Fig. 5: Activation of p-Jun through the ERK signaling pathway contributes to the maintenance of primed chromatin accessibility.

a Schematic structure of the JUN protein sequence showing the DNA-binding and dimerization domains of JUN (bZIP) and the JNK phosphorylation sites (Ser⁶³ and Ser⁷³). For JUN-AA, point mutations were introduced to convert Ser⁶³ and Ser⁷³ to Ala. For JUN-EE, point mutations were introduced to convert Ser⁶³ and Ser⁷³ to Glu. For JUN-EE-Δbasic, the DNA-binding domain was truncated based on JUN-EE. For JUN-EE-Δleucine, the heterodimerization domain with other AP-1 family members was truncated based on JUN-EE. b Normalized mean fluorescence intensity of immunostaining signals of p-JUN in cells cultured on the soft matrix with or without JUN-EE mutant, and the signals of p-JUN in cells cultured on the stiff matrix with or without JUN-AA mutant. signals of p-JUN in cells cultured on the soft matrix with or without JUN-EE mutant. About 15 pairs of cells within the same imaging view were analyzed for each independent experiment. Paired Wilcoxon test. c Immunostaining of p-JUN showing the fluorescence intensity of p-JUN induced by the mutant. The merged image of DAPI and GFP indicating the cell expressed mutant JUN-AA or JUN-AA (outlined by red circle) and the control cell (outlined by white circle). Scale bars, 5 μm. d Normalized ATAC-seq signal profiles in the primed chromatin regions for cells cultured on the soft matrix following expression of JUN-EE, JUN-EE-Δbasic, and JUN-EE-Δleucine are shown in (a). e Normalized ATAC-seq signal profiles in the primed chromatin regions for cells cultured on the stiff matrix following expression of JUN-AA are shown in Fig. 5a. f Relative mRNA expression of α-SMA on the soft or stiff matrices for 4 days with or without mutants JUN. The expression level was normalized with GAPDH, three independent experiments, the separate one-way ANOVA analysis was done in each figure. The results are shown as mean ± SD. g Immunofluorescence images of soft matrix-induced cytoskeletal structures reconstructed following expression of JUN-EE. Scale bar, 10 μm. And Immunofluorescence images of stiff matrix-induced cytoskeletal structures reconstructed following expression of JUN-AA Scale bar, 10 μm. The image was collected from cells on different matrices for 4 days. h Mouse model of liver fibrosis induced by CCl₄ administration and the CCl₄ treatment schedule. n = 3 biologically independent samples per group. Created in BioRender. Zhao, W. (2025) https://BioRender.com/j7b1xe3. i Visualization of the degree of liver fibrosis by Masson’s trichrome staining, where collagen fibers are stained blue. Scale bar, 200 μm. IHC characterizes the expression levels of p-JUN in fibrotic mouse liver cells. Consecutive sections from fibrotic liver (j) and control liver (k) are stained with α-SMA antibody to mark activated hepatic stellate cells, and with the p-JUN. Scale bar, 200 μm. Enlarged views of the areas enclosed by the dashed boxes are shown below, scale bar, 100 μm. The black arrows in the images emphasize the regions where hepatic stellate cells are located.