Novel modulators of p53-signaling encoded by unknown genes of emerging viruses
Fig 2
Expression of the p53-signaling components in the presence of ZIKV NS2A.
(A) Cells were transfected with empty vector (EV) and pOME0303R (ZIKV NS2A) or left untransfected (UN). At 18h after transfection, the cells were stimulated with 10 μM etoposide for 6h. The cell lysates were analyzed by SDS-PAGE and immunoblotting with indicated antibodies. (B) Shows protein levels by immunoblotting (IB) normalized to beta-actin relative to empty vector (% EV) for three different experiments that were calculated based on pixel density of each protein band measured using ImageJ software. Bars indicate the standard error of mean (s.e.m., n = 3). P-values calculated using Student t-test are shown as ‘ns’ (P>0.05), * (P≤0.05), **(P≤0.01), or *** (P≤0.001). (C) Shows protein levels in ZIKV NS2A expressing cells by immunofluorescence (IF) relative to untransfected cells (%UN) that were calculated based on pixel density measured using ImageJ software. Bars indicate s.e.m. (n≥10). P-values calculated using Student t-test are shown as ‘ns’ (P>0.05), * (P≤0.05), **(P≤0.01), or *** (P≤0.001). (D and E) U2OS cells were transfected with ZIKV NS2A-Flag. At 18h after transfection the cells were stimulated with 10 μM etoposide for 1.5 hrs, fixed, and stained with anti-Flag, anti-phospho-p53Ser15, or anti-p21 antibodies. DAPI was used to delineate the nucleus. The images were subjected to a digital deconvolution. Each image represents an individual optical section. The scale bar is 50 μm. Arrows point at the cell expressing ZIKV NS2A.