PCTAIRE1-Knockdown Sensitizes Cancer Cells to TNF Family Cytokines
Fig 2
Targeting PCTAIRE1 using an inducible shRNA vector in PPC1 cells.
(A) PPC1 cells stably containing inducible shRNAs targeting different sites on PCTAIRE1 mRNA (shRNA#1, #2) or scramble-control were cultured for 48 hours with doxycycline (Dox, 100 ng/ml). Protein lysates were generated, normalized for total protein concentration, and analyzed by SDS-PAGE/immunoblotting using antibodies for PCTAIRE1 (top) and beta-actin (bottom). (B, C) PPC1 cells were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with various concentrations of either anti-Fas antibody CH-11 (B) or TRAIL (C). After 24 hours, cellular ATP levels were measured, and the data expressed as a ratio relative to cells cultured without anti-Fas or TRAIL (mean ± SD; n = 3). (D, E) Clonogenic survival assays. PPC1 cells stably containing inducible shRNAs (scramble or shRNA#2) were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with anti-Fas antibody (CH-11, 10 ng/ml) or TRAIL (20 ng/ml). Cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye.