Figure 5
An unexpected antiviral contribution of Mdm2 revealed by silencing/transient expression experiments. (A) Knock-down of p53 and/or Mdm2 mRNA expression in A549 cells differentially modulates levels of IAV production (Left panel). Forty-eight hours after si-RNA transfection using a control si-RNAs (si-Ctrl) or specific siRNAs (si-p53/si-Mdm2), A549 cells were infected by A/Moscow/10/99 (H3N2) at a MOI of 0.01 and the level of viral production at 24 h post-infection was assessed using three different experiments: (i) RT-qPCR (log10 RNA copies/mL, measured in three independent experiments. An asterisk indicates a significant difference compared with the results for si-Ctlr treated cells (*** and **** for respectively P-values < 0.001 and <0.0001), (ii) determination of infectious viral titers of supernatants (TCID50/mL) by endpoint titration in MDCK cells (measured in quadruplicate in 2 independent experiments), and (iii) western blot. Ku80 was used as a loading control; (B) Transient expression/co-expression of p53 and/or Mdm2 in H1299 cells decreases the level of viral production (Right panel). Forty-eight hours after transfection, H1299 cells were infected by A/Moscow/10/99 (H3N2) at a MOI of 0.01 and the level of viral production at 24 h post-infection was assessed using similar methods. An asterisk indicates a significant difference compared with the results obtained for cells transfected with the empty vector (*** and **** for respectively P-values < 0.001 and <0.0001) (C) Comparative viral kinetics in the context of transient expression of Mdm2, Mdm2 RING mutant C462A or Mdmx in H1299 cells. Forty-eight hours after transfection, H1299 cells were infected by A/Moscow/10/99 (H3N2) at a MOI of 0.01 and the level of viral production at 24 h and 48 h post-infection was assessed using determination of infectious titers of supernatants (log10 TCID50/mL) by endpoint titration in MDCK cells (measured in quadruplicate in 2 independent experiments). An asterisk indicates a significant difference compared with the results obtained for cells transfected with the empty vector (*** for P-value < 0.001).
