Figure 4. The ERK1/2 signaling pathway mainly contributes to PD-L1 expression after cisplatin treatment.

(A,B) T24 and 5637 BC-derived cell lines were treated with different concentrations of cisplatin for 24 h (6.25, 12.5, or 25 μM, respectively); total protein was extracted and phosphorylation of ERK1/2, p38 and JNK was detected by Western blot. Total ERK1/2, p38, and JNK were used as the internal controls. (C–F) T24 and 5637 BC-derived cell lines were pretreated with different ERK1/2 pathway inhibitors (PD98059, 10 μM; U0126, 10 μM) for 30 min then cisplatin (25 μM) for 24 h. Total protein was extracted, then ERK1/2 activation and expression levels of PD-L1 were detected by Western blot. β-Actin was used as the internal control. (G,H) T24 and 5637 BC-derived cell lines were pretreated with 10 μM of a JNK inhibitor (SP600125) for 30 min then with cisplatin (25 μM) for 24 h. Total protein was extracted and JNK activation and expression levels of PD-L1 were detected by Western blot. β-Actin was used as the internal control. (I,J) T24 and 5637 BC-derived cell lines were pretreated with 10 μM of p38 inhibitor (SB203580) for 30 min then with cisplatin (25 μM) for 24 h. Total protein was extracted, then p38 activation and expression levels of PD-L1 were detected by Western blot. β-Actin was used as the internal control. (K) T24 BC-derived cell lines were pretreated with different pathway inhibitors (PD = PD98059; U = U0126; SP = SP600125; SB = SB203580) for 30 min, followed by cisplatin (25 μM) incubation for 24 h, total mRNA was extracted from the cells and expression levels of PD-L1 were detected by qPCR. The efficacy of inhibitors was provided in Supplementary Figure S1. Results are expressed as the mean ± S.D of triplicate samples. *P<0.05 compared with the control group and #P<0.05 compared with the cisplatin-treated group.