Length‐independent telomere damage drives post‐mitotic cardiomyocyte senescence ARepresentative images of rat neonatal CMs 4 days following transfection.

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Length‐independent telomere damage drives post‐mitotic cardiomyocyte senescence ARepresentative images of rat neonatal CMs 4 days following transfection with a FLAG‐tagged TRF1‐FokI‐D450A (top row) or TRF1‐FokI (middle and bottom row) fusion protein (cell treatments the same for all subsequent panels in Figure; red—telo‐FISH; green—γH2A.X). Images are z‐projections of 0.1 μm stacks taken with 100× objective. White arrows indicate co‐localisation between telomeres and γH2A.X, with co‐localising foci amplified in the right panels (taken from single z‐planes where co‐localisation was found). Scale bar represents 3.5 μm. Scale bar in magnified images showing individual co‐localisation 500 nm. B, C% of γH2A.X foci co‐localising with telomeres (B) and mean number of telomere‐associated foci (TAF) (C) in FLAG‐tagged TRF1‐FokI‐D450A‐ and TRF1‐FokI‐expressing CMs. Data are mean ± SEM of n = 4 independent experiments. > 50 cells were analysed per condition. Statistical analysis was performed by two‐tailed t‐test ***P < 0.001. DHistograms displaying telomere intensity for telomeres co‐localising or not co‐localising with γH2AX foci. Red dotted lines represent median. Mann–Whitney test shows no significant difference in telomere intensity between TAF and non‐TAF. EMean % of FLAG‐labelled CMs positive for SA‐β‐Gal activity. Data are mean ± SEM of n = 3 independent experiments. > 100 cells were quantified per condition. Statistical analysis performed using two‐tailed t‐test; **P < 0.01. FExpression of p21 mRNA (as a function of β‐actin and Gapdh) by real‐time PCR in TRF1‐FokI‐D450A and TRF1‐FokI‐expressing CMs. Data are mean ± SEM of n = 6 independent experiments. Statistical analysis performed using two‐tailed t‐test; ***P < 0.001. GMean ± SEM cell surface area (μm2) of FLAG‐labelled CMs expressing TRF1‐FokI‐D450A and TRF1‐FokI. Statistical analysis performed using two‐tailed t‐test; ***P < 0.001. Scale bar represents 50 μm. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 38, Issue: 5, First published: 08 February 2019, DOI: (10.15252/embj.2018100492)