Fig. 3: Nintedanib induces G1-phase cell cycle arrest without inducing cell death in nonsenescent HDFs.

A Nonsenescent (NS) HDFs were treated with nintedanib (0.5, 1, 2, 5, and 10 μM) for 3 days, and then CCK-8 assays were performed to evaluate cell viability and proliferation. n = 8. B Nonsenescent (NS) HDFs were treated with DMSO or nintedanib (5 μM) for one day and flow cytometry analysis of PI staining was performed to determine the number of cells in each cell cycle phase. n = 5. C Nonsenescent (NS) HDFs were treated with DMSO or nintedanib (5 μM) for one day, and then western blot assays using anti-CDK2, anti-p53, and anti-p21 antibodies were performed to investigate the protein expression of cell cycle regulators. n = 4–9. D Volcano plot (top) and GOBP analysis (bottom) of up- (yellow) and downregulated (blue) DEGs in nintedanib-treated senescent cells. E Volcano plot (top) and GOBP analysis (bottom) of up- (yellow) and downregulated (blue) DEGs in nintedanib-treated nonsenescent cells. F Experimental scheme for testing the restoration of cell proliferation after nintedanib removal. The ‘control’ group was treated with DMSO and the ‘Nin n day’ group was treated with nintedanib for n days. The ‘withdrawal’ group was treated with nintedanib for 3 days and then cultured in complete growth medium from 3 to 10 days after drug removal. G Nonsenescent HDFs were treated according to the experimental scheme, and the number of cells was monitored to investigate the effect of drug removal on cell proliferation. H Nonsenescent HDFs were treated according to the experimental scheme, and then western blot assays using anti-cyclin D1, anti-CDK2, anti-p16, and anti-p21 antibodies were performed to investigate the restoration of cell proliferation after drug removal. n = 3. I Correlation matrix for the transcriptome of all samples shown in G. The data normalized to those for DMSO-treated cells are shown as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc test.