Fig 2: SB225002 retards cellular senescence and mitochondrial dysfunction in TGF-ß1-treated tubular cells in vitro. (A–F) Representative micrographs of western blot and quantitative statistical data showing protein expressional levels of (B) active ß-catenin, (C) TOMM20, (D) CPT1A, (E) p16INK4A and (F) FN in each group. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3. (G) Representative micrographs show the protein expressional level of FN, ?-H2AX and TOMM20 in each group, as indicated. Frozen sections were stained with FN, ?-H2AX and TOMM20 antibodies, respectively. Arrows indicate positive staining. Scale bar = 50 or 25 µm. (H–K) Graphical representations show the relative mRNA level of (H) CXCR2, (I) p14, (J) FN and (K) p21 in each group. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3. (L–N) Graphical representations show the relative mRNA level of CXCR2 after siRNA transfection (L). ***p < 0.001, n = 3. Representative (M) Western blots and graphical representations of (N) FN expression in four groups. HKC-8 cells were transfected with siCXCR2 or siNC and then treated with TGF-ß1 (2 ng/ml) for 24 h.

Fig 3: ICG-001 inhibits CXCR2-aggravated tubular senescence and mitochondrial dysfunction in vitro. (A–F) Representative micrographs of western blot and quantitative statistical data show protein levels of (B) active ß-catenin, (C) PGC-1a, (D) p16INK4A, (E) ?-H2AX and (F) FN in a given group. *p < 0.05, **p < 0.01, n = 3. (G) Graphical representations show the relative abundance of PGC-1a mRNA in each group. *p < 0.05, **p < 0.01, n = 3. (H) Representative micrographs showing the mitochondrial ROS assessed by mitoSOX staining, and mitochondrial morphology via transmission electron microscopy (TEM) detection, and the expression of ß-catenin, ?-H2AX and TOMM20 in different groups, as indicated. Arrows indicate positive staining. Scale bar = 50, 10, 25 or 1 µm, as indicated.

Fig 4: Replicative senescence in endothelial cells. HUVEC of passage 1 (young) and passage 20–25 (senescent) were subjected to analysis of senescence markers. (A) Senescence-associated-ß-galactosidase (SA-ß-gal) staining. Representative picture out of n = 3, scale bar = 200 µm. (B) Quantification of SA-ß-gal-positive cells (n = 3, * p < 0.05, unpaired Student’s t-test with Welch’s correction). (C,D) Representative western blots of senescence markers p16 and p21 (upper panels) and densitometric quantification of p16 and p21 after normalization to vinculin (lower panels, n = 5, * p < 0.05, for p16: unpaired Student’s t-test with Welch’s correction; for p21: unpaired Student’s t-test). All data represent mean + SD.

Fig 5: FCCP induces premature senescence. Primary non-senescent HUVEC were left untreated (control, ctrl) or treated with 5 µM of FCCP for 6 days. (A) Senescence-associated-ß-galactosidase (SA-ß-gal) staining. Scale bar = 200 µm. (B) Quantification of SA-ß-gal-positive cells (n = 4, * p < 0.05, paired Student’s t-test). (C,D) Representative western blots of senescence markers p16 and p21 (upper panels) and densitometric quantification of p16 and p21 after normalization to vinculin (lower panels (n = 4, * p < 0.05, paired Student’s t-test). All data represent mean + SD.