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. 2026 May 6;48(1):2666452. doi: 10.1080/0886022X.2026.2666452

Figure 2.

Multi-panel figure displaying Western blots and immunofluorescence evaluating USP11, a-SMA, and Collagen I under various treatments. This figure contains several panels analyzing protein expression and immunofluorescence. **Panel A**: Western blot comparing USP11, a-SMA, and Collagen I levels in Scramble and USP11 siRNA cells with and without UA. **Panel B**: Bar graphs quantifying relative protein abundance, highlighting significant differences. **Panel C**: Immunofluorescence images show fibronectin and vimentin (red) staining, juxtaposed with DAPI-stained nuclei (blue). **Panel D**: Western blot on protein levels in response to Methotrexate (MTX) concentrations. **Panel E**: Bar graphs quantifying relative protein abundance under MTX treatment. Subsequent panels focus on additional Western blots and quantitative analysis for USP11, a-SMA, and Collagen I across conditions.

USP11 contributes to RIF activation in the UA-stimulated NRK49F. NRK49F cells were seeded to 70–80% confluence in the antibiotic-free medium and grown followed by transfection with USP11 siRNA or USP11-pcDNA 3.0 plasmid. After transfection, the medium was changed to DMEM with F12 containing 0.5% FBS for starvation and then cells were incubated with or without uric acid (800 μM) for an additional 36 hours before being harvested for analysis. Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (A, F, H). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (B, G, I). Representative images and quantitative analysis of immunofluorescence staining for Fibronectin and Vimentin with DAPI nuclear counterstaining in each group (C). Then NRK49F cells were starved for 24 h with DMEM containing 0.5% FBS before they were exposed to uric acid in the presence or absence of MTX (1, 5, and 10 μM). Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (D). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (E). Data were expressed as mean±SD (n = 4 for each group). N.S.: no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bars = 100μm.