Figure 2. pVHL suppresses progerin expression. (A) pVHL suppresses progerin (PG) expression. Instead, the expression of other Lamin family proteins is not affected by pVHL. C2 cells were co-transfected with pVHL and the indicated GFP-Lamin A (LMA), Lamin B (LMB), Lamin C (LMC), or progerin (PG) vectors for 24 h. Because we used the GFP-fused vectors, the expression of each gene was examined with the GFP antibody. EV indicates empty-vector transfection. (B) Si-VHL induces progerin (PG) expression. C2V cells were transfected with indicating vectors or si-RNA for 24 h. “L. E” and “S. E” indicate longer exposed and shorter exposed results, respectively. (C) Endogenous progerin (PG) is reduced by pVHL overexpression. A704 and A498 cells were transfected with pVHL for 24 h. (D) Si-VHL induces endogenous progerin (PG) expression in C2V. C2V cells were transfected with si-C or si-VHL for 24 h and incubated with Nocodazole (Noc) or colcemide (Col) for 12 h. (E) pVHL suppress the half-life of progerin. 293 cells were transfected with progerin and pVHL or si-VHL for 24 h. After washing, cells were incubated with CHX (de novo protein synthesis inhibitor) for the indicated time. In pVHL-transfected cells, progerin’s half-life was reduced to 3.5 h, whereas si-pVHL extended it up to 24 h. We also provided the graphic analysis data in Figure S4B. (F) Proteasome inhibitor can block pVHL-induced progerin suppression. 293 cells, transfected with indicating vectors, were incubated with ALLN (20 μM) for the indicated time. In pVHL- or EV-transfected cells, progerin expression was increased. However, ALLN did not show this effect in si-pVHL-transfceted cells. (G) pVHL promotes progerin (PG) ubiquitination. 293 cells were co-transfected with HA-Ub, progerin and/or pVHL for 24 h and incubated with ALLN for additional 12 h. Cell lysates were subjected to IP with GFP Ab and WB with HA-Ub. (H) pVHL mutants do not suppress progerin expression. HA-tagged wild-type (WT-VHL) or mutant pVHL (C162F; 162, R167W; 167) were co-transfected with GFP-progerin (GFP-PG) for 24 h. (I) pVHL can ameliorate progerin-induced nuclear deformation. 293 cells were co-transfected with VHL and non-tagged progerin for 24 h. Differently from EV-transfected cells, WT-pVHL blocks progerin-induced nuclear deformation. Cells were fixed with Me-OH and stained with Lamin A/C (green) and HA (red). DAPI staining was used to visualize the DNA.