FIG 4.

MDM2 gene silencing induces some p53-regulated genes. (A to C) MDMs were transfected with siCtrl or siMDM2 for 48 h, which corresponds to the time of virus infection in previous figures. (A) qRT-PCR analysis was performed to monitor the relative mRNA expression for MDM2, TP53, CDKN1A, FAS, and BAX genes. Each symbol represents a different donor. The dotted line represents the gene expression of the siCtrl condition. Statistical analyses were performed using the paired Student's t test (ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n = 10). (B) Western blot analyses were carried out to quantify the amount of MDM2, p53, p21, total SAMHD1, p-T592 SAMHD1, and actin. Total SAMHD1 and p-T592 SAMHD1 were probed on a different membrane using the same samples and are shown with their respective actin loading controls. Some samples were treated with MG-132 for 6 h before cell lysis. Donor shown is representative of 4 distinct donors. (C) Apoptotic cells at the time of infection were measured by flow cytometry with annexin V and 7-AAD. siRNAs have only a mild effect on apoptosis compared to that of siCtrl. This donor is representative of a total of 2 donors.