Fig. 4: Reversal of activated PSC and ECM reduction in vitro.

a Western blot for phosphorylated p65 (p-p65) and total p65 (p65) protein in whole cell lysates of T3M4 cells and PSC cultured with various treatments in standard culture medium or conditioned medium (CM) at pH 6.5 for 48 h. The normalized intensities of the p-p65 relative to p65 are presented. PSC-CM, CM from PSC; T3M4-CM, CM from T3M4 cells. b Immunofluorescence (IF) staining of p65 (green) in T3M4 cells after treatment with the indicated formulations for 48 h at pH 6.5. Nuclei were labeled with DAPI (blue). Scale bar, 10 μm. c Quantification of the normalized nucleus p65 protein expression in T3M4 cells (n = 6 independent experiments). d IF staining of p65 (red) in PSC treated with the indicated formulations for 48 h at pH 6.5. Nuclei were labeled with DAPI (blue), and cell skeletons were labeled with phalloidin (green). Scale bar, 25 μm. e Quantification of the normalized nucleus p65 protein expression in PSC (n = 6 independent experiments). f IF staining of α-SMA (green), fibronectin (green), and Sirius red staining (red) of deposited collagen of PSC treated with the indicated formulations for 48 h at pH 6.5. Nuclei were labeled with DAPI (blue). Scale bars, 25 μm. Quantification of the normalized fluorescence intensities of α-SMA (g), fibronectin (h), and collagen (i) in PSC. Cells were treated with various formulations (AsiG: 50 nM; TPCA-1: 20 μM for T3M4 cells and 10 μM for PSC, n = 3 independent experiments). Immunostaining experiments were independently repeated three times. The data in (c, e), and (g–i) are shown as the mean ± SEM. Two-way ANOVA with Bonferroni’s multiple comparisons test was used for statistical significance analysis. Source data are provided as a Source Data file.