Figure 3

Increased P53 activation in ZIKV-infected hNPCs. (a) Control (CTRL) and infected (ZIKV) NSCi90 cells were analyzed 48 h after infection by immunofluorescence with anti-tubulin, anti-cleaved caspase-3 (C-cas 3) and anti-flavivirus antigens (4G2), and counterstained with DAPI (4',6-diamidino-2-phenylindole). The field shown in figure contains a caspase-positive/4G2-intense cell (yellow arrowhead in the merge), a caspase-positive cell with low 4G2 signal (green arrowhead) and a caspase-negative/4G2-positive cell (red arrowhead). Scale bar=5 μm. (b) The first two bars of the histogram represent a quantification of the average frequency of caspase-3-positive cells in control and infected cells. The third bar represents the relative levels of 4G2-positive (red) and 4G2-negative (white) cells in caspase-3 positive cells of the infected samples. Error bars=S.E.M. (n=4000). (c) Representative image of ZIKV-infected cells stained with anti-cleaved caspase-3 and antiphospho-Ser15 P53. Positive nuclear stain with the latter antibody was observed in essentially all cells positive for cleaved caspase-3 (n=180). Scale bar=10 μm. All data shown in the figure are representative of three independent infections. (d) Western blot analysis of cleaved caspase-3, normalized on histone H3. (e) IF analysis of γH2Ax foci in control and ZIKV-infected cells 24 h after infection. Cells with multiple foci are magnified in the inset. Scale bar=10 μm. (f) The average number of foci per nucleus was automatically quantified. ***P<0.001; **P<0.01 (two-tailed unpaired Student's t-test)