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. 2019 Sep 23;9(24):7168–7183. doi: 10.7150/thno.36830

Figure 5.

Figure 5

LRP-1/ERK/c-JUN signaling is required for PAI-1-dependent PSC activation. A, After PAI-1 (200 ng/mL) treatment, α-SMA expression in RLT-PSC and PSC kdLRP cells was determined by Western blotting. B, After coculture with PANC-1 cells for 72 hours or treatment with PAI-1 (200 ng/mL) for 24 hours, RLT-PSC and PSC kdLRP cells were stained with anti-α-SMA (green) and oil red O (red). Magnification: 400x, scale bar: 20 μm. C, The migration of RLT-PSC cells and PSC kdLRP cells was analyzed using transwell assays after incubation with CM derived from PANC-1 or fresh medium containing PAI-1 (200 ng/mL) for 24 hours. The bar graph indicates the average number of migrating cells. *** P < 0.001 versus scrambled control cells, unpaired t-test. D and E, Protein and mRNA expression of collagen I and fibronectin in RLT-PSC cells and PSC kdLRP cells was determined by IF staining and q-PCR, respectively. The bar graph depicts relative mRNA expression of collagen I and fibronectin. ** P < 0.01; *** P < 0.001, significant difference between groups, one-way ANOVA. F, After coculture of cancer cells with RLT-PSC cells or PSC kdLRP cells, the stiffness of 3D coculture gels was measured by AFM. The bar graphs indicate the average values of Young's modulus of organotypic gels.** P < 0.01; *** P < 0.001, significant difference between groups, one-way ANOVA. G, After treatment with PAI-1 (200 ng/mL) for the indicated times in RLT-PSC cells and PSC kdLRP cells, expression of the indicated proteins was determined by Western blot analysis. H, RLT-PSC cells and PSC kdLRP cells were pretreated with U0126 (20 μg/mL) for 1 hour followed PAI-1 stimulation for 0.5 and 3 hours. Cell lysates were subjected to Western blotting with antibodies against the indicated proteins. I, RLT-PSC cells were transiently transfected with either nontargeting control siRNA or JUN siRNA. Forty-eight hours after transfection, cells were treated with PAI-1 (200 ng/mL) for 0.5 and 3 hours, and then cell lysates were subjected to Western blotting using antibodies against the indicated proteins.