony forming ability of cancer cells treated with the indicated small molecule inhibitor was determined using clonogenic assay as described before^{26 (link)}. Briefly, 4 × 10⁴ cells were seeded in 2 ml culture medium in 12-well flat-bottom tissue culture plate, followed by subsequent serial dilution (1:1 to 1:32) in the presence of PTC-209 (5.0 μM), palbociclib (5.0 μM), or combination of PTC-209 and palbociclib (5.0 μM each) compared to DMSO vehicle control. The media was changed with fresh media supplemented with the appropriate inhibitor every 3–4 days. On day 10, the plates were washed and stained with Diff-Quik Staining Kit (Siemens Healthcare Diagnostics, USA), and were subsequently scanned and the number of colonies were observed under inverted microscope.