Fig 2: SSBP1 is inhibited by pterostilbene to suppress DNA-PK/p53 pathway in the alleviation of high fructose-induced glomerular podocyte ferroptosis. (A) Rats were assigned into control group with a standard water and high fructose group fed with 10% fructose solution(W/V) for 16 weeks. After 8-week fructose intake, high fructose-fed rats were randomized into 5 subgroups, receiving water (vehicle), 10, 20 and 40 mg/kg pterostilbene, and 4 mg/kg pioglitazone for the next 8 weeks. Western blot analysis of the protein levels of SSBP1, p-DNA-PKcs(S2056), p-p53(S15), nucleus p53 and SLC7A11 in rat glomeruli (n = 6 per group). (B) Podocytes were cultured with or without fructose (5 mM), pterostilbene (10 µM) and pioglitazone (10 µM). Western blot analysis of the protein levels of SSBP1, p-DNA-PKcs(S2056), p-p53(S15), nucleus p53 and SLC7A11 in podocytes (n = 6 per group). (C) Lipid peroxidation in podocytes was analyzed by C11-BODIPY (581/591) staining and measured by flow cytometer (n = 6 per group). (D–E) The MDA level was measured by assay kit in rat glomeruli and podocytes (n = 6 per group). (F) Cell death was analyzed by TUNEL staining in rat glomeruli. Green fluorescence in pictures represented positive signals (cell death) (scale bras: 20 µm) (n = 3 per group). (G) Podocyte death was analyzed by propidium iodide staining and measured by flow cytometer (n = 6 per group). Data are plotted as mean ± SEM. P-values were acquired by one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001 vs control vehicle group, +P < 0.05, ++P < 0.01, +++P < 0.001 vs fructose vehicle group.

Fig 3: SSBP1 activates DNA-PK to phosphorylate p53 in high fructose-induced glomerular podocytes ferroptosis. (A) Lysates from high fructose-cultured podocytes were incubated with anti-SSBP1, and the co-eluted proteins were examined using DNA-PKcs antibody (n = 3 per group). (B–C) Podocytes were transfected with SSBP1 plasmid and negative control. DNA-PK activity in podocytes was measured by assay kit (n = 6 per group). Western blot analysis of the protein level of DNA-PKcs S2056 phosphorylation in podocytes (n = 6 per group). (D–E) Podocytes were transfected with SSBP1 siRNA and negative control. DNA-PK activity in podocytes was measured by assay kit (n = 6 per group). Western blot analysis of the protein level of DNA-PKcs S2056 phosphorylation in podocytes (n = 6 per group). (F) Western blot analysis of the protein level of DNA-PKcs S2056 phosphorylation in high fructose-stimulated rat glomeruli (n = 6 per group). (G) DNA-PK activity in high fructose-cultured podocytes was measured by assay kit (n = 6 per group). (H) Western blot analysis of the protein level of DNA-PKcs S2056 phosphorylation in high fructose-cultured podocytes (n = 6 per group). (I–J) Podocytes transfected with SSBP1 siRNA or negative control and then cultured with or without fructose (5 mM). DNA-PK activity was measured by assay kit in podocytes (n = 6 per group). Western blot analysis of the protein level of DNA-PKcs S2056 phosphorylation in podocytes (n = 6 per group). (K) Flag-SSBP1, Myc-p53 and HA-DNA-PKcs were expressed alone or in combination in podocytes and Co-IP assays were performed using anti-HA antibody (n = 3 per group). (L–Q) Podocytes were cultured with or without fructose (5 mM) in the presence or absence of vehicle or DNA-PK inhibitor LTURM34. Western blot analysis of the protein levels of p53 S15 phosphorylation, nucleus p53 and SLC7A11 in podocytes (n = 6 per group). Lipid peroxidation in podocytes was analyzed by C11-BODIPY (581/591) staining and measured by flow cytometer (n = 6 per group). The MDA level was measured by assay kit in podocytes (n = 6 per group). Podocyte death was analyzed by propidium iodide staining and measured by flow cytometer (n = 6 per group). Data are plotted as mean ± SEM. P-values were acquired by one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: SSBP1 interacts with p53 and facilitates p53 S15 phosphorylation to increase accumulation of nuclear p53 in high fructose-stimulated glomerular podocyte ferroptosis. (A) Lysates from high fructose-cultured podocytes were incubated with anti-SSBP1, and the co-eluted proteins were examined using p53 antibody (n = 3 per group). (B–E) Podocytes were transfected with SSBP1 plasmid or siRNA and the respective negative control. The mRNA level of p53 was measured by qPCR (n = 6 per group). Western blot analysis of the protein level of total p53 protein in podocytes (n = 6 per group). (F–I) Western blot analysis of the protein level of total and nucleus p53 in high fructose-stimulated rat glomeruli and podocytes (n = 6 per group). (J) Immunofluorescence staining of SSBP1 and p53 in high fructose-cultured podocytes (scale bras: 20 µm) (n = 3 per group). (K–L) Western blot analysis of the protein level of p53 S15 phosphorylation in high fructose-exposed rat glomeruli and podocytes (n = 6 per group). (M) Podocytes were transfected with Myc-Wt p53 or Myc-Mut p53 and cultured with or without 5 mM fructose for 96 h. Podocytes were then used to isolate whole cell lysates (WCL), cytoplasmic (Cyto) and nuclear (Nucl) fractions, respectively. Aliquots relative to same cell number per component were immunoblotted using anti-Myc antibody (n = 3 per group). Lamin B1 and GAPDH were used as the control of nuclear and cytoplasmic fraction, respectively. (N–P) Podocytes were transfected with SSBP1 siRNA as well as negative control and then cultured with or without fructose (5 mM). Western blot analysis of the protein level of p53 S15 phosphorylation and nucleus p53 in podocytes (n = 6 per group). Immunofluorescence staining of p53 in podocytes. (scale bras: 20 µm) (n = 3 per group). Data are plotted as mean ± SEM. P-values were acquired by one-way ANOVA, n.s., no significance, *P < 0.05, **P < 0.01, ***P < 0.001.