Figure 3

Combined treatment with the CDK4/6 inhibitor abemaciclib (ABE) and SI-2 can be strongly cytotoxic without inducing PD-L1 expression. (A) E0771 cells were treated with SI-2 or ABE or both for 72 hr at the indicated concentrations. Increased concentrations of ABE result in reduced PD-L1 expression compared to SI-2 alone, on protein (top) and mRNA (bottom right) levels. Compared to SI-2, cytotoxic concentrations of ABE alone do not bring about strong induction of PD-L1 mRNA (bottom right) or protein levels (top right, bottom left). (B) Addition of up to 625 nM ABE to SI-2 does not induce ATF4 expression compared with SI-2 alone. Western analysis of ATF-4 protein expression was determined 24 hr after treatment with the indicated concentrations of SI-2 and/or ABE. (C) Exposure of E0771 cells to cytotoxic concentrations of ABE for 24 hr only moderately induces the expression of ATF4. ***P < 0.005, **P < 0.05, two-tailed Student’s t-test. For each group of WB figures (A–C): all the blots were performed in the same experimental setup and processed in parallel. All gels were loaded with identical amount of protein sample. Dashed separating lines indicate that the blots were run in parallel but on separate gels. Full length blots are available in the supplementary information.