Fig. 3: p16 increases the stability of PD-L1 protein in senescent cells.
From: p16-dependent increase of PD-L1 stability regulates immunosurveillance of senescent cells
a–c, Flow cytometry analysis of PD-L1 expression in DNA damage-induced senescence (D-Sen) (a and b), RIS (b) and in cells with p16 overexpression (p16 OE) (c) compared with control cells. Primary mouse lung fibroblasts (CCL-206) (a and c) and primary human lung fibroblasts (IMR-90) (b) were used in these experiments (n = 3–8). d,e, PD-L1 protein expression in growing IMR-90 cells treated with CDK4/6 inhibitors Palbociclib (Palbo) (d), Abemaciclib (Abem) (e) or vehicle (control) (n = 6–7). Gr, growing. f, PD-L1 protein expression in D-Sen treated with non-targeting small interfering RNA (siControl) or small interfering CDKN2A (siCDKN2A) (n = 9). g, ELISA-based measurement of PD-L1 protein levels in D-Sen treated with siControl or siCDKN2A and CHX (n = 3). h–k, Immunoblot analysis of whole cell lysates derived from Gr and D-Sen cells (h), D-Sen cells treated with siControl and siCDKN2A (i), Gr and D-Sen cells treated with MG132 or vehicle (negative control) (k) (n = 3), and immunoblot (IB) analysis of ubiquitin in immunoprecipitated (IP) PD-L1 protein from Gr and D-Sen cells (j). l, Quantification of p16+, PD-L1+ and p16+PD-L1+ cells in normal lung tissue (n = 3) and human lung pathologies: emphysema (n = 3), fibrosis (n = 3), adenocarcinoma (n = 3) and squamous cell carcinoma (n = 3). m, Representative immunofluorescence image of p16 (red) and PD-L1 (green) staining in emphysema patient. Blue, nuclei stained by DAPI. Scale bar, 10 μm. The image is representative of n = 4 emphysema lung specimens. PD-L1 expression in a–f was quantified by flow cytometry analysis as median fluorescent intensity. Two-tailed unpaired Student’s t-test (a, c and g) and two-tailed paired Student’s t-test (d, e and f) was used. Error bars, mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA (b and l) was also used for statistical analysis; error bars, mean ± s.e.m. **P < 0.01, ****P < 0.0001. In a–k, experiments were repeated three times independently with similar results. Source numerical data and unprocessed blots are available in Source data.
