Fig. 4

IL-1β induced CPCs senescence time dependently, resulting in vicious IL-1β accumulation. P1 CPCs, both in monolayer and cell pellet culture, were treated with 20 ng/ml IL-1β for 0 h, 0.5 h, 1 h, and 2 h per day for 7 days. Afterwards, a series of examinations were performed. a Western blotting analysis of P53 level. GAPDH was used as a loading control. b, c Representative β-gal staining (b) and β-gal positive cells counting (c) (three random fields were selected, N = 3 repetitions per group). d CCK8 assay (N = 3 repetitions per group). e, f Representative macroscopic photos (e) and quantitative analysis (f) of colony formation assay (N = 3 repetitions per group). (g) Representative Saf-O (top) and Col 2 (bottom) staining of cell pellets. h, i Quantitative analysis of pellet scores (h) and Col 2 levels (i) of cell pellets (n = 6 sections/pellet, N = 3 pellets per group). j ELISA assay of the IL-1β level in the supernatant during cell pellet culture (N = 3 repetitions per group). b Scale bar 50 μm. g Scale bar 200 μm. d Values are shown as mean ± SD. **P < 0.01, ****P < 0.0001, two-way ANOVA with Sidak’s multiple comparisons test. c, f, h–j Values are shown as mean ± SD. NS, no significance; **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test