Extended Data Fig. 2. PD-L2 protein levels are upregulated in human and murine senescent cells.
(a) CRISPR-Cas9 genome editing of the human PDCD1LG2 locus, specifying the sgRNA binding site in exon 3. The sequence corresponds to the single clone of edited SK-MEL-103 cells used in the experiments labelled as PD-L2-KO SK-MEL-103. (b) Gating strategy for definition of PD-L2 positive populations in culture and (c) representative example (1 out of n = 3) of histogram for PD-L2 protein levels upon generation of a PD-L2-KO SK-MEL-103 cell line, in control and senescent conditions, measured by flow cytometry. The gating strategy in (b) applies to Figs. 1h, 1j and panel (d) in the present figure. (d) PD-L2 protein levels as measured by flow cytometry upon induction of senescence with palbociclib, bleomycin and doxorubicin in SK-MEL-103 cells. N = 3 independent experiments. Data are presented as mean ± SEM. 1-way ANOVA was applied. (e) Double staining of PD-L2 and active caspase-3 in Saos-2 and U2OS cell pellets, after treatment with palbociclib. (f) Double staining of PD-L2 and p21 (above) and PD-L2 and caspase-3 (below) in SK-MEL-103 cell pellets, after treatment with doxorubicin. Double stainings in (e) and (f) were performed once. Scale bars = 100 μm. For all the experiments in culture, senescence was induced with 200 nM doxorubicin for 48 h, 5 μM palbociclib for 7 days (unless otherwise indicated) or 12 mU bleomycin for 48 h. Senescence was evaluated at day 7 unless otherwise indicated.