Fig. 3: HFHS diet and tumor growth induce liver senescence, steatosis, and fibrosis.

A Liver triglycerides levels. B Histopathology of liver steatosis. Representative liver sections from CR, Chow, and HFHS tumor-free (control) and tumor-bearing mice (T-B mice) were stained with hematoxylin and eosin (scale bar = 30 μM). C Steatosis score. D Liver cholesterol levels. E, F AST and ALT liver enzyme levels. G Collagen staining with Picrosirius red stain. Scale bar = 20 μM. H Silver impregnation to detect reticulin fibers. Scale bar = 20 μM. I Reticular fibers quantification. J Interleukin 6 (Il6) mRNA expression. K–N Liver fibrosis markers evaluated by qPCR. K Vimentin (Vim), L Transforming growth factor beta (Tgfb), M Matrix metalloproteinase-9 (Mmp9), and N Keratin 18 (Krt18). O–Q Senescence markers: p21(Cdkn1a) (O), p16 (Cdkn2a) (P) and Telomerase reverse transcriptase (TERT) (Q) mRNA expression. R Representatives immunoblot analysis of Sirtuin 1 (SIRT1) expression, phosphorylated p53 (S15) and total p53 expression, phosphorylated Retinoblastoma protein (pRB) (S807/811) expression, and Proliferating cell nuclear antigen (PCNA) expression. GAPDH was used as a loading control. The results represent three independent experiments (n = 3). Western blot densitometries are in S3. Each test was performed with at least nine animals (n = 9–20) and plotted values are mean ± S.E.M. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001. For all panels, a two-way ANOVA was performed, followed by Tukey’s post-test.