Fig 1: The expression of RNF168 and the activation of NF-κB signaling pathway are impaired by inhibition of podocyte autophagy. Protein expression of p-p65 (phospho S536) and t-p65 in podocytes assessed by Western blot analysis in response to 3-MA (a) or Atg5-shRNA (d). Levels of TNF-α and IL-1β in the culture medium of podocytes assessed by ELISA in response to 3-MA (b) or Atg5-shRNA (e). Immunofluorescence staining of RNF168 and p-p65 (phospho S536) in podocytes in response to 3-MA (c) or Atg5-shRNA (f). Cell experiment was repeated 3 times independently. *p < 0.05 versus untreated podocytes from MRL/MpJ mice or MRL/lpr mice.
Fig 2: The accumulation of RNF168 is abrogated and the repair of DNA damage is accelerated by up-regulation of A20. a mRNA expression of A20 in control tissues (n = 25) and LN renal tissues (n = 40) determined by RT-qPCR. b Protein expression of A20 in control tissues (n = 25) and LN renal tissues (n = 40) determined by Western blot analysis. c Immunohistochemical staining of positive expression of A20 protein in control tissues (n = 25) and LN renal tissues (n = 40). d mRNA expression of RNF168 and A20 in podocytes in response to pcDNA3 or pcDNA3-A20 evaluated by RT-qPCR. e Protein expression of RNF168 and A20 in podocytes evaluated by Western blot analysis. f Protein expression of γH2Ax, p53, and p21 in podocytes in response to pcDNA3 or pcDNA3-A20 evaluated by Western blot analysis. g DNA damage pattern and tail moment in the podocytes in response to pcDNA3 or pcDNA3-A20 at 24 h after treatment measured by the neutral comet assay. h Immunofluorescence staining of γH2Ax positive nuclear expression in the podocytes in response to pcDNA3 or pcDNA3-A20 at 24 h after treatment. Cell experiment was repeated 3 times independently. *p < 0.05 versus untreated podocytes from MRL/MpJ mice or MRL/lpr mice.
Fig 3: Repair of DNA damage is promoted by RNF168 silencing in the podocytes of LN mice. The podocytes of MRL/lpr mice were transfected with RNF168-shRNA plasmids, with those transfected with NC-shRNA plasmids as the control. Podocytes of MRL/lpr mice without transfection were evaluated with those untreated podocytes from MRL/MpJ mice as the control. a Protein expression of γH2Ax, p53, and p21 in the podocytes at 24 h after treatment determined by Western blot analysis. b DNA damage pattern and tail moment in the podocytes at 24 h after treatment detected by the neutral comet assay. c Immunofluorescence staining of γH2Ax positive nuclear expression in the podocytes at 24 h after treatment. Cell experiment was repeated 3 times independently. *p < 0.05 versus the podocytes from MRL/MpJ mice or the podocytes transfected with NC-shRNA plasmids.
Fig 4: Schematic diagram of the mechanism by which podocyte autophagy affects the repair of DNA damage and LN progression. Podocyte autophagy promotes the degradation of A20 and the RNF168 translocation into nuclei, activating the NF-κB signaling pathway, suppressing the repair of DNA damage, and aggravating LN.
Fig 5: The activation of the NF-κB signaling pathway is disrupted by the inhibition of RNF168 expression in podocytes of LN mice. a Protein expression of p-p65 (phospho S536) and t-p65 in the podocytes assessed by Western blot analysis. b Levels of TNF-α and IL-1β in the culture medium of podocytes assessed by ELISA. c Immunofluorescence staining of RNF168 and p-p65 (phospho S536) translocation into the nuclei of the podocytes. Cell experiment was repeated 3 times independently. *p < 0.05 versus the podocytes from MRL/MpJ mice or podocytes transfected with NC-shRNA plasmids.
Supplier Page from Abcam for Anti-RNF168 antibody