Fig 2: Effects of GA on TNF-α-stimulated adhesion molecule protein and mRNA levels in VSMCs.(A and B) The mouse VSMCs, MOVAS-1 cells, were incubated with TNF-α (10 ng/mL) in the presence or absence of GA (25 and 50 μM) for 8 h. The CAM protein levels of whole cells were investigated by western blotting. (C and D) MOVAS-1 cells were stimulated with TNF-α (10 ng/mL) in the presence or absence of GA (25 and 50 μM) for 4 h. The mRNA level of VCAM-1 and ICAM-1 was investigated by qRT-PCR. GAPDH served as the internal control. (E and F) The human primary cells, HA-VSMCs, were incubated with TNF-α (10 ng/mL) in the presence or absence of GA (25 and 50 μM) for 24 h. Results are shown as means ± SEM from a representative experiment (n = 5). *p<0.05 significantly different from the group treated with TNF-α.

Fig 3: Nrf2 deletion prevents successful renal repair after IRI(A) Experimental workflow for testing Nrf2 function in regulating PT cell fate after IRI. Nrf2 cKO mice and their littermate controls (control) were subjected to the same ischemic stress (20 min) and tamoxifen treatment. Nrf2 is deleted in Sox9-lineage cells after IRI with tamoxifen administration. Kidneys were harvested on day 21 post-IRI.(B and C) Immunostaining for indicated proteins. Representative images are shown. Arrowheads, SOX9+/VCAM1+ cells. See Figure S8F for KIM1 quantification.(D) Real-time PCR analyses of indicated gene expression. n = 6.(E) Quantification of SOX9+/VCAM1+ cells (arrowheads in C). n = 4–5.(F and G) Immunostaining for 4-HNE and ACSL4. Quantifications are shown in (G). n = 4–5. Arrowheads, ACSL4+ cells.(H and I) TUNEL staining for evaluating cell death. Quantification of TUNEL+ cells is shown in (I). n = 4. Arrowheads, TUNEL+ cells.One-way ANOVA with post hoc multiple comparisons test for (D), (G), and (I) and t test for (E). n.s., not significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bars: 50 μm in (B), (C), and (F) and 20 μm in (H). Data are represented as mean ± SEM.

Fig 4: Pharmacological inhibition of ferroptotic stress blunts the accumulation of damage-associated PT cells and cell death.(A) Schematic representation of experimental workflow. All mice (cKO and control littermates) were subjected to the same ischemic stress (ischemic time, 22 min, unilateral IRI) and tamoxifen treatment. The same volume of vehicle was administered to the control groups (control vehicle and cKO vehicle). Kidneys were harvested on day 21 post-IRI. (B) Liproxstatin-1 prevents renal atrophy. Relative size of post-IRI kidneys compared to contralateral kidneys (CLK) was quantified. Control, littermate control. N = 4–5. (C and D) Immunostaining for KIM1 and KRT8. IRI kidneys from cKO are shown. Quantification of immunostained area over the DAPI+ area is shown in (D). N = 4–5. (E and F) Immunostaining for SOX9 and VCAM1. Quantification of VCAM1+EMCN–F4/80– area over the DAPI+ area is shown in (F). Arrowheads indicate damage-associated PT cells. (G) Real-time PCR analyses of indicated gene expression. Whole kidney lysates were used. N = 4–5. (H) and (I) TUNEL staining for evaluating cell death. Quantification of TUNEL-positive nuclei is shown in (I). N = 4–5. Red arrowheads indicate TUNEL+ tubular epithelial cells. Scale bars, 50 μm in (C) and (E); and 20 μm in (H). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, unpaired t-test for (D), (F). and (I); One-way ANOVA with post hoc multiple comparisons test for (G). (J) Liproxstatin-1 improves renal repair after IRI.

Fig 5: Rb2 reverses the effect of miR-216a on the Smad3/IκBa signaling pathway. (A) Protein expression levels of Smad3 and IκBα with or without Rb2 treatment in PDL8 HUVECs transfected with miR-216a mimics (n=5). *P<0.05; #P<0.05. (B) mRNA expression levels of Smad3, ICAM1 and VCAM1 after silencing Smad3 in PDL8 HUVECs with or without Rb2 treatment (n=5). (C) Protein expression levels of Smad3, ICAM1 and VCAM1 after silencing Smad3 in PDL8 HUVECs with or without Rb2 treatment (n=5). *P<0.05, **P<0.01. miR-216a, microRNA-216a; NC, negative control; VCAM1, vascular cell adhesion molecule 1; Rb2, ginsenoside Rb2; ICAM1, intercellular adhesion molecule; si, small interfering RNA.