Figure 3.

PCNA is sequestered by progerin in HGPS cells. A) XPA nuclear focus formation in normal BJ cells after PCNA knockdown by PCNA-specific siRNA. Western blot analysis demonstrated the efficiency of PCNA knockdown. B) Formation of XPA foci and colocalization with γ-H2AX in BJ cells with increasing passage number. One hundred cells were randomly chosen and counted. Cells that had at least 3 XPA foci were regarded as focus-positive cells. Statistical analysis of the data indicated that the changes were statistically significant between passage numbers 16 and 33 and between 16 and 44 (χ² = 79; P < 0.01). Nuclear XPA is significantly associated with the chromatin in later passage BJ cells. Progerin accumulation with increasing passage number in BJ cells was analyzed by Western blot analysis of the whole-cell extracts. C) PLA using PCNA-specific and either lamin A- or progerin-specific antibodies in old-passage HGPS cells. A significant majority of PCNA was associated with progerin, not lamin A. After primary antibody incubation, secondary antibodies conjugated to complimentary oligonucleotides that were bound, ligated, and amplified to generate a red signal indicating interaction of the respective protein pairs. Interaction-positive foci were quantified and plotted. D) HGPS cells were subjected to a Co-IP assay with pulldown by the anti-progerin antibody. The immunoprecipitated progerin was analyzed for PCNA association by Western blot analysis. More PCNA pulldowns with progerin in untreated old HGPS cells (P18) than in younger HGPS cells (P9). The bead control contains HGPS cell lysate without antibody.