Fig 2: Nuclear rupture kinetics can be experimentally modulated.(a) Blebbistatin (BLEB) reduces rupture frequency in both L2 HT-LKO and C4 HT-WT cells (p < 0.001); (b) Expression of a dominant-negative KASH construct (DN-KASH) does not significantly affect rupture frequency in L2 HT-LKO or C4 HT-WT cells; (c) Remodelin (REMOD) significantly increases the circularity (p < 0.001) in L2 HT-LKO or C4 HT-WT cells. The Y-axis has been cropped for clarity; (d) Remodelin significantly reduces the coefficient of variation (CoV) for the circularity across time L2 HT-LKO cells (p < 0.01) but not in C4 HT-WT cells; (e) Remodelin reduces rupture frequency in L2 HT-LKO cells (p < 0.05) but not in C4 HT-WT cells; (f) siRNA-mediated knockdown of CHMP4B (siCHMP4B) causes >95% reduction (expressed as log2 fold change of ddCt value) of CHMP4B transcript levels as compared to the non-targeting control siRNA (siNT); (g) siRNA-mediated knockdown of CHMP4B causes >50% reduction of CHMP4B protein levels as compared to the non-targeting control siRNA; (h) siRNA-mediated knockdown of CHMP4B increases nuclear recovery halftimes (p < 0.05). Bar graphs reflect mean ± standard error (n = number of cells).

Fig 3: ESCRT-III is recruited to the sites of membrane proliferation in fibroblasts from an HGPS patient. (a) Confocal microscope images of normal (AG03512) or HGPS fibroblasts (AG11513) transfected with the pEGFP-CHMP4B for 48 h were stained with anti-lamin A/C antibody. The right panels are the magnified image of the boxed area in the left panels. Bars, 20 µm. Images are representative of 3 independent experiments. (b) The number of CHMP4B dots on the NM in the experiment in (a) was measured for 29 (normal) or 23 (HGPS) transfected cells. Data are shown as the mean ± SEM and are representative of 3 independent experiments. (c) Lysates of normal or HGPS cells transfected with the pEGFP-CHMP4B for 48 h were analyzed by immunoblotting with anti-lamin A/C, anti-EGFP and anti-a-tubulin antibodies. Images are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S5. (d) Quantitation of the amount of CHMP4B-EGFP, as indicated, in the immunoblot bands shown in panel (c) relative to the amount in the a-tubulin band. Each value is the mean ± standard error of 3 independent experiments. n.s. not significant (by the unpaired Student’s t test).

Fig 4: Effects of ectopic expression of CHMP4B in HeLa cells. (a) HeLa/puro, HeLa/CHMP4BKO/puro and HeLa/CHMP4BKO/CHMP4B-EGFP cells were transfected with the TagRFP-progerin expression vector for 24 h. The number of nuclear tube-like structures was measured in 100 transfected cells. Data are shown as the mean ± SEM and are representative of 3 independent experiments. (b) Lysates of HeLa/puro, HeLa/CHMP4BKO/puro and HeLa/CHMP4BKO/CHMP4B-EGFP cells transfected with the TagRFP-progerin expression vector for 24 h were analyzed by immunoblotting with anti-TagRFP, anti-CHMP4B and anti-a-tubulin antibodies. Images are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S2.

Fig 5: CHMP4B alleviates the level of necroptosis after TBI. A, Protein levels of RIP3 and p-MLKL obtained from TBI group and TBI + CHMP4B group. GAPDH was used as the loading control. B, Bar graphs show the results of analysis (by band density analysis) of RIP3 and p-MLKL (n = 5 mice; data are presented as the means ± SEM). C, Immunohistochemistry of RIP3 and p-MLKL in TBI group and TBI + CHMP4B group. (Scale bar = 50 µm.) D, Transmission electron microscopy (TEM) images of tissues. Translucent cytoplasm mitochondrial swelling and destruction of membrane integrity were observed in TBI neurons. (Scale bar = 5 or 1 µm.) *P < .05