Fig. 4. Nintedanib inhibits tyrosine phosphorylation of JAK2 and STAT3 in senescent HDFs.

A Senescent (S) HDFs were treated with DMSO or nintedanib (5 μM) for one hour, and then western blot assays using anti-p-JAK2, anti-JAK2, anti-p-STAT3, and anti-STAT3 antibodies were performed to identify the signaling pathways affected by nintedanib. B Senescent (S) HDFs were transfected with scramble siRNA or STAT3-specific siRNA three days prior to nintedanib (5 μM) treatment. Then, after drug exposure for three days, western blot assays using anti-STAT3, anti-caspase-9, anti-caspase-3, and anti-caspase-7 antibodies were performed to evaluate apoptosis induction. C Senescent (S) HDFs were treated with DMSO, nintedanib (5 μM), or WP1066 (5 μM, a STAT3 kinase inhibitor) for three days, and a flow cytometry analysis with Annexin V/PI staining was performed to identify apoptotic cells. D Nonsenescent (NS) HDFs were treated with DMSO, nintedanib (5 μM), or WP1066 (5 μM, STAT3 kinase inhibitor) for three days, and flow cytometry analysis with Annexin V/PI staining was performed to identify apoptotic cells. E Nonsenescent HDFs were infected with lentiviral particles having LacZ (as control), STAT3-wt, STAT3-Y705E, or STAT3-Y705F encoded gene and then CCK-8 assays were performed after nintedanib treatment for 3 days to assess cell viability. n = 3. 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.