Figure 2.
PR induces a rapid upregulation of Slug, an EMT and chemotherapy resistance marker, independent of the TGFβ1/Smad pathway. Representative immunoblots (A) and (B) show Slug expression in AsPC-1 and BxPC-3 cells after time course treatment with platelet releasate (PR). 2 × 105 cancer cells per well were seeded in a 12-well plate for 24 h, serum starved for 6 h, then PR was added to the culture media (final concentration of PR was equivalent to releasate from 5 × 108 platelets/mL) for 10 min, 30 min, 2 h, 6 h, 18 h and 24 h. Representative immune blots (C) and (D) show Slug and pSmad2/3 expression in AsPC-1 and BxPC-3 cells after treatment with PR ± 10 µM SB431542 (TGFβ1 receptor inhibitor) or 0.1%DMSO (vehicle control, Sigma-Aldrich, St. Louis, MO, USA) for 2 h. Cell lysates were separated by SDS-PAGE and immunoblotted using specific antibodies for Slug (upper panel), p-Smad2/3 (middle panel) and loading control protein α-actinin or β-tubulin (lower panel). Each of the immunoblots (A)–(D) is representative of two independent experiments with similar results. Representative immune blots (E) and (F) and the associated bar graphs show Slug expression in AsPC-1 and BxPC-3 cells after 2 h PR ± gemcitabine treatment. The expression level of the protein of interest was quantified relative to the loading control. The graph columns represent fold changes in protein expression level compared to vehicle-treated cells (n = 4 for (E) and n > 3 for (F)). One way ANOVA with post.-hoc Bonferroni’s Multiple Comparison Test was used to examine the significance of the mean (** p < 0.001, * p < 0.05).