Script Author Manuscript Author Manuscript Author ManuscriptNature. Author manuscript; readily Chalcone Purity available in PMC

Script Author Manuscript Author Manuscript Author ManuscriptNature. Author manuscript; readily Chalcone Purity available in PMC 2019 January 18.Mirman et al.PageAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptFigure 1. Shieldin and CST counteract resection at dysfunctioinal telomeresa, Left: Schematic showing POT1b-bound CST counteracting resection of telomere ends. Ideal: Depiction of telomeres All sglt2 Inhibitors medchemexpress lacking TPP1, POT1a, and POT1b as a proxy for DSB resection. Telomeres lacking TPP1 undergo ATR-dependent hyper-resection which is repressed by 53BP1. b, Immunoblots displaying loss of Rev7 and Stn1 inside the indicated TPP1F/F Rev7+/+ MEFs and TPP1F/F Rev7-/- (CRISPR) clones treated with Cre (96 h) and/or Stn1 shRNA as indicated. Chk1-P serves as a proxy for TPP1 deletion. c, Quantitative evaluation of telomere finish resection within the cells shown in (b) working with in-gel hybridization to detect the 3 overhang (major) followed by rehybridization for the denatured DNA inside the same gel (bottom) to determine the ratio of ss to total TTAGGG signal. Representative of 4 experiments. d, Quantification of resection detected as in (c), determined from 4 independent experiments (distinct shades of gray) displaying implies and SDs. 3 independent Rev7 KO clones have been used (distinct symbols). e, Telomeres lacking TRF2 as a model for resection upon ATM activation. f, Immunoblots showing Cre-mediated deletion of TRF2 from TRF2F/F Lig4-/- cells, CRISPR deletion of Rev7, shRNA-mediated reduction of Stn1, and Chk2 phosphorylation. Asterisk: non-specific. g and h, Telomere end resection evaluation around the cells in (f) as in (c) and (d). Signifies and SDs from four independent experiments employing two clones of every single genotype. Note that the order in the samples is unique in (h) versus (f) and (g). All information panels within the figure are representative of four experiments. All means areNature. Author manuscript; accessible in PMC 2019 January 18.Mirman et al.Pageindicated with center bars and SDs with error bars. All statistical analysis based on twotailed Welch’s t-test. , p0.05; , p0.01; , p0.001; , p0.0001; ns, not important.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptNature. Author manuscript; readily available in PMC 2019 January 18.Mirman et al.PageAuthor Manuscript Author Manuscript Author ManuscriptFigure two. 53BP1- and Shieldin-dependent localization of CST to dysfunctional telomeresAuthor Manuscripta, Left: Representative IF-FISH for 6myc-tagged Ctc1 (red) at telomeres (false-colored in green) in TPP1F/F MEFs just before and immediately after Cre (96 h). Arrowheads: Ctc1 at telomeres. POT1b -/- cells handle for spurious telomere-Ctc1 co-localization. Right: The same nuclei showing -H2AX (red) at telomeres lacking TPP1. The -H2AX and Ctc1 signals are both falsecolored in red. Arrows: telomeres with Ctc1 and -H2AX. b, Quantification on the of telomeres co-localizing with Ctc1 detected as in (a). Every single dot represents a single nucleus from the indicated TPP1F/F cell lines with and without Cre and/or ATRi. Means and SDs fromNature. Author manuscript; obtainable in PMC 2019 January 18.Mirman et al.Pagethree independent experiments. c, As in (b) but using TPP1F/F cells treated with a Shld2 or perhaps a manage sgRNA. Suggests and SDs as in (b). d, Immunoblots for POT1 deletion, ATR knockdown, and HA-Stn1 in conditional POT1 KO HT1080 cells. Asterisk: non-specific band. e, IF-FISH showing telomeric DNA co-localizing with Stn1 in cells as in (d) treated with Cre (96 h) and ATR shRNAs. f, Quantification of Stn.