Fig. 6: EPRS1 inhibitor suppresses fibroblast activation and proliferation in NRK-49F cells.

a Representative confocal image (left) and quantitative data (right) of EPRS1 (red) and Pan-cadherin (green) in NRK-49F cells induced with TGF-β (5 ng/ml) for 24 h. The yellow arrow indicates the plasma membrane portion. Scale bar = 20 μm. Colocalization analysis was performed according to the methods provided in ZEN software. b Representative Western blot (top) and quantitative data (bottom) of the membrane and cytosol of NRK-49F cells treated with or without TGF-β (n = 3). Mem, membrane fraction; Cyto, cytosolic fraction. c Relative mRNA levels of Col1a1 and Col3 were analyzed via qPCR and normalized to Gapdh to assess the effect of the EPRS1 inhibitor (n = 3). d Representative Western blot (left) and quantitative data (right) of FN, COL1A1, and α-SMA in the whole lysate of EPRS1i-treated NRK-49F cells under TGF-β treatment conditions (n = 3). e Representative confocal images (top) and quantitative data (bottom) of COL1A1 and α-SMA in EPRS1i-treated cells under TGF-β treatment conditions for 24 h. The α-SMA-positive area and COL1A1-positive area were quantified by ImageJ analysis (n = 6). In particular, quantitative data for α-SMA were analyzed according to the area under the curve (AUC). The yellow line represents the value of the surface plot profile and the calculated area under the curve. f Membrane and cytosol of EPRS1i-treated NRK-49F cells incubated in the absence or presence of TGF-β for 24 h. g mRNA levels of Eprs1 were analyzed by qPCR and normalized to Gapdh (n = 3). h Representative Western blot (left) and quantitative data (right) of EPRS1 expression in EPRS1i-treated cells under TGF-β treatment conditions for 24 h (n = 3). i Representative Western blot (left) and quantitative data (right) of protein (p-Smad3/Smad3) expression in EPRS1i-treated cells after 24 h of TGF-β treatment (n = 3). j The level of hydroxyproline in cultured cells was quantified via colorimetric assay (n = 3). k Proliferation of cells treated with the indicated concentrations of EPRS1i under TGF-β treatment conditions determined by a WST-1 assay. l Representative confocal images (left) of Ki67 in EPRS1i-treated cells under TGF-β treatment conditions for 24 h. The Ki67 ratio (right) was quantified by ImageJ analysis (n = 5). The data are presented as mean ± standard deviation. Statistical data were analyzed by ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001.