Fig. 2

TGF-β1 treatment increases SMAD2/3 signaling activation and α-SMA expression levels more in SHR-CF than in WKY-CF. a Results obtained by ImageStreamX (IMX) technology, on SMAD2 nuclear translocation in WKY- and SHR-CF before and after 3 h of TGF-β1 treatment. IMX microscopy images were obtained on fixed and permeabilized CF, hybridized with anti-SMAD2-FITC antibody (green), and stained with DRAQ5 for the nucleus (red). Merged signal (yellow) indicates SMAD2 signal after nuclear translocation. b IMX fluorescence-activated cell sorting analysis on data collected from 10,000 events/sample. Bar graph indicates the similarity dilate index of WKY-CF (white bars) and SHR-CF (red bar), without and with TGF-β1 treatment. Scale bar = 10 μm. c Phospho-SMAD2/3 and total SMAD2/3 expression levels in WKY- and SHR-CF after 30 min of treatment with 5 ng/ml of recombinant TGF-β1. α-SMA protein (d) and gene (e) expression in WKY- and SHR-CF after treatment with 5 ng/ml TGF-β1. Western blot quantification data are expressed as mean ± SD after β-tubulin normalization, qRT-PCR data are expressed as fold ± SD normalized with GAPDH gene, n = 5/group. The experiments on cells were performed in triplicate. 2-way ANOVA with Bonferroni’s post-test: *p < 0.05, ***p < 0.001