Extended Data Fig. 2: Regulation of PD-L1 expression by p130-E2F axis.

a, Immunoblotting of lysates from quiescent (Quie), replicative senescent (r-Sen), nutlin3a senescent (n-Sen), d-Sen, and proliferative (Prolifer) HCA2 cells using the indicated antibodies. b, qPCR analysis of PD-L1 and CDC20 expression in sorted G1, S, and G2/M HCA2 cells. The results are shown as relative fold changes. c, Immunoblotting of lysates from HCA2 cells expressing mock, p130, and Flag-E2F97 using the indicated antibodies. d, qPCR analysis of PD-L1 and CDC20 expression in HCA2 cells as in c. e, Immunoblotting of lysates from proliferative and d-Sen HCA2 cells expressing doxycycline (Doxy)-inducible 3xFlag-E7 in the presence or absence of Doxy (1 μg/ml) using the indicated antibodies. The duration of senescence induction by doxorubicin were shown (Day 3 or Day 12). f, A volcano plot of differentially expressed genes (DEGs) between PD-L1⁺ and PD-L1⁻ d-Sen HCA2 cells. Blue dots indicate the DEGs identified by FDR < 0.05. A red arrow indicates Cd274 (PD-L1). g, TF candidates enriched on promoter regions of PD-L1 positively correlated genes (R² > 0.95) in RNA-seq results as in f. The threshold of normalized enrichment score was greater than 3. The transcriptional regulatory network was showed in the right panel. (Pink circle: targeted correlated gene; Blue circle: untargeted correlated gene; Green circle: TF candidate; Red box: E2F1 and CD274) h, Immunoblotting of lysates from sorted PD-L1⁻ and PD-L1⁺ d-Sen HCA2 cells using the indicated antibodies. i, Gating strategy for the experiments shown in Fig. 1e. Relative quantified values of PD-L1 was labeled. Data are presented as means ± SEM of three independent experiments. All immunoblotting results were repeated independently for three times. One-way ANOVA with post-hoc Tukey HSD test (b) and Dunnett’s test (d). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The precise p-values were described in Methods.