Imaging software

Tumor cells were plated in 6 cm dishes and treated with cisplatin, H-4073 or a combination of both. After 48 hours of treatment, 4×10³ viable cells from each group were plated in 6 cm dishes and cultured for additional 10 days. The colonies were fixed with methanol and stained with crystal violet. Photomicrographs were taken and the number of colonies was counted by Alpha Innotech imaging software (San Leandro, CA).

Western blots were performed as previously described [23 (link)] using specific antibodies against TP63 (EPR5701, Abcam), SOX2 (D6D9), SOX4 (Millpore), NOTCH1 (D1E11), NOTCH3 (D11B8), NANOG (D73G4), OCT4, KLF4 (D1F2), CD44 (EPR1013Y, Abcam), and ABCG2; GAPDH (1:2000, 14C10) and α-Tubulin (1:2000, 11H10) were used as internal references. All antibodies were purchased from Cell Signaling Technology, except where indicated, and used at 1:1000 dilutions. The full scans of the Western blots were quantified using the Alpha Innotech imaging software (San Leandro, CA, USA).

Total proteins were collected with RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and protein concentrations were measured following the BCA method (Beyotime, Jiangsu, China), and 40ug protein were subjected to 4–20% SDS gel electrophoresis (Sigma) and were then transferred to PVDF membranes (Roche, Indianapolis, IN, USA). Then, 5% milk blocked membranes were incubated with TWIST1 (ab50581, Abcam, Cambridge, MA, USA), E-cadherin (ab1416, Abcam) or Vimentin (ab92547, Abcam) primary antibody and subsequently incubated with matched secondary antibodies (#7074 and #7076, Cell signaling Technology, Beverly, MA, USA). Then, signals for each protein expression were detected with the Bio-Rad Gel imaging system (Bio-Rad, Hercules, CA, USA). GAPDH (G8140, US Biological, Swampscott, MA, USA) was used as a loading control. Alpha Innotech (San Leandro, CA, USA) imaging software was used to quantify western blotting data.