Fig 2: Hypoxia induces stress fiber formation through MKL1 nuclear translocation. (a) ACTA2 was significantly upregulated in hypoxia (n = 8 biological replicates). (b) Cross-section of AT constructs, fluorescent confocal microscopy images were taken at least 20 µm above the glass coverslip. (c) Fluorescent confocal microscopy images of the mono-culture AT constructs after 7 days in normoxia (top left panel) and 1% oxygen (bottom left panel), arrows point to ⍺-SMA signal and stress fiber morphology (scale bar-50 µm, insets-20 µm). (d) ⍺-SMA protein expression was significantly increased in 1% oxygen (data are presented for the mono-culture condition, cropped blot is shown, full length blot is presented in Supplementary Fig. S5). (e) Fluorescent confocal microscopy images of the mono-culture AT constructs and the orthogonal view in normoxia (top right panel) and 1% oxygen (bottom right panel), arrows point to MKL1 signal localization (scale bar- 20 µm, insets- 10 µm). (f) Nuclear to cytoplasmic ratio for MKL1 was significantly increased in 1% oxygen (n = 5 biological replicates, 20 cells per replicate). (g) MKL1 nuclear (N) to cytoplasmic (C) protein ratio was significantly increased in 1% oxygen, arrows point to the quantified MKL1 protein bands (data are presented for the mono-culture condition, n = 3 biological replicates, cropped blot is shown, full length blot is presented in Supplementary Fig. S6). (h) Hypoxia induces actin stress fiber formation via MKL1 nuclear translocation. Data are presented as means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document}± SEM. Comparisons between groups and statistical analysis were performed using two-way ANOVA with Tukey post hoc test or unpaired Mann–Whitney test with two-tailed p-values (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: Compound 145 significantly reduced TGF-β1-induced MRC-5 transition into myofibroblasts. MRC-5 were pre-incubated for 1 h with the studied compounds (10 µM) and then cultured for 24 h (A, C) or 48 h (B, D–F) in TGF-β1 (5 ng/mL). (A, C) qPCR was carried out to analyze ACTA2, MYH11, SM22, COL1A1, TNC, FN1, and VCAN transcript levels. Samples were run three times in duplicates. Cellular α-smooth muscle actin (α-SMA) (B) and collagen I (D) protein level was determined by in-cell ELISA; n = 8. (E) To visualize α-SMA positive stress fibers, MRC-5 were fixed, permeabilized, blocked, and labeled with anti-α-SMA antibody, followed by Alexa Fluor 488 conjugated antibody, nuclei were stained with Hoechst 33342 dye. Microphotographs were taken using a Leica DMiL LED Fluo microscope, 40× objective, bar = 50 µm. (F) Myofibroblasts (MRC-5 positive for α-SMA) were counted in 20 randomly selected fields of view and expressed as a percentage of the entire MRC-5 population. Each bar represents the mean value (± S.E.M.). The results were considered statistically significant at the p level of 0.05, against control (#) and TGF-β1 (*).

Fig 4: Effect of vitamin D on the fibrogenic activation of myofibroblasts. a RT-qPCR result: expression of a representative profibrogenic cluster of genes. Cells were pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 μg/mL for 6 h) and then treated with 5 nmol/L vitamin D or its vehicle. Analysis was performed 72 h post-shock. Values are folds of induction over control cultures (treated with bleomycin vehicle: PBS); veh.: vitamin D vehicle. The results presented in the figures are means ± SEM. Significance of the analysis is indicated as ***, P < 0.001. b Detection of VIM (vimentin) and ACTA2 (α-SMA) in myofibroblasts cell extracts. Cells were pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 μg/mL for 6 h) and then treated with 5 nmol/L vitamin D or its vehicle. Cell extracts were obtained at 72 h post-shock. PBS is the bleomycin vehicle; veh.: vitamin D vehicle. TUBA1A (tubulin α) was used as loading control. KDa: kilodaltons. Bottom panel: densitometry analysis of bands; a.u.: arbitrary units. c Representative micrographs of indirect immunofluorescence detection of ACTA2 (α-SMA fibers) and PTK2 (focal adhesion kinase), two markers of the profibrogenic activation. Cells were pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 μg/mL for 6 h) and then treated with 5 nmol/L vitamin D or its vehicle. Cells were processed for immunofluorescence 72 h post-shock. d Quantification of the immunofluorescence result (% of activated fibroblasts showing clear expression of α-SMA fibers and PTK2)

Fig 5: MCL-1 targeting influences transcriptional programs involved in actomyosin organization/contractility in viable bCAFs.A Three different primary cultures of CAFs were treated with ABT-199 (1 µM), A1331852 (100 nM), ABT-199 (1 µM) +A1331852 (100 nM) or ABT-737 (1 µM) with or without S63845 (500 nM) for 72 h in DMEM containing 1% FBS, apoptosis was measured by Annexin-V flow cytometry. Data are means ± SEM from three independent experiments. Two-way ANOVA, ****P < 0.0001, ns: not significant. B Caspase 3/7 activity was measured by caspase Glo assay and expressed in arbitrary unit. Data are means ± SEM from four independent experiments. Two-way ANOVA. *P < 0.05, ns: not significant C Volcano plot displaying differential expressed genes between S63845 treated and non-treated primary culture of CAFs from luminal B (n = 3) and triple negative (n = 3) breast cancers. The vertical axis (y-axis) corresponds to the mean expression value of log 10 (q-value), and the horizontal axis (x-axis) displays the log2 fold-change value. The red dots represent the upregulated expressed transcripts; the blue dots represent the transcripts whose expression is downregulated. D A hierarchically clustered heatmap showing the expression patterns of the 23 most extreme DE mRNA. Red and blue represent up- and downregulated expression in CAFs treated or not with S63845. Color density indicating levels of fold-change was displayed. E qRT-PCR of MYH10, MYH2, ACTA2 andITGA11 mRNA in CAFs Sg control or sg MCL-1 treated or not by S63845 500 nM for 18 h. Mean and SEM of three independent experiments are represented as fold-change of mRNA level related to untreated Sg control. Two-way ANOVA, *P < 0.1, **P < 0.01, ****P < 0.0001, ns: not significant. F CAFs sg control or silenced for MCL-1 (sg MCL-1) were treated or not with S63845 (500 nM), A1331852 (100 nM) or S63845 (500 nM) + A1331852 (100 nM) for 48 h in DMEM containing 1% FBS. Apoptosis was measured by Annexin-V flow cytometry. Data are means ± SEM from three independent experiments. P-value was determined by two-way ANOVA. ****P < 0.0001, ns: not significant.