Figure 3

Treatment by poly(I:C) increases c-IAP2 gene ( BIRC3 ) transcription in human malignant cells. Inhibition by Bafilomycin A1 and knock-down of TRIF. A) Cell concentrations of BIRC3 messenger RNAs are measured by quantitative RT-PCR in several types of malignant cells mock-treated or treated with poly(I:C) for 16 h at 500 ng/ml. Concentration of BIRC3 transcripts measured in basal conditions were chosen as a reference for each tested cell type and arbitrarily set at 1. B) Kinetics of the increase in the concentration of BIRC3 messenger RNAs in HeLa cells treated with poly(I:C) 500 ng/ml for increasing time intervals from 1 to 48 h. C) The influence of poly(I:C) on BIRC3 transcripts is neutralized by Bafilomycin A1, an inhibitor of endosome acidification and TLR3 signalling. Prior to RNA extraction, HeLa cells are incubated for two hours in the presence of poly(I:C)(500 ng/ml) or recombinant TNF α (20 ng/ml) in combination with Bafilomycin A1 (BFA) 100 nM or 2-aminopurin (2-AP) 5 mM or without additional compound (control condition). D) The induction of c-IAP2 by poly(I:C) in Hela cells is suppressed when the TRIF adaptor protein is knocked-down using 3 distinct specific siRNA whereas a non-specific (NS) RNA used as a negative control has no inhibitory effect. Upper panel: quantitative PCR assessment of the BIRC3 transcripts in the absence or in the presence of specific siRNAs. Lower panel: absence of TRIF protein expression detectable by western blot in Hela cells treated with specific siRNA (1, 2 and 3); in contrast TRIF expression is not altered by a negative control non-specific siRNA (NS). Data are representative of two similar experiments. The stars indicate a statistical difference from respective controls (p < 0.05).