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. 2020 Oct 19;10:17649. doi: 10.1038/s41598-020-74806-2

Figure 2.

Figure 2

P-AscH− decreases invadopodia-mediated ECM degradation and down regulates MMP-9 and MMP-2 expression. (A) P-AscH− attenuates the degradation of fluorescent gelatin matrices by invadopdia of BxPC-3 PDAC cells. BxPC-3 cells were treated with 1 mM ascorbate with or without catalase (200 U/mL) for 1 h. Following treatment, cells were re-plated on coverslips with Oregon Green-conjugated gelatin for 24 h. Cells were fixed, permeabilized, and stained for actin using TRIC-Phalloidin. Fluorescence images were acquired to determine foci of degraded matrix visible as black spots in the bright fluorescent gelatin matrix. (B) P-AscH− decreased the area of degradation of BxPC-3 cells compared to control. The addition of catalase reversed the decrease seen with ascorbate. Data represent the mean area of degradation compared to control ± SE (n = 3, *p < 0.05; one-way ANOVA with Bonferroni’s multiple comparisons). (C) MMP-9 expression was decreased in MIA PaCa-2 and PANC-1 cells following ascorbate treatment (1 mM MIA PaCa-2, 5 mM Panc-1 for 1 h) and subsequent Western blot analysis. Incubation with catalase (200 U/mL) reversed the decrease seen with ascorbate. GAPDH was used as a loading control (n = 3). Loading controls for Fig. 2C were run on a separate gel from samples derived from the same experiment. Original unprocessed blots can be found in Supplementary Fig. S2. (D) MMP-2 expression was decreased in 339 and PANC-1 cells following ascorbate treatment (5 mM for 1 h) and subsequent Western blot analysis. Incubation with catalase (200 U/mL) reversed the decrease seen with ascorbate. GAPDH was used as a loading control (n = 3). Original unprocessed blots can be found in Supplementary Fig. S2.