Novel modulators of p53-signaling encoded by unknown genes of emerging viruses
Fig 5
Induction of p53, p53 pSer15, and p21 by KSHV orf10.
(A) Cells were transfected with empty vector (EV) and pOME0004 (KSHV orf10) or left untransfected (UN). At 18h p.t., the cells were stimulated with 10μM etoposide for 6h or mock treated (No Drug). The cell lysates were analyzed by SDS-PAGE and immunoblotting with indicated Abs. (B) Shows relative protein expression levels (fold changes compared to EV) by IB cumulative of two different experiments that were calculated based on pixel density of each protein band measured using ImageJ software. Bars indicate the s.e.m. (n ≥ 3). P-values calculated using Student t-test (n = 3) are shown as ‘ns’ (P>0.05), * (P≤0.05), **(P≤0.01), or *** (P≤0.001). (C) Shows protein levels in orf10 expressing cells by IF relative to mock transfected cells (fold changes compared to 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 (n = 3) are shown as * (P≤0.05), **(P≤0.01), or *** (P≤0.001), **** (P≤0.0001). (D-G) U2OS cells were transfected with KSHV orf10-Flag. At 18 hrs p.t., the cells were stimulated with 10 μM etoposide for 1.5 hrs (D, F) or left untreated (E, G), fixed with methanol, and stained with indicated Abs. The scale bar is 50 μM. Arrows point at the cells expressing KSHV orf10.