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. 2016 Jan 13;7(4):3709–3725. doi: 10.18632/oncotarget.6915

Figure 6. IFNα/BV6-induced cell death depends on TRAIL signaling in A172 cells.

Figure 6

Figure 6

A.-C. A172 cells were transiently transfected with 5 nM siRNA targeting TRAIL (siTRAIL1, siTRAIL2) or control siRNA. TRAIL mRNA levels were analyzed by qRT-PCR and are shown as fold increase with mean + SD of three (A172) or five (HT-29) independent experiments performed in duplicate, 28S rRNA was used as loading control A.. Cell death was determined after treatment for 72 hours with 5 ng/ml IFNα and/or 1 μM BV6 by PI staining using flow cytometry, mean + SD of five independent experiments performed in duplicate are shown; *, P < 0.05; **P < 0.01 B.. Cell viability was determined by MTT assay and data are shown as percentage of untreated control cells with mean + SD of five independent experiments performed in triplicate; *P < 0.05; **P < 0.01 C..D.-G. A172 cells were transiently transfected with 5 nM siRNA targeting DR5 (siDR5-1, siDR5-2) or control siRNA. Protein expression of DR5 was analyzed by Western blot analysis, GAPDH was used as loading control D., and by immunostaining of DR5 surface expression using flow cytometry showing the isotype control (ISO Ctrl) in grey E.. Cell death was determined after treatment for 72 hours with 5 ng/ml IFNα and/or 1 μM BV6 by PI staining using flow cytometry and mean + SD of three independent experiments performed in duplicate are shown (F). Cell viability was determined by MTT assay and data are shown as percentage of untreated control cells with mean + SD of three independent experiments performed in triplicate; *P < 0.05; **P < 0.01 (G). H. A172 cells were transiently transfected with 5 nM siRNA targeting DR5 (siDR5-2), TRAIL (siTRAIL-1) or control siRNA and treated with 5 ng/ml IFNα and 1 μM BV6 for 18 hours. Caspase-8 was immunoprecipitated using an anti-caspase-8 antibody and detection of RIP1, FADD and caspase-8 was done by Western blotting, GAPDH served as loading control.