Fig 2: The a-TAT1 gene contains functional CArG boxes. (A) Schematic representation of the a-TAT1 gene with an indication of the putative CArG boxes (CArG elements 1–4) present in the region examined and the fragments (Prom1, Prom2, and Int1) used in our analysis. (B) The indicated constructs were transfected in control and INF2 KD RPE-1 cells. The luciferase activity was measured and was expressed relative to that of cells transfected with minP. (C) The four CArG boxes present in the constructs Prom1, Prom2, and Int1 were placed in tandem upstream from a minimal promoter (a-TAT1 CArGs1–4). A similar construct was made in which the four central nucleotides of each box were mutated in such a way that all of them were C or G (a-TAT1 CArGs1–4m). Control cells were transfected with the indicated constructs in the presence or absence of the MRTF-A ?N100 construct, and the luciferase activity of the cells was assayed. The histogram represents the luciferase activity obtained in each case relative to that of cells transfected with minP. Data in B and C represent the mean and SEM of seven and four independent experiments, respectively; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3: Both Ser 214 and Ser 431 residues of ABLIM1 are involved in the nuclear accumulation of MKL1 and activation of Arp3. (A) HCCLM3 cells transfected with a series of plasmids expressing ABLIM1 mutants were stained with anti-MKL1 antibodies, followed by incubation with Alexa488-conjugated secondary antibodies. Representative images were photographed using confocal fluorescence microscopy. (Scale bar: 20 µm). (B) The ratio of MKL1 immunofluorescence intensity in nucleus (N), nucleus and cytoplasm (N+C), or cytoplasm (C) among total cells was quantified by chi-square test. At least 50 cells were counted. ***: p<0.0001. (C) Real-time quantitative PCR was carried out to evaluate the relative mRNA levels of ABLIM1 and Arp3 in ABLIM1-KO cells transfected with indicated dominant negative mutants of ABLIM1. Differences between the different groups were quantified by one-way ANOVA test. (D) Schematic diagram illustrating a working model for ABLIM1 phosphorylation induced by Rictor involving in actin polymerization. In control cells, Rictor interacts with ABLIM1 and phosphorylates ABLIM1, thereby leading to actin polymerization and accumulation of MKL1 in the nucleus, where it promotes expression of Arp3. In HCC cells with Rictor knockdown, phosphorylated levels of ABLIM1 are dramatically decreased, which results in reduction of F-actin formation. Free G-actin can bind to MKL1 in the cytosol, resulting in its cytoplasmic retention. As a consequence, MKL1 fails to be translocated into the nucleus and activate Arp3 expression.

Fig 4: MKL1 deletion in resident fibroblasts attenuates renal fibrosis. (A) Schematic diagram of unilateral ureteral obstruction (UUO)-induced renal fibrosis in MKL1f/f and MKL1?Fib mice. Tamoxifen (50 mg/kg/d) was injected into mice by intraperitoneal injection (i.p.) for 10 days. The mice were then subjected to UUO surgery for 14 days. (B) Masson’s trichrome staining. Scale bar, 20 µm. N = 5 mice. (C) Picrosirius red staining under white light or polarized light. Scale bar, 20 µm. N = 5 mice. (D) Hydroxylproline level were detected by test kit. N = 8 mice. (E) Pro-fibrogenic genes were measured by RT-qPCR. N = 9 mice. (F) Urine urea nitrogen (BUN) levels. N = 5 mice. (G) Urine creatinine (Cr) levels. N = 5 mice. Data represent mean ± SD. * p < 0.05, two-tailed t-test.

Fig 5: 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).