Skip to main content
. 2022 Jul 21;64:101558. doi: 10.1016/j.molmet.2022.101558

Figure 3.

Figure 3

Insulin worsens stress-induced senescence in human hepatocytes. (A) Representative immunoblots of respective proteins in Ctrl and DOX-treated IHH cells, stimulated with or without insulin (100 nM) for 72 h. Bar graphs showing the relative protein quantification, normalized to GAPDH (n = 8). (B) Representative immunofluorescent images of Ctrl and DOX-treated IHH cells, stimulated with or without insulin (100 nM), stained for CCND1 (green) and nuclei (DAPI, blue). Scale bar represents 20 μm. A scatter plot displays fluorescence intensities quantified using ImageJ and normalized to number of nuclei (n = 3, 8–10 randomly chosen fields from each experiment). (C) RT-qPCR analysis of senescence markers (p21, CCND1) and SASP factors (IL8, IL18 and IL32) in Ctrl and DOX-treated IHH cells, stimulated with or without insulin (n = 5). (D) Representative images of flow cytometry analysis by plotting forward scatter-area (FSC-A, cell size) versus 7AAD staining, in Ctrl and DOX-treated IHH cells, stimulated with or without insulin. The lower figure shows result quantitation. On the left, values are presented as percentage (%) of cells that are rich in DNA (Q2+Q3). On the right, values are presented as percentage (%) of the DNA rich cells with increased cell size. Data are shown as mean ± SEM. Dots represent individual level data. Statistical significance was determined by Student's t-test or one-way ANOVA followed by Bonferroni post-hoc test or Kruskal–Wallis with post-hoc Dunn's test. ∗,#p < 0.05, ∗∗,##p < 0.01, ∗∗∗,###p < 0.001. ∗vs. DOX-treated cells; #vs. Ctrl cells. a.u., arbitrary unit.