Fig 2: Characterization of control- and TRAIL-R4 knockdown Colo357 cells. (A) Colo357 cells were transduced with either control lentiviral shRNA or TRAIL-R4 specific shRNA to stably knockdown TRAIL-R4. The protein level of TRAIL-R4 was detected in the whole cell lysate of the transduced cells via western blot. Additionally the mRNA level of TRAIL-R4 in TRAIL-R4 KD clones [TR4 KD (a) and (b)] was measured via qPCR (B), shown is the mean of three biological replicates. (C) Furthermore, cell surface expression of TRAIL-R4 in the TRAIL-R4-KD clones (a) and (b) (gray filled histograms; thin lines, isotype control) was detected by using flow cytometry. Shown is a representative histogram out of six biological replicates and an overlay of control- (gray filled histograms) vs. TR4-KD Colo357 cells (bold lines). (D) The viability of cells under TRAIL treatment (50 ng/mL) was determined by EZ4U assay. Shown is the mean of three biological replicates. Student t-test was done to determine significance (* < 0.05 p-value). (E) Ten thousand control- (light green line) or TRAIL-R4-knockdown (TR4-KD) Colo357 cells [TR4-KD (a), dark blue line; TR4-KD (b), orange line] were cultured in complete medium for 95 h on E-plates. The bottom of the E-plates were covered with electronic sensors that measured the impedance of these adherent tumor cells expressed as an arbitrary unit called cell index (CI) every 5 min. The arrows mark the addition of substances after 26 h as follows: medium [light green line for control KD Colo357 cells, dark blue line for TR4-KD- (a), orange line for TR4-KD (b) Colo357 cells], 100 ng/mL recombinant TRAIL [dark green line for control KD cells, red line for TR4-KD (a), and light blue line for TR 4 KD (b) cells] or Triton-X-100 to induce maximal lysis (black line). CI was then measured for additional 69 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 is shown.

Fig 3: Impact of TRAIL-R4 KD on the expression of adhesion- and costimulatory molecules as well as TNF-receptor family members on Colo357 cells. Flow cytometric analysis of the indicated (A) adhesion- and costimulatory surface receptors and (B) CD95, TNF-R1, TNF-R2, TRAIL-R1, -R2, and -R3 expressed on control KD - and TRAIL-R4 KD (TR4 KD) Colo357 cells determined by staining the cells with the appropriate monoclonal antibodies (gray histograms) and appropriate isotype controls (open black lines). The expression was calculated as the fold change of the median of specific antibody signal relative to the isotype control. Median of fold changes of one representative histogram out of four independent experiments is shown.

Fig 4: 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

Fig 5: 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.