Fig. 5. UBR2 protection towards CICD depends on MAPK/Erk signaling pathway.

A HeLa cells were treated with Actinomycin D (1 µM) and qVD-OPH (20 μM) for 4 days and the MAPK/Erk pathway was assessed by immunoblots. Activation was assessed by looking at the phosphorylation state of Mek1/2, Erk1/2, and p90Rsk. Hsp60 was used as a loading control. % of PI positive cells are indicated below the immunoblots. B HeLa cells were treated as previously or in combination with the MEK1/2 inhibitor U0126 (10 μM) for 24 h. Cell death was measured by flow cytometry using a DAPI staining. C HeLa cells were transfected with the three independent siRNA targeting UBR2. 48 h later, whole-cell lysates were analyzed for p.Erk1/2, Erk1/2, p.p90RSK, and RSK1/2/3 by immunoblotting. Hsp60 was used as a loading control. D HeLa cells were transfected to transiently overexpress the control vector (pcDNA₃) or UBR2-His-tagged. After 24 h, cells were cultivated without serum for 16 h and the next day were stimulated with 10% FBS for the indicated times. Whole cell lysates were analyzed for p.Erk1/2 and Erk2 (used as a loading control). E HeLa cells were transfected to transiently overexpress (OE) the control vector (pcDNA₃) or UBR2-his-tagged and were treated with Actinomycin D (1 µM) + qVD-OPH (20 μM) alone or in combination with the MEK1/2 inhibitor U0126 (10 μM) for 3 days. Cell death was measured by flow cytometry using a DAPI staining. Data are expressed as mean ± s.d (n = 3) immunoblots are representative of 3 individual experiments. *p < 0.05, ****p < 0.0001, N.S: non-significant according to a two-way ANOVA.