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. 2017 Oct 24;9(10):142. doi: 10.3390/cancers9100142

Figure 3.

Figure 3

Platelets modulate the expression of Slug, human equilibrative nucleoside transporter 1 (hENT1) and cytidine deaminase (CDD) in PDAC cells. Representative immunoblots (A) and (B) show the expression of Slug, hENT1 and CDD in AsPC-1 and BxPC-3 after treatment with platelets (Plt), platelet releasate (PR) or degranulated platelets (DG Plt) for 24 h. PR and degranulated platelets (DG Plt) were isolated from activated platelets. Briefly, platelets (1 × 109/mL) were aggregated by incubating with 1 µg/mL CRP for 30 min then the supernatant (i.e., PR) and the pellet (i.e., DG Plt) were separated by centrifugation (5000 g for 10 min). The pellet was resuspended in Tyrode’s buffer, using the initial volume. The cancer cells were seeded in a 6-well plate at 3 × 105 per well for 24 h, then incubated with Plt, PR or DG Plt to the final concentration equivalent to 1 × 108 platelets/mL for 24 h in serum-free media. Cell lysates were separated by SDS-PAGE and immunoblotted using specific antibodies for Slug, hENT1, CDD and loading control protein α-actinin. Bar graphs (C) and (D) show the changes in the expression of Slug, hENT1 and CDD in AsPC-1 and BxPC-3 after different treatments. The expression level of the protein of interest was quantified relative to the loading control and normalised to the negative control group (n ≥ 3). Data are presented as mean ± SEM. One way ANOVA with post-hoc Bonferroni’s Multiple Comparison Test was used to examine the significance of the mean. *** p < 0.0001, ** p < 0.001, * p < 0.05.