Quantification of Tumor Cell Response

3x10⁵ Tumor cells per well were seeded on 24mm square cover slips in 6-well plates and allowed to grow for 24 hours. Tumor cells were then treated with RCM1, VCR or RCM1 + VCR for 24 hours. Tumor cells were then fixed and stained as previously described (36 (link)). To visualize the nucleus, Hoechst 33342 (ThermoFisher Scientific) was used as a counter stain. For quantification, 5 random fields were acquired per sample and quantified using ImageJ. To perform immunostaining of tumor tissue, paraffin embedded Rd76-9 subcutaneous tumor sections were stained as described previously (37 (link)). 5 Random fields per sample were acquired and quantified using ImageJ. Antibodies used for immunostaining were anti-Ki-67 (Invitrogen, MA5-14520), anti-phospho-histone H3 (Santa Cruz, sc-374669 (C-2)), anti-CD31 (R&D, AF3628), anti-Cleaved-Caspase 3 (R&D, MAB835), anti-CHAC1 (Novus Biologicals, OTI1E2).