Figure 7. Knock down of SUN1 alleviated HGPS-associated loss of NURD complex and cellular senescence.
(A) Normal (AG03512) and HGPS (AG11498) skin fibroblasts were stained for heterochromatin markers (RBBP4 or H3K9me3; green) and SUN1 (red). Yellow arrow heads point to high-SUN1 cells; white arrow heads denote low-SUN1 cells. See also Figure S6.
(B) Expression levels of RBBP4 or H3K9me3 and SUN1 in two normal and three HGPS skin fibroblasts were quantified by MetaMorph software. Each dot represents fluorescence intensity (in Log10 scale) in a single cell of RBBP4 (left) or H3K9me3 (right) vs. SUN1. Linear curve fitting and correlation coefficient (r) are indicated. In HGPS cells, RBBP4 and H3K9me3 expression correlates negatively with SUN1 expression.
C) HGPS fibroblasts (AG03513) treated with control or SUN1 siRNA for 72 hours were stained with antibodies for SUN1 (red) and RBBP4 (green). Increased RBBP4 expression was observed in SUN1 siRNA-treated cells compared to control siRNA-treated cells. Graphic quantification of the staining intensities of RBBP4 vs. SUN1 in individual HGPS fibroblasts treated with control (blue) or SUN1 (brown) siRNA is shown (right); each dot represents a single cell (154 control and 157 SUN1 RNAi treated cells were quantified).
(D) Visualization (Left) and quantification (Right) of acidic senescence associated β-galactosidase (SA-β-Gal) in normal (AG03257) and HGPS (AG11498 at passage 8) fibroblasts transfected with Control- or SUN1-RNAi for 96 hours. Standard deviations are from three independent assays counting 1200 to 2000 cells in each experiment. Cell scoring was performed in a blinded fashion by an independent investigator. P value (Chi-square) is indicated.
(E) Cell proliferation in normal (AG03257) and HGPS (AG11498) cells transfected with control or SUN1 RNAi. Cells at ~50% confluency were transfected. When cells reached confluency, equal numbers were seeded into dishes and quantified for proliferation using Cell Counting Kit-8 24 hours after cell seeding (day 0) and after another 4, 8, 10, 12 days. Relative absorbance at 460nm was obtained by [(Absorbance460nm-background Absorbance460nm) at day N]/[(Absorbance460nm-background Absorbance460nm) at day 0]. Standard deviations are from triplicate experiments.