Panel A shows a schematic of the experimental setup. Panel B includes representative microscopy images and a bar graph quantifying migrated renal fibroblasts under different treatments. Panel C displays Western blot results for various proteins. Panel D presents bar graphs of mRNA levels for specific genes. Panel E outlines another experimental setup. Panel F shows microscopy images and a bar graph of migrated renal fibroblasts with different treatments. Panel G includes Western blot results. Panel H presents bar graphs of mRNA levels. Panel I shows a schematic of a different experimental setup. Panel J includes microscopy images and a bar graph of migrated renal fibroblasts. Panel K displays Western blot results. Panel L presents bar graphs of mRNA levels. Panel M outlines another experimental setup. Panel N shows microscopy images and a bar graph of migrated renal fibroblasts. Panel O includes Western blot results. Panel P presents bar graphs of mRNA levels. Each panel compares different conditions and treatments, showing the effects on renal fibroblast activity and protein expression.
Renal TEC-derived IFN-λ drives renal fibrosis by promoting renal fibroblast activation and migration. (A–D) Primary renal fibroblasts from WT and Ifnlr1−/− mice were treated with 100 ng/ml of IFN-λ2 or PBS for 24 h (A). n = 4 per group. (B) Representative images and quantification of the migrated renal fibroblasts. (C) The indicated protein levels were measured by western blot. (D)Acta2, fibronectin, vimentin, and Tgf-β mRNA levels in fibroblasts were detected by RT-qPCR. (E–H) Renal TECs were transfected with 1 μg/ml of poly(I:C) in the presence of 10 μg/ml of anti-IFN-λ2/3–neutralizing antibody (anti–IFN-λ2/3) or IgG2b isotype control (anti-IgG2b) for 12 h. The supernatants were collected and applied to primary renal fibroblasts for 24 h (E). n = 4 per group. (F) Representative images and quantification of the migrated renal fibroblasts. (G) The indicated protein levels were detected by western blot. (H)Acta2, fibronectin, vimentin, and Tgf-β mRNA levels in fibroblasts were measured by RT-qPCR. (I–L) Renal TECs were transfected with or without 1 μg/ml poly(I:C) for 12 h. The supernatants were then collected and added to the culture medium of primary renal fibroblasts from WT and Ifnlr1−/− mice for 24 h (I). n = 4 per group. (J) Representative images and quantification of the migrated renal fibroblasts. (K) Western blot was used to quantify the indicated proteins in renal fibroblasts. (L)Acta2, fibronectin, vimentin, and Tgf-β mRNA levels in renal fibroblasts were assessed by RT-qPCR. (M–P) Renal TECs from WT and Mavs–/– mice were transfected with 1 μg/ml of poly(I:C) for 12 h. Supernatants were collected and added to primary renal fibroblasts for 24 h (M). n = 4 per group. (N) Representative images and quantification of the migrated renal fibroblasts. (O) Western blot was utilized to quantify the indicated proteins in renal fibroblasts. (P)Acta2, fibronectin, vimentin, and Tgf-β mRNA levels were measured by RT-qPCR. Scale bar = 50 μm (B, F, J, and N). Data in A–P are representative of two independent experiments. Data are presented as mean ± SEM. *P < 0.05, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test. ns, no significant difference. Source data are available for this figure: SourceData F7.