Fig 2: Validation of BAF-specific antibodies in lamin-KO MEFs, the effect of BAF overexpression on accumulation kinetics of the LC mutants at the rupture sites. (A and B) Validation of anti-BANF1 (3F10-4G12) for immunofluorescence after different fixation methods. Representative immunofluorescence images of single confocal sections from WT, Lmna−/−, Lmnb1−/−, and Lmnb2−/− MEFs fixed with 4% PFA only (A) or 4% PFA containing 0.1% Triton X-100 (B) and stained with the anti-BANF1, followed with Cy5-labeled anti-mouse IgG, and Hoechst 33342 for DNA. Bars: 20 μm. (C and D) The effect of BAF overexpression on accumulation kinetics of the LC mutants at the rupture sites. Halo-BAF (lower panels) with mEmerald-LC full-length, ∆417-422 (∆NLS) and ∆432-572 (∆Tail; all, upper panels) were expressed in Lmna-KO MEFs. (C) Dynamics of mEmerald-LC full-length, ∆417-422 (∆NLS), ∆432-572 (∆Tail), and Halo-BAF. The right four columns are magnified views of orange boxes, and the rupture sites are indicated with yellow arrowheads. Bars: 5 μm (the first column) and 2 μm (the second column to others). (D) Relative fluorescence intensities of the mEmerald-LC mutants (means ± SEM; n = 10 cells; **, P < 0.001 from full-length by a linear mixed model).

Fig 3: Lamin A abnormality destabilizes METTL14 protein. (a, b) Representative immunofluorescence images showing METTL14/3 and Lamin A expression in skin fibroblasts derived from individual HGPS patients (HG122 and HG155) and normal human skin fibroblasts (F2S). Scale bar, 50 µm. (c) Western blot analysis of METTL3/14 protein levels in Zmpste24 +/+, Zmpste24 -/-, Lmna +/+, and Lmna -/-MEFs. The data represent three independently derived sets of MEFs in separate experiments. (d, e) Western blot analysis of METTL3/14 protein levels in HG122, HG155, and F2S cells at different passages. (f, g) Western blot analysis of METTL3/14 expression in Lmna -/- (f) and Zmpste24 -/- (g) MEFs compared with WT cells. Cells were treated with cycloheximide (CHX) and/or MG132 (MG) for indicated time. Quantification was performed by Image J®. The data represent the means ± SEM. *p < 0.05

Fig 4: Effect of lamin depletion on localization of BAF and cGAS. (A and B) The localization and phosphorylation of BAF in WT, Lmna−/−, Lmnb1−/−, and Lmnb2−/− MEFs was analyzed by immunofluorescence (A) and immunoblotting, respectively (B). (A) Single confocal sections of the indicated cells stained with anti-BANF1/BAF (EPR7668), followed with Alexa Fluor 488-labeled anti-rabbit IgG, and Hoechst 33342 for DNA. (B) Whole cell lysates from the indicated cells were probed with anti-LA/C, anti-LB1/2, anti-GAPDH (as loading control), and anti-BANF1/BAF (EPR7668). (C and D) The localization and phosphorylation of BAF in WT MEFs expressing scrambled control, shLA, shLC or a combination of shLA and shLC was analyzed by immunofluorescence (C) and immunoblotting, respectively (D). (C) Single confocal sections of the indicated cells stained with anti-BANF1/BAF (EPR7668), followed with Cy5-labeled anti-rabbit IgG, and Hoechst 33342 for DNA. (D) Whole cell lysates from the indicated cells were probed with anti-LA/C, anti-GAPDH (as loading control), and anti-BANF1/BAF (EPR7668). (E and F) The localization and the expression levels of cGAS in WT, Lmna−/−, Lmnb1−/−, and Lmnb2−/− MEFs was analyzed by immunofluorescence (E) and immunoblotting, respectively (F). (E) Single confocal sections of the indicated cells stained with anti-cGAS, followed with Alexa 488-labeled anti-rabbit IgG, and Hoechst 33342 for DNA. (F) Whole cell lysates from the indicated cells were probed with anti-cGAS and anti-GAPDH (as loading control). (A–F) At least two independent experiments were performed. (A, C, and E) Bars: 20 μm. (B, D, and F) Positions of the size standards are shown on the right. The values on non-phospho-BAF, phospho-BAF and LA/C are densitometric quantitation normalized to GAPDH. Source data are available for this figure: SourceData F6.

Fig 5: Effect of lamin depletion on BAF and cGAS accumulation at the rupture sites. (A) Single confocal sections of WT, Lmna−/−, Lmnb1−/−, and Lmnb2−/− MEFs fixed within 10 min after laser microirradiation and stained with anti-BANF1 (3F10-4G12) and anti-cGAS, followed with Alexa Fluor 488-labeled anti-rabbit IgG, Cy5-labeled anti-mouse IgG, and Hoechst 33342 for DNA. Yellow arrowheads indicate laser microirradiation-induced NE rupture sites. Blue arrowheads indicate spontaneously produced NE protrusions. Bar: 20 μm. (B and C) The max intensities of BAF (B) and cGAS (C) signals at the rupture sites. The plotted points are from two independent experiments (n = 36, 35, 32, and 34 for WT, Lmna−/−, Lmnb1−/−, and Lmnb2−/−, respectively; horizontal dotted lines show the mean values, *, P < 0.05 from others by a Steel-Dwass multiple comparison). (D–F) Relative fluorescence intensities of the NLS-sfCherry in the nucleus, Halo-Baf and cGAS-sfGFP at the rupture site in WT and Lmna−/− MEFs (means ± SEM; n = 20 cells from two independent experiments; **, P < 0.001; ns, P > 0.05 from WT by a linear mixed model). See Fig. S7 B for microscopic images.