Fig 2: Enhanced Vδ1 T cell-mediated lysis of tumor cells compared to Vδ2 T cells is diminished by TRAIL-R4 knockdown. (A) After culturing 104 control KD- (green lines) or two TRAIL-R4 KD Colo357 cells [TR4 KD (a), dark blue lines and TR4 KD (b), orange lines] in complete medium for 30 h, impedance of these adherent tumor cells expressed as cell index (CI) was measured every 5 min. The CI was normalized to 1 shortly before the addition of substances as follows: Triton-X-100 to induce maximal lysis (black line), Vδ2 γδ T cell line (brown lines) or Vδ1 γδ T cell line (pink lines) of healthy donor #1 (E/T ratio 25:1) with 50 IU/mL rIL-2. CI was then measured every minute for additional 23 h. The loss of tumor cell impedance and thus a decrease of CI correlated with lysis of tumor cells. The average of triplicates and standard deviation were calculated; one representative experiment out of three for Vδ2 γδ T cell lines and eight for Vδ1 γδ T cell lines is shown. (B) Ten thousand control KD (ctrl) or the indicated TRAIL-R4 (TR4) KD tumor cells were cultured under the same conditions as described under (A), and also cocultured with Vδ2- or Vδ1 γδ T cell lines (E/T ratio 25:1) in the presence of 12.5 IU/mL rIL-2 in the RTCA. Percentage lysis was analyzed from RTCA data by calculating the normalized impedance of spontaneous lysis (cell growth of tumor cells in medium alone) in relation to the maximal lysis induced by 1% Triton-X-100. The mean of six (for Colo357 cells) to three (for the other tumor cells) individual samples cultured as triplicates plus standard deviation are shown 4 h after the addition of γδ T cells. Significances are shown as P-value; *P < 0.05 and **P < 0.01.

Fig 3: MAP Kinases ERK1/ERK2 regulate COX-2 level in Colo357 cells. Control KD- as well as TRAIL-R4 KD [TR4 KD (a)] Colo357 cells were treated with four different inhibitors [MG-132 (proteasome inhibitor), U0126 (MEK inhibitor), MK2206 (AKT1,2,3 inhibitor)] for 8 h. (A) The protein levels of COX-2, phospho-ERK1/2 and ERK were detected in whole cell lysates via western blot. (B) The basal level of the different proteins in both TR4 KD- (a, b) and control Colo357 cells is shown. (C) The correlation between COX-2, phospho-ERK1/2 and ERK1/2 in control KD and TR4 KD (b) cells is presented.

Fig 4: Enhanced PGE2 release and COX-expression of TRAIL-R4 knockdown Colo357 cells in comparison to control cells. (A) PGE2 release was determined in the supernatant of control KD–and two different TRAIL-R (TR) 4 KD (a) or (b)—Colo357 cells after overnight culture. (B,C) In parallel, cells were intracellularly stained with anti-COX-1 and anti-COX-2 mAb and analyzed by flow cytometry. The expression is shown as fold change in median fluorescence intensity (MFI) relative to isotype control. Bars represent mean ± SD of three independent experiments. Significances are shown as P-value; * = P < 0.05 and ** = P < 0.01.

Fig 5: Altered recruitment of inhibitory components to the DISC promotes caspase-8 activation in uPA knockdown cells. (a) Time-course analysis of procaspase-8 cleavage. BJELR cells were transfected either with pooled siRNAs targeting PLAU mRNA (‘siPLAU') or non-targeting scramble siRNAs (‘scramble'). Forty-eight hours after transfection, cell populations were either left untreated or challenged with 1 μg/ml TRAIL during 1, 3 or 7 h and the percentage of cells displaying cleaved caspase-8 (‘C8') was determined by flow cytometry. Images from one representative experiment out of three independent replicates are shown. Percentage of cleaved C8-positive cells is indicated. (b) DISC composition in uPA-depleted cells. BJELR cells were transfected either with pooled siRNAs targeting PLAU mRNA (‘siPLAU') or non-targeting scramble siRNAs (‘scr'), and 48 h after transfection, cell populations were either left untreated or challenged with TRAIL (1 μg/ml) during 30 min. Immunoprecipitation of Death Receptor 5 (IP-DR5) was performed and co-immunoprecipitation of cognate DISC components, uPA and uPAR, was analyzed by western blot analysis. Immunoprecipitation using isotypic IgG1 (‘IgG1') was used as background control. (c) Surface levels of TRAIL-Rs upon uPA knockdown. BJELR cells transfected either with pooled siRNAs targeting PLAU mRNA (‘siPLAU') or non-targeting scramble siRNAs (‘scr'). Forty-eight hours after transfection, surface levels of Death Receptor 5 (‘DR5'), Death Receptor 4 (‘DR4'), Decoy Receptor 1 (‘DcR1') and Decoy Receptor 2 (‘DcR2') were analyzed by flow cytometry. Isotypic IgG1 labeling was used as control for background fluorescence in scramble- (‘IgG1 scr') and siPLAU- (‘IgG1 siPLAU') transfected cells. (d) Antiapoptotic role of DcR2. BJELR cells were transfected either with pooled siRNA targeting Death Receptor 5 mRNA (‘siDR5'), Decoy Receptor 2 mRNA (‘siDcR2') or non-targeting scramble siRNAs (‘scr'), and 48 h after transfection, cell populations were either left untreated or challenged with TRAIL (1 μg/ml) during 3 h. Apoptosis was determined as the percentage of cells with positive labeling for cleaved PARP by flow cytometry. Histograms represent the mean±S.D. of three independent biological replicates. Statistical significance was calculated by applying two-tailed, unpaired Student's t-test, *P-value<0.05; **P-value<0.005. (e) Efficiency of DR5 and DcR2 knockdown at 48 h after transfection analyzed by western blot analysis. α-Tubulin, loading control