Fig. 5: GL-V9 inhibits the reorganization of the actin cytoskeleton and affects the activity of MRTF and YAP.

The primary lung fibroblasts from mice were subjected to serum starvation for 24 h, followed by treatment with 5 ng/mL TGF-β1 and GL-V9 for various times. NIH/3T3 cells were treated with 10 ng/mL of TGF-β1 in a serum-free culture medium for 24 h and treated with GL-V9. A The protein expression of α-SMA, collagen I, and fibronectin in primary lung fibroblasts (48 h). B The content and distribution of Collagen I and α-SMA in primary lung fibroblasts (48 h) were examined via immunofluorescence. Scale bar: 20 μm. C The mRNA content of ACTA2, COL1A1, and FN1 in primary lung fibroblasts (24 h). D The protein expression of α-SMA and collagen I in NIH/3T3 cells. E The mRNA content of ACTA2 and COL1A1 in NIH/3T3 cells. F The nuclear localization of MRTF-A and the F-actin cytoskeleton in primary lung fibroblasts (24 h). Scale bar: 20 μm. The proportion of nuclear MRTF-A was quantified. G The distribution of MRTF-A within the primary lung fibroblasts was assessed through nucleocytoplasmic fractionation experiments (24 h). H The effect of CA MRTF-A expression on the content of ACTA2 mRNA in NIH/3T3 cells. Differences between groups were analyzed using Welch’s ANOVA. I The nuclear localization of YAP in primary lung fibroblasts (12 h). Scale bar: 20 μm. The proportion of nuclear YAP1 was quantified. J The phosphorylation levels of YAP1 and LATS in primary lung fibroblasts (12 h). K The protein level of YAP1 and LATS in primary lung fibroblasts (24 h). L Dual-luciferase assay to measure TEAD transcriptional activity. Expression of a YAP/TAZ-responsive synthetic promoter driving firefly luciferase, along with a control Renilla luciferase, in NIH3T3 cells. M Dual immunohistochemistry of α-SMA and P-YAP1 in lung tissue of mice with bleomycin-induced pulmonary fibrosis, with quantification of P-YAP1 fluorescence intensity. Scale bar for the main image: 200 μm; enlarged section scale bars: 25 μm. Data are shown as mean ± SEM. ns p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.