FIG 4.

Silencing of the TP53-i9 alternatively spliced β and γ isoforms impairs IAV replication. (A) A549 cells were treated twice with either the control nonspecific si-RNA (si-ctrl) or a specific si-RNA targeting the p53β and p53γ spliced isoforms. (B) Twenty-four hours after the last si-RNA treatment, cells were infected with H3N2 virus at an MOI of 0.1 or 0.01. Supernatants were harvested at 24-h intervals over 3 days, and the viral replication was determined by endpoint TCID₅₀ titration in MDCK cells (measured in quadruplicate in two independent experiments). (C) Cell lysates were harvested before infection (T = 0) or at 72 hpi to quantify p53 total and p53β mRNA expression levels by RT-qPCR, normalized to actin expression. (D) Cellular and viral proteins were detected by Western blotting. # and ## indicate short and long exposures, respectively. Data represent independent experimental duplicates. Mean values ± standard deviations are shown, and statistical tests compared each condition with the si-ctrl T = 0 condition using two-way ANOVA (*, P < 0.05; **, P < 0.01).