Figure 2. Impact of lamin B1, mechanical stress, and progerin farnesylation on NM ruptures.

(A) Bar graph comparing the effects of prelamin A (PreA), progerin (Prog), lamin B1 knockdown (B1 KD), and nf-Prog on NM ruptures in static (white bars) and stretched (black bars) SMCs (mean ± SEM, n = 5 experiments). Differences were compared with static SMCs expressing prelamin A by 2-way ANOVA (*P < 0.005, **P < 0.0001). The total numbers of cells examined are shown in parentheses above each bar. (B) Bar graph showing transcript levels for genes involved in NM repair (mean ± SEM, n = 3 experiments; Student’s t test, all nonsignificant). (C) Confocal fluorescence microscopy images showing SMCs expressing Nuc-RFP (red) and either GFP-KASH2ext (green) or GFP-KASH2 (green). Scale bar: 20 μm. (D–F) Characterization of NM ruptures in live PreA-SMCs and Prog-SMCs expressing KASH2ext (white bars) or KASH2 (blue bars). Bar graphs show the percentage of cells with a NM rupture; the number of NM ruptures per cell; and the percentage of cells with a NM rupture that die (mean ± SEM, n = 3 experiments; Student’s t test, *P < 0.01, **P < 0.005). (G) Confocal fluorescence microscopy images showing increased phosphorylated-STING (p-STING; green) in Progerin-SMCs. SMCs were examined in both static (left) and stretched (right) conditions. PreA-SMCs were included as a control. Lap2β staining is shown in red. Scale bar: 20 μm. (H) Bar graph showing the percentage of cells staining positive for p-STING in PreA-SMCs (white bars) and Prog-SMCs (black bars) in static and stretched conditions in a representative experiment. The ratios above each bar show the number of cells positive for p-STING over the total number of cells assessed.