Script Author Manuscript Author Manuscript Author ManuscriptNature. Author manuscript; offered in PMC 2019 January 18.Mirman et al.PageAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptFigure 1. Shieldin and CST counteract AZD5718 Biological Activity resection at dysfunctioinal telomeresa, Left: Schematic showing POT1b-bound CST counteracting resection of telomere ends. Proper: Depiction of telomeres lacking TPP1, POT1a, and POT1b as a proxy for DSB resection. Telomeres lacking TPP1 undergo ATR-dependent hyper-resection that’s repressed by 53BP1. b, Immunoblots displaying loss of Rev7 and Stn1 within 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 end resection within the cells shown in (b) working with in-gel hybridization to detect the three overhang (major) followed by rehybridization towards the denatured DNA inside the exact same gel (bottom) to figure out the ratio of ss to total TTAGGG signal. Representative of 4 experiments. d, Quantification of resection detected as in (c), determined from four independent experiments (various shades of gray) displaying means and SDs. 3 independent Rev7 KO clones were utilised (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 finish resection evaluation around the cells in (f) as in (c) and (d). Means and SDs from 4 independent experiments using two clones of every single genotype. Note that the order of the samples is different in (h) versus (f) and (g). All data panels in the figure are representative of 4 experiments. All suggests areNature. Author manuscript; available in PMC 2019 January 18.Mirman et al.Pageindicated with center bars and SDs with error bars. All statistical analysis according to twotailed Welch’s t-test. , p0.05; , p0.01; , p0.001; , p0.0001; ns, not considerable.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptNature. Author manuscript; offered in PMC 2019 January 18.Mirman et al.PageAuthor Manuscript Author Manuscript Author ManuscriptFigure 2. 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 before and just after Cre (96 h). Arrowheads: Ctc1 at telomeres. POT1b -/- cells handle for spurious telomere-Ctc1 co-localization. Proper: The identical nuclei showing -H2AX (red) at telomeres lacking TPP1. The -H2AX and Ctc1 signals are each falsecolored in red. Arrows: telomeres with Ctc1 and -H2AX. b, Quantification with the of telomeres co-localizing with Ctc1 detected as in (a). Every single dot A-3 site represents one particular nucleus in the indicated TPP1F/F cell lines with and with no Cre and/or ATRi. Signifies and SDs fromNature. Author manuscript; out there in PMC 2019 January 18.Mirman et al.Pagethree independent experiments. c, As in (b) but utilizing TPP1F/F cells treated using a Shld2 or possibly a handle 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 displaying telomeric DNA co-localizing with Stn1 in cells as in (d) treated with Cre (96 h) and ATR shRNAs. f, Quantification of Stn.
