Fig 1. PCTAIRE1 knockdown sensitizes PPC1 cells to anti-Fas antibody and TRAIL.

(A) PPC1 cells were transfected with scrambled RNA or three different siRNAs targeting PCTAIRE1 (siRNAs 1472, 1566, 1656). Lysates from cells at 48 hours post-siRNA transfection were prepared, normalized for total protein content, and aliquots were analyzed by immunoblotting using anti-PCTAIRE1 (top) or anti-alpha-tubulin (bottom) antibodies. (B, C) PPC1 cells were transfected with control RNA (purple “x”) or various siRNAs targeting PCTAIRE1 (blue diamonds, 1472; red squares, 1566, green triangles, 1656). After 48 hours, cells were stimulated with either anti-Fas antibody (CH-11) (B) or TRAIL (C) at various concentrations as indicated. After 24 hours, cellular ATP levels were measured as a surrogate indicator of cell viability using Cell Titer Glo reagents, and the data are expressed as the ratio between cells cultured with and without anti-Fas (B), and TRAIL (C). (D) Apoptosis analyses were performed using an annexinV kit. PPC1 cells were transfected with scramble RNA of siRNAs targeting PCTAIRE1. After 48 hours, cells were treated with anti-Fas antibody (100 ng/ml) or TRAIL (50 ng/ml) for 4 hours. *P < 0.05, ***P < 0.001 by t-test. All data represent mean ± SD (n = 3). (E, F) Clonogenic survival assays. PPC1 cells were seeded in 6 well (35 mm) dishes at 1.0 x 10⁵ cells per well and then reverse-transfected with scramble-control or PCTAIRE1-targeting siRNAs as indicated. After 48 hours, anti-Fas antibody (E, 10 ng/ml) or TRAIL (F, 20 ng/ml) was added and cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye. (G, H) PPC1 cells were transfected with control RNA or three different siRNAs targeting PCTAIRE1. After 48 hours, the cells were stimulated with Fas (G, 10 ng/ml) or TRAIL (H, 10 ng/ml). After 3 hours, cell lysates were prepared, normalized for total protein content, and analyzed by immunoblotting using an antibody specific for cleaved caspase-8. The partially cleaved 43/41 kDa bands and fully cleaved 18 kDa band are indicated, as are molecular weight markers (kDa). The blot was reprobed with beta-actin antibody as a loading control. (I) PPC1 cells were transfected with scramble-control or siRNA targeting PCTAIRE1 (si-1472). After 48 hours, cells were or were not stimulated with anti-Fas antibody (CH-11, 10 ng/ml). After 3 hours, cell lysates were harvested and immunoprecipitated with anti-caspase 8 antibody, followed by immunoblotting with the indicated antibodies. The red arrowheads indicate non-specific bands.