Fig 2: Chromatin decompaction induced by HMGN5 overexpression leads to nuclear blebbing(a) Cells overexpressing GFP-HMGN5, but not cells overexpressing GFP-HMGN5S17,21E (HMGN5-SE), show loss of heterochromatin (right panels) and nuclear blebs (arrows) that stain with lamin A/C, but not lamin B1. Quantitative microscopy verified that similar amounts of WT or mutant HMGN5-GFP were expressed (Supplementary figure 1c). Scale bar: 10 µm. (b) HMGN5 mediated chromatin decompaction does not alter the levels of histone modifications indicative of compact chromatin. Numbers in parenthesis shows GFP/GFP-HMGN5 signal intensity. (c) Western blot showing that HMGN5 mediated chromatin decompaction does not alter the levels of Lamin A/C or Lamin B1. Numbers in parenthesis shows GFP/GFP-HMGN5 signal intensity. (d) Frequency of blebbed nuclei in MEFs and CAT413 cells. For each cell type, 750 to 2,000 cells were examined. (e) Severity of blebs: (+) small extruded nuclear membrane, (++) large bleb, (+++) more than 2 blebs. Data shown derived from 261 (MEF) and 239 (CAT413) GFP-HMGN5 positive cells. (f) Synergistic effects of HMGN5 overexpression and loss of Lamin A on nuclear blebbing. Shown is the frequency of blebs in MEFs isolated from littermate Lmna+/+ and Lmna-/- mice that either do, or do not overexpress HMGN5. Data derived from analysis of a total of over 2000 cells.

Fig 3: The nucleus becomes stiffer and acts as a mechanical barrier in vertical confinement.(A) Nuclear entry time of H2B-mCherry–labeled LMNA-KD (knockdown) or scramble control HT-1080 cells (n = 143; three independent experiments) in lateral and vertical channels. (B) Representative heat map of Brillouin shift for 2D, vertically, and laterally confined cells. Scale bars, 10 µm. (C) Nuclear Brillouin shift for scramble control, LMNA-KD–treated, blebbistatin (2 µM)–treated, and TSA (100 ng/µl)–treated HT-1080 cells in 2D, lateral, and vertical confinement (n = 13 cells; two or more independent experiments). (D) Nuclear Brillouin shift of cells in the vertical and lateral segments of contiguous microchannels, where cells experienced vertical and subsequently lateral confinement (n = 9 cells; two independent experiments). (E) Heat map of Brillouin shift for an individual cell migrating, first, through the vertical and, subsequently, the lateral segment of a contiguous microchannel. Scale bars, 10 µm. Values represent the mean ± SD; **P < 0.01 relative to lateral control; ##P < 0.01 relative to vertical control; §§P < 0.01 relative to 2D control.

Fig 4: HGPS aging defects are most pronounced in mechanically stressed tissues. (A) HGPS disproportionately affects stiff and mechanically stressed tissues (e.g. skeletal/cardiac muscle) while soft tissues (e.g. brain, marrow) appear normal, regardless of lineage or developmental origin. (B) Post-translational processing of normal lamin-A/C and progerin. The truncated mutant, progerin, retains a C-terminus farnesyl group. Upper right box: A-type lamin isoforms differ in molecular weight (MW) by ~5 kDa. Lower right: Venn diagram of unique and common A-type lamin peptides. The majority of the peptides are shared by all three isoforms, with the exception of isoform-specific peptides at the C-terminus. ‘FTase’ = Farnesyl transferase; ‘RCE1’ = Ras converting CAAX endopeptidase 1; ‘ZMPSTE24’ = Zinc metallopeptidase STE24; ‘ICMT’ = Isoprenylcysteine carboxyl methyltransferase.

Fig 5: Expression of NANOG reverses cellular senescence in myoblasts isolated from progeria mice.(A) Representative picture of a heterozygous LAKI progeria mouse (LmnaG609G/+) and his WT sibling (Lmna+/+) both at the age of 10 months. (B) Immunostaining for NANOG and lamin A/C confirmed the expression of NANOG upon addition of Dox (1 µg/ml) in the medium (LAKIN) and the presence of nuclear blebs (white arrows) in both LAKI and LAKIN condition due to LMNA mutation. (C) Quantification of NANOG expression. Data shown as means ± 95% CI for 500 cells in one representative experiment and means ± SD for three independent experiments. (D) Quantification of the percent cells with nuclear blebs. Data shows the means ± SD for three independent experiments. (E) Immunostaining for phosphorylated form of histone H2AX (?H2AX). (F) Quantification of ?H2AX intensity. Data shown as means ± 95% CI for 500 cells per condition in one representative experiment and means ± SD for three independent experiments. (G) Quantification of the DNA DSBs measured as the tail DNA percent in single-cell electrophoresis (Comet) assay. Data shown as means ± SD for three independent experiments. (H) Representative image of comets for each condition. (I) Representative image of the TMRM fluorescence intensity, a measure of mitochondrial membrane potential. (J) Quantification of TMRM intensity. Data shown as means ± 95% CI for 200 cells per condition in one representative experiment and means ± SD for three independent experiments. (K) Intracellular ATP concentration as a metric for cellular energetics. Data shown as means ± SD for three independent experiments. $ denotes P < 0.05 as compared to all other samples. Photo credit: Aref Shahini, University at Buffalo, The State University of New York.