Fig 2: MACC1 promotes PC cell migration and invasion by upregulating FN1.A Heatmap of the upregulated gene expression clusters in control (Ctrl) and MACC1-overexpressing (OE) SUIT-2 cells. B, C Quantitative real-time PCR validation of selected differentially regulated genes after overexpression of MACC1 in PANC-1 and SUIT-2 cells. **p < 0.01, ***p < 0.001, ****p < 0.0001. D Quantitative real-time PCR validation of selected differentially regulated genes after knockdown of MACC1 in BxPC-3 cells. *p < 0.05, **p < 0.01, ****p < 0.0001. E–G Western blot for FN1, PTGS2 and MMP10 after overexpression of MACC1 in PANC-1 and SUIT-2 cells or knockdown of MACC1 in BxPC-3 cells. H, I GO analyses (H) and KEGG pathway analyses (I) of the upregulated genes in MACC1 overexpressing cells. J, K MACC1 and FN1 mRNA levels in primary tumor tissues and matched adjacent normal tissues from 10 PC patients. *p < 0.05, **p < 0.01, ****p < 0.0001. L MACC1 and FN1 protein levels in primary tumor tissues and matched adjacent normal tissues from 10 PC patients. M Correlation between MACC1 and FN1 mRNA levels in PC tissues from the TCGA datasets. R, Pearson correlation coefficient. N–P Immunofluorescence staining was performed in PANC-1, SUIT-2 and BxPC-3 cells using an anti-FN1 antibody. Representative images of immunofluorescent staining in PANC-1 and SUIT-2 cells stably expressing empty vector (lv-ctrl) or MACC1 (lv-MACC1) or in BxPC-3 cells stably expressing shNT, shMACC1#1 or shMACC1#2. Data are representative of at least three independent experiments. Scale bars, 20 μm. D–F, J, K, Data represent the mean ± SD. of three biologically independent experiments (two-tailed t test). G–I, L, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments.

Fig 3: miR-302d suppresses TGFB-induced production of FN1.(A) ICC staining and (B) representative Western blot of FN1 in transfected hRPE cells after 3 d incubation of TGFB1. The scale bar represents 50 μm. (C) The relative expression of FN1 normalized to total proteins (S1 Raw images). The values were further normalized to the "wo TGFB1" group. One-way ANOVA with Tukey’s multiple comparisons tests (n = 3) were performed. The error bars represent SD. ns, non-significant, *p < 0.05.

Fig 4: Rolipram restores ERK1/2 signaling to block TGF-β1 stimulated LF fibroblast fibrosis. (A) Different drugs were used to treat fibroblasts stimulated by TGF-β1. WB detects the expression of TGF-β1. a, Rolipram (50 nM) is a PDE4 inhibitor.; b, WAY-262611 (1 μM) is the Wnt/β-catenin pathway activator.; c, LY294002 (1 μM) is an inhibitor of the PI3K pathway; d, SCH772984 (1 μM) inhibits ERK1/2 signal transduction. (B) The expression of FN1 and TGF-β1 was up-regulated after exogenous TGF-β1 stimulation. Using Rolipram or SCH772984 blocked this change. (C) Western blot analysis reveals the expression levels of p-ERK1/2 and ERK1/2 in LF fibroblasts treated with TGF-β1 (5 ng/ mL) and Rolipram (50 nM, 24 h). β-tubulin served as an endogenous loading control. *p < 0.05

Fig 5: Rolipram blocks TGF-β1-induced LF fibroblast fibrosis. (A) a and b, Picture of fibroblasts (40x). c and d, staining with Sirius Red makes the fibroblasts easy to observe (100x). (B) Cell scratch test in LF fibroblasts (100×). (C-G) After fibroblasts were stimulated by TGF-β1 and treated with Rolipram, the expression of Col I, Col3A1, FN1, α-SMA and TGF-β1. GAPDH or β-tubulin served as an endogenous loading control. *p < 0.05, **p < 0.01, ***p < 0.001