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. 2018 May 4;9(1):230–245. doi: 10.1080/19491034.2018.1460185

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© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Low nuclear tension increases A-type lamin phosphorylation and degradation, but mechanosensitivity is lost with passage in HGPS iPS-MSCs. (A) (i) Full-length Western blots of cells rounded and detached from their substrate using low concentrations of trypsin, probed with anti-lamin-A/C (left) and anti-pSer22 (right). A-type lamins (LA, P, and LC) in the intact MW range (60-80 kDa) decrease with cell rounding, which anti-correlates with the increase in phosphorylation and the increase in low MW degradation fragment bands (≤ 40 kDa). Bottom plot: Normalized phosphorylation (‘pSer22/LMNA’) of intact LA, P, and LC vs rounding time. (ii) Sensitivity to low tension (cell/nuclear rounding) is blunted with cell passage (P7). (B) Immunoblot of P7 HGPS iPS-MSCs cultured on stiff vs soft gels. Lower bar graph: fold change in densitometry signal (soft/stiff) of LA, P, and LC. Gray bar (right) indicates fold change of total A-type lamins in early passage primary hMSCs. (C) Cartoon and Immunofluorescence images of HGPS iPS-MSCs on rigid culture plastic treated with blebbistatin (25 μM, 2h) or DMSO control. (D) Nucleus 2D projected area decreases with blebbistatin treatment. (E) (i) Fraction (%) of cells with nuclear blebs. (ii) Nucleus circularity measurements. Nuclei with circularity < ∼0.65 (red box) correspond to those with more than one bleb. (F) Immunofluorescence measurements of LA/P/LC (A-type lamin) abundance in DMSO vs blebbistatin treated iPS-MSCs. LB1/B2 (B-type lamins) remain unaffected by myosin-II inhibition. (G) (i) Normalized phosphorylation (as ‘pSer22/LMNA’) measured in DMSO vs blebbistatin treated iPS-MSCs. (ii) Fraction (%) of ‘2N’ vs ‘4N’ cells (by DNA content) remain unchanged with blebbistatin treatment. (H) Representative immunofluorescence images of LA/C, LB1/2, and γH2AX in HGPS iPS-MSCs (P1 and P4) and normal primary hMSCs. Scale bar = 10 μm. (I) Lamin-A:B ratio (total A : B-type lamin ratio) and cell/nuclear size increase with higher passage in iPS-MSCs. (J) Quantification of progerin intensity (using anti-progerin, top) and % cells with >1 γH2AX foci (bottom) determined by immunofluorescence.