Fig. 1. PD-L2 is upregulated in human and murine cancer cell lines on induction of cellular senescence.
a,b, Representative images of SA-β-gal staining (a, scale bar, 50 μm) and PD-L1 and PD-L2 mRNA expression in human cancer cell lines treated with palbociclib (b, n = 3 experiments). c, PD-L1 and PD-L2 mRNA expression in murine cancer cell lines after treatment with doxorubicin (n = 3 experiments). d, PD-L1 and PD-L2 mRNA expression in SK-MEL-103 xenograft tumors in nude mice, treated with 100 mg kg−1 oral palbociclib for 10 days and euthanized after the treatment. Control (n = 6 tumors); palbociclib-treated tumors (n = 7). e, PD-L1 and PD-L2 mRNA expression in HCmel3 tumors in C57BL/6 mice treated with 5 mg kg−1 doxorubicin (days 7, 10 and 17), analyzed after day 19. Control (n = 3 tumors); doxorubicin-treated tumors (n = 6). f, PD-L1 and PD-L2 mRNA expression in SK-MEL-28 cells treated with doxorubicin and then with the IKK inhibitor BAY 11-7082 (3 μM, 24 h, n = 3 experiments). g, PD-L1 and PD-L2 mRNA expression in SK-MEL-28 cells treated with TNF-α (100 ng ml−1, 3 days, n = 3 experiments). h, Quantification of PD-L2 protein levels using flow cytometry on the generation of a PD-L2 KO SK-MEL-103 cell line, under control and senescent conditions (n = 3 experiments). i, PD-L2 staining of WT and PD-L2 KO SK-MEL-103 cells in culture (as cell pellets) and as xenograft tumors, untreated and treated with palbociclib. Scale bar, 100 μm. Insets: high-magnification images. Scale bar, 50 μm. Representative images of n = 3 experiments. j, PD-L2 protein levels measured using flow cytometry after induction of senescence with palbociclib, bleomycin or doxorubicin in different cancer cell lines on day 7 after induction. MFI, mean fluorescence intensity (n = 3 experiments). Data are presented as the mean ± s.e.m. Two-sided t-tests or a one-way analysis of variance (ANOVA) with Tukey post-hoc test were used. k, Representative images of cell pellets stained for p21 and PD-L2 in Saos-2 and U2OS. Double staining was performed once. Scale bar, 100 μm. For all the experiments in culture, senescence was induced with 200 nM doxorubicin for 48 h, 5 µM palbociclib for 7 days or 12 mU bleomycin for 48 h. Senescence was evaluated at day 7.