Fig 2: Pafah1b1 knockdown reverses the pro-growth of miR-181c/d inhibited murine Sertoli cells. The murine SCs were transfected with NC siRNA or Pafah1b1 siRNA. a Immunofluorescence staining of Ki67 (red) in Pafah1b1 siRNA treated murine SCs. Scale bar: 100 µm. b Quantification of Ki67-positive cells in Pafah1b1 siRNA treated murine SCs. c CCK-8 assay performed in Pafah1b1 siRNA treated murine SCs. d Western blot analysis of PAFAH1B1, PCNA, BAX, and BCL2 in Pafah1b1 siRNA treated murine SCs. The quantification of protein level is shown in the bar graph (e). f Annexin V-FITC/PI and flow cytometry analysis was used to examine cell apoptotic rate in Pafah1b1 siRNA treated murine SCs. g The quantification of cell apoptotic rate in Pafah1b1 siRNA treated murine SCs. Five co-transfection treatments were constructed in this experiment, including inhibitors NC + NC siRNA, miR-181c inhibitors + NC siRNA, miR-181d inhibitors + NC siRNA, miR-181c inhibitors + Pafah1b1 siRNA, and miR-181d inhibitors + Pafah1b1 siRNA. h-j Ki67 staining (h) and CCK-8 (j) assay were performed in murine SCs treated with co-transfections. Quantification of Ki67-positive murine SCs treated with co-transfections (i). Scale bar: 100 µm. k Western blot analysis of PAFAH1B1, PCNA, BAX, and BCL2 in murine SCs treated with co-transfections. The quantification of protein level is shown in the bar graph (l). m Annexin V-FITC/PI and flow cytometry analysis was used to examine cell apoptotic rate in murine SCs treated with co-transfections. n The quantification of cell apoptotic rate in murine SCs treated with co-transfections. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05; **p < 0.01; ns, not significant

Fig 3: PDIA6 promotes proliferation of PC cells. (A) The knockdown/overexpression efficiency of PDIA6 in PC cells was assessed by Western blot. (B) CCK-8 detection of cell viability in cells with PDIA6 overexpression or knockdown. (C) Analysis of cell colony formation capacity in cells with PDIA6 overexpression or knockdown. (D) PCNA, c-MYC, and cyclin D1 protein levels were assessed by Western blot after PDIA6 inhibition or overexpression. N = 3. *P < 0.05, **P < 0.01, and ***P < 0.001. Data were mean ± SD. Unpaired Student's t test, one-way ANOVA and two-way ANOVA with Tukey's post-tests.

Fig 4: DSS repressed the proliferation and migration of NIH3T3 cells induced by TGF-β1. NIH3T3 cells were treated with TGF-β1 (5 ng/mL) in the absence or presence of DSS for 24 h. (a) The effect of DSS (25, 50, 100, 200 and 400 μM) on the cell viability was detected by CCK-8 assay. (b) The effect of DSS (25, 50, 100, 200 and 400 μM) on the cell viability induced by TGF-β1 was detected by CCK-8 assay. (c) Effects of DSS (50 μM) on the cell migration were detected by wound healing analysis. Scale bar: 200 μm. (d) The calculation of wound healing ratio. (e, f) The expression of PCNA was detected by western blot assay. GAPDH was conducted as a loading control. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant

Fig 5: PDIA6 activates the ß-catenin pathway. (A) P-ß-catenin (Ser33/Ser37/Thr41) and ß-catenin protein expression was determined by Western blot after the downregulation or overexpression of PDIA6. (B) Immunofluorescence analysis of ß-catenin expression and location in PDIA6-downregulated or -overexpressing cells. (C) The ß-catenin activity was assessed by luciferase reporter assay after the downregulation or overexpression of PDIA6. (D-F) The effects of ß-catenin overexpression on viability, migration, and invasion were detected in PDIA6-downregulated cells. (G) The c-MYC, PCNA, MMP2, and MMP9 protein expression was detected in PDIA6-downregulated cells transfected with ß-catenin overexpression plasmids. N = 3. *P < 0.05, **P < 0.01, and ***P < 0.001. Data were mean ± SD. Unpaired Student's t test and one-way ANOVA and two-way ANOVA with Tukey's post-tests.