Fig 1: Effect of juglone-NaSe on expression of CDH1, COL4A3 and ITGB3 genes in PANC-1 cells (a) and in BxPC-3 cells (b) for 24 h determined by qPCR analysis. Values represent the mean ±SD at three independent experiments. * p<0.05 compared to the control group.
Fig 2: Characterization of Col4a3 transgenic mouse model. (a) Western blot analysis of type IV collagen α3 chain level in glomeruli of Col4a3 transgenic mice and wild type (WT) mice. The transgenic group (Mut) had a significantly lower type IV collagen α3 chain level compared with the WT group. (b) Sanger sequencing of Col4a3 mutant mice constructed by CRISPR/Cas9 technology. (c)–(e) The mutant and WT mice treated with or without NOX4 inhibitor at 28 weeks of age, quantification of urinary ACR ([c] each group N = 4), 24 hour proteinuria ([d] each group N = 3), serum creatinine ([e] each group N = 3, and serum creatinine was transformed as Ln[Scr] because of skewed distribution). ∗∗P < 0.05, ∗∗∗P < 0.001. (f) Real-time PCR validation show the expression of NOX4 and MMP-2 mRNA was higher in mutant group compared with that in WT group. N = 4; ∗∗P < 0.01. (g) Representative light micrographs of HE stained kidney sections from WT and mutant mice at 28 weeks of age. Light micrographs represent focal adhesion of glomeruli (red arrow), tubular cast (yellow arrow), interstitial inflammatory cell infiltration (yellow triangle) in mutant group. N = 4. (h) Electron micrographs of kidney tissue from WT and mutant mice at 28 weeks of age. Electron micrographs show partial podocyte foot process effacement (red star), segmental thickening of GBM (red arrow) in mutant group. N = 4. ACR, albumin-to-creatinine ratio.
Fig 3: Type IV collagen α3 chain expression in glomeruli of patients with ADAS or MCD. (a) Expression of type IV collagen α3 chain in glomeruli of patients with ADAS and patients with MCD using immunofluorescence. Representative immunofluorescence image shows α3 chain expression in glomeruli of a ADAS patient caused by COL4A3 mutation (p.C1616Y), and α3 chain expression in glomeruli of MCD patients as control. (b) Comparison of fluorescence intensity of α3 chain in glomeruli of patients with ADAS versus patients with MCD. Fluorescence intensity was calculated from 3 patients with ADAS and 5 control patients with MCD. The results are presented as the mean ± SEMs, 2-tailed t-test was used to analyze the data, ∗∗P < 0.01.ADAS, autosomal dominant Alport syndrome; MCD, minimal change disease.
Fig 4: Validation of the relation between NOX4 and MMP-2. (a) In vitro validation, Western blot analysis of NOX4, MMP-2, and apoptosis-related protein cleaved caspase 3 in COL4A3 transgenic podocytes. (b) In vivo analysis, Western blot verification showed increased expression of NOX4, MMP-2, and cleaved caspase 3 in glomeruli of Col4a3 transgenic mouse. (c) Measurement of podocyte H2O2 before and after NOX4 inhibition. (N = 4). (d) Densitometric analysis represented Western blot analysis from (a). (N = 4) (e) Densitometric analysis represented Western blot analysis from (b). (N = 4) ∗P < 0.05, ∗∗ P < 0.01.
Fig 5: Microarray analysis of patients with ADAS with COL4A3 mutation followed by validation of DEGs from both transcriptomic and proteomic level. (a) The heat map of gene expression analysis in glomeruli of patients with ADAS and patients with MCD. The line highlighted represents top highest DEG, MMP-2. Red indicates upregulated. Green indicates down-regulated. Black indicates no significant difference between the 2 groups. F1–F3 indicated patients with ADAS with COL4A3 mutation. M1–M5 indicates patients with MCD as control. (b)–(c) Pathways of genes upregulated in patients with ADAS compared with patients with MCD, using Kyoto Encyclopedia of Genes and Genomes analysis (b), and Enrichr Gene Ontology Biological Process program (c). (d) Real-time PCR validation of NOX4 and MMP-2 in glomeruli of 3 additional patients with ADAS and 7 additional patients with MCD. (e) Immunohistochemical staining of MMP-2 in glomeruli of patients with ADAS and patients with MCD. (f) Histogram of immunohistochemical staining from (e) showed a significant increase in the expression of MMP-2 in glomeruli of patients with ADAS compared with that in glomeruli of patients with MCD. ∗P < 0.05, ∗∗P < 0.01. N = 3. ADAS, autosomal dominant Alport syndrome; MCD, minimal change disease.
Supplier Page from Abcam for Anti-COL4A3 antibody