FIG 3.

The SASP upregulates PD-L1 through the JAK-STAT pathway. (A) The conditioned media from etoposide or HRasV12-induced senescent IMR90 cells were harvested and incubated with low-passage-number proliferating IMR90 cells. The protein lysates were analyzed for PD-L1. (B) The conditioned media were harvested from control cells, or IR-induced senescent cells with sh-NTC or cGAS knockdown, as described in the legend to Fig. 2A. These conditioned media were used to incubate low-passage-number proliferating IMR90 cells for 24 h. The cell lysates were probed for PD-L1. (C) IMR90 cells were induced to senescence with HRasV12 for 1 week. The cell lysates and the media were harvested and probed for indicated antibodies. (D) Low-passage-number proliferating IMR90 cells were stably inactivated with shRNA encoding NTC or two independent STAT3 hairpins. The cells were then incubated with conditioned media from HRasV12-induced senescent IMR90 cells for 24 h. The cell lysates were then analyzed with indicate antibodies. (E, F) Similar to panel D, low-passage-number proliferating IMR90 cells were stably inactivated with shRNA encoding NTC or two independent JAK2 hairpins. The cells were then incubated with conditioned media from HRasV12-induced senescent IMR90 cells for 24 h. The cell lysates were probed with indicated antibodies. (G) Cells as in panels D to F were analyzed by RT-qPCR. The results were normalized to lamin A/C and were presented as mean values with SEM (n = 3). The P values were calculated from two-tailed Student’s t test. (H, I) Low-passage-number proliferating IMR90 cells stably expressing shRNA against NTC, JAK1, or STAT1 hairpins were incubated with conditioned media from HRasV12-induced senescent IMR90 cells for 24 h. The cell lysates were probed for indicated antibodies. (J, K) IMR90 cells with indicated hairpins were subjected to IR (20 Gy) and cultured for 14 days. The cell lysates were analyzed as indicated.