Extended Data Fig. 2: Doxorubicin-induced senescence in IHH cells.
From: BMP4 and Gremlin 1 regulate hepatic cell senescence during clinical progression of NAFLD/NASH
(a) Brightfield microscopy images of control and DOX-treated cells assayed for SA-β-Gal (blue). Scale bar represents 100 µm. (b-d) Representative immunofluorescence images of control and DOX-treated cells stained for p53 (green) (b), p21 (green) (c), γH2AX (green) (d), and nuclei (DAPI, blue). Scale bar represents 20 µm. (e) Bar graph displays fluorescence intensities quantified using ImageJ and normalized to number of nuclei (n = 3, 6-10 randomly chosen fields from each experiment). (f) After 48 h, DOX-treated IHH cells were stimulated with OA for 24 h. Cells were stained for lipid droplet accumulation (ORO, red) and nuclei (DAPI). Scale bar represents 100 µm. (g) Intracellular lipid accumulation was quantified by spectrophotometry and normalized to number of nuclei (n = 3). (h) RT-qPCR analysis of genes involved in lipid metabolism in DOX-treated cells (n = 4). (i) Representative transmission electron micrographs of control and DOX-treated hepatocytes. Scale bar represents 1 μm. (n = 3 biologically independent experiments). (j) Representative fluorescence images of control and DOX-treated cells stained with Mitotracker Red dye (mitochondria, red), which detects mitochondrial polarization status. Scale bar represents 20 μm. Bar graph displays fluorescence intensities of Mitotracker Red, normalized to number of nuclei (n = 3, 6-10 randomly chosen fields from each experiment). Values are mean ± SEM. Statistics: 2-tailed, Unpaired t-test for (e) (p21 & γH2AX), Mann-Whitney test for (e, j) (p53 & Mitotracker), and one-way ANOVA followed by Bonferroni’s/Dunnett’s post-hoc test or Kruskal-Wallis with post-hoc Dunn’s test (g, h). Black arrows indicate normal mitochondria and red arrows indicate mitochondria with disrupted cristae. a.u., arbitrary unit.
