Figures & data
Table 1. Primers used in the study.
Figure 1. SMURF1, cGAS and STING were markedly upregulated in the serum of LN patients, while YY1 was downregulated. (A–D) SMURF1, YY1, cGAS and STING mRNA levels in the serum of LN patients (n = 22) and healthy people (n = 22) were determined by qRT-PCR. (E-F) The correlation between, SMURF1, YY1 and cGAS was analyzed using Pearson correlation analysis. Data were expressed as mean ± SD. ***p < 0.001.
Figure 2. YY1 overexpression alleviated LN-IgG-induced ERS and apoptosis in podocytes. (A) YY1 expression in MPC5 cells after vector or OE-YY1 transfection was examined by qRT-PCR. MPC5 cells were treated with serum IgG from LN patients (LN-IgG) and YY1 overexpression was induced. (B-C) YY1 level in cells was determined using qRT-PCR and western blot. (D) CCK8 assay was performed to determine cell viability. (E) Cell apoptosis was analyzed by flow cytometry. (F) Bax and Bcl-2 protein levels were measured by western blot. (G) Immunofluorescence was employed to analyze GRP78 level in cells. (H) GRP78, CHOP, ATF6 and PERK protein levels in cells were assessed using western blot. Data were expressed as mean ± SD. All our data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3. SMURF1 inhibited YY1 expression by regulating YY1 protein ubiquitination. (A) YY1 and SMURF1 expressions in MPC5 cells after vector or OE-SMURF1 transfection were examined by western blot. (B) The binding relationship between SMURF1 and YY1 was verified using Co-IP assay. (C) YY1 protein level in SMURF1-overexpressing podocytes after MG132 treatment was analyzed by western blot. (D) The degradation of YY1 in SMURF1-overexpressing podocytes after CHX treatment was analyzed by western blot. (E) YY1 ubiquitination was analyzed by ubiquitination analysis. Data were expressed as mean ± SD. All our data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4. SMURF1 knockdown inhibited LN-IgG-induced ERS and apoptosis in podocytes by mediating ubiquitination of YY1. (A) SMURF1 or YY1 expression in MPC5 cells after sh-NC or sh-SMURF1/sh-YY1 transfection was examined by qRT-PCR. Both SMURF1 knockdown and YY1 knockdown were induced in LN-IgG-treated podocytes. (B-C) YY1 expression level in cells was assessed using qRT-PCR and western blot. (D) Cell viability was measured using CCK8 assay. (E) Cell apoptosis was detected using flow cytometry. (F) Bax and Bcl-2 protein levels in cells were examined using western blot. (G) GRP78 level in cells was analyzed using immunofluorescence. (H) GRP78, CHOP, ATF6 and PERK protein levels in cells were determined using western blot. Data were expressed as mean ± SD. All our data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5. YY1 alleviated podocytes injury by transcriptional inhibition cGAS/STING/IFN-1 signal axis. (A) The potential binding sites between YY1 and cGAS promoter region was predicted by JASPAR. (B-C) ChIP and dual-luciferase reporter gene assays were performed to analyze the interaction between YY1 and cGAS. (D) cGAS, STING and IFN-1 protein levels in podocytes following vector or OE-YY1 transfection were assessed by western blot. (E) cGAS mRNA level in MPC5 cells after sh-NC or sh-cGAS transfection was examined by qRT-PCR. Both cGAS knockdown and YY1 knockdown were induced in LN-IgG-treated podocytes. (F) cGAS, STING and IFN-1 protein levels in podocytes were detected by western blot. (G) CCK8 assay was employed to analyze cell viability. (H) Cell apoptosis was examined using flow cytometry. (I) Bax and Bcl-2 protein levels in cells were assessed by western blot. (J) GRP78 level in cells was analyzed using immunofluorescence. (K) GRP78, CHOP, ATF6 and PERK protein levels in cells were examined by western blot. Data were expressed as mean ± SD. All our data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6. SMURF1 knockdown inhibited cGAS/STING/IFN-1 signal axis by targeting YY1 to suppress LN progression in mice. sh-SMURF1 lentivirus were injected into LN mice (MRL/lpr mice). (A) The level of urinary protein was shown by urinary Albumin/Creatinine. (B) The representative pictures of HE staining of kidney tissues. (C) SMURF1 and YY1 mRNA levels in kidney tissues were detected using qRT-PCR. (D) Western blot was conducted to analyze cGAS, STING and IFN-1 protein levels in kidney tissues. (E) GRP78 protein level in kidney tissues was determined using IHC. (F) Cell apoptosis was examined using TUNEL staining. Data were expressed as mean ± SD. n = 8. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplemental Material
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The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.