Fig 2: Identified compound 841 as a selective CELF1 inhibitor. (A) Schematic of in well protein RNA-binding assay. (B) The signal-to-noise ratio of the in well protein RNA-binding assay was confirmed by depleting strepavidin, probe, protein lysate and anti-Flag one by one. (C) Unlabelled GU RNA (Cold probe) efficiently displaced Biotin-GU probe in a dose-dependent manner. (D) The selectivity of compounds 27, 838 and 841 was performed on in-well protein RNA-binding assay. (E) Quantitative PCR analyses of COL1A1, ACTA2 and IFN-? from LX-2 cells pre-treated with or without compound 841 for 6 h and then co-treated with 5 ng/ml TGF-ß1 for 12 h. The data are representative of three independent experiments (mean ± SD). *P< 0.05, **P< 0.01, ***P< 0.001 by Student's t-test.

Fig 3: Immunoblot analyses of E-cadherin: a-SMA and MMP-2.NRK-49F Cells were treated with ß-HB (10 mM) in 5% FCS for 48 h, followed by treatment with DA water extract for a further 24 h. For Western blot analysis (A), cell extracts were subjected to SDS-PAGE and immunoblotted with primary antibodies against E-cadherin, a-SMA, and MMP-2. ß-actin was the internal control. B–D: Densitometry analysis from A is shown. It is evident that ß-HB induced an increase in a-SMA and MMP-2, and caused a decrease in E-cadherin expression; DA reversed the ß-HB -induced increase in a-SMA and MMP-2, and also reversed the decrease in E-cadherin expression. The experiment was duplicated with similar results observed. # P<0.05, **P<0.01, *P<0.05.

Fig 4: Regulation of myofibroblast differentiation by the NUAKs. A and B, AG1523 cells were subjected to double transfection with negative control or siRNA targeting NUAK1 (A) or NUAK2 (B) and treated with TGFß (1 ng/ml) for 24 h. Samples were subjected to immunoblotting for NUAK1, aSMA, calponin, and a-tubulin, which serves as a loading control (A), or for NUAK2, aSMA, calponin, SMAD3, and SMAD4, with Ponceau-S staining included as a loading control (B). A star shows nonspecific protein bands. Molecular size markers in kDa are shown. C, aSMA immunofluorescence microscopy of AG1523 cells after NUAK1, NUAK2, or negative control siRNA transfection using individual and distinct siRNAs. Cells were starved in 0.01% FBS/DMEM and incubated with TGFß (1 ng/ml) as indicated. Representative photomicrographs are shown. Bar, 20 µm. D, collagen gel contraction assay with AG1523 cells transfected with control or individual and distinct NUAK1 siRNAs (left panels) or individual and distinct NUAK2 siRNAs (right panels). Two days after the first transfection, TGFß (5 ng/ml in 0.01% FBS/DMEM) was added, and the contracted lattice surface was measured 24 h post-treatment. Surface area was measured by using the ImageJ software and is illustrated in the corresponding graphs. Representative pictures of contracted lattices are shown. Bar graphs show average values derived from triplicate determinations and the corresponding S.E. (error bars). Each independent experiment was repeated at least three times. E, mRNA levels of calponin, ACTA2, and SM22a from the samples used in C and D. After performing real-time qRT-PCR, values were normalized to HPRT1 mRNA. Asterisks illustrate significant differences between the conditions indicated and respective control: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 5: Calcitriol inhibits the expression of ACTA2 in dose- or time-dependent manner. (A) mRNA expression and (C) protein level of ACTA2 after pre-treatments with three chosen concentrations of 1,25(OH)2D3 and treatment with 10 ng/mL of TGF-ß. (B) mRNA expression and (D) protein level of ACTA2 after pre-treatments with 1,25(OH)2D3 for three chosen incubation times and treated with 10 ng/mL of TGF-ß. The results of experiments (n = 3) are presented in relation to GAPDH as a mean ± SEM. Western blots are shown for genes, in which the statistical significance has been observed at the mRNA level. Original Western blots can be found at Figure S1. # p < 0.05, ## p < 0.01, #### p < 0.0001 versus NT group; $ p < 0.05, $$$$ p < 0.0001 versus TGF-ß group; & p < 0.05 and &&&& p < 0.0001 versus MT group. One-way analysis of variance (ANOVA) or Kruskal-Wallis test followed by corresponding post hoc was used to determine differences in several groups, and Student’s t-test or Welch’s t-test was used to determine statistically significant differences between two means. RQ—relative quantification of genes expression normalized to GAPDH, NT—non-treatment cells, TGF-ß—cells treated with TGF-ß, MT—cells treated with 0.1% of methanol and TGF-ß.