F the extent of resection detected in (b) as in Fig. 1d. Suggests (center bars) and SDs (error bars) from 3 independent experiments. All statistical analysis as in Fig. 1.Nature. Author manuscript; readily available in PMC 2019 January 18.Mirman et al.PageAuthor Manuscript Author Manuscript Author ManuscriptExtended Data Figure five. CST interacts with ShieldinAuthor Manuscripta, Immunoprecipitation of individual mouse CST subunits or the three subunit complex (every subunit bearing a Myc-tag) with Flag-tagged mouse Shld1 co-expressed in 293T cells. Flag-tagged POT1b and POT1a serve as optimistic and adverse controls for CST binding, respectively. Representative of two experiments. b, Two-hybrid analysis of CST-Shieldin interaction. Yeast cultures were grown overnight in synthetic total medium lacking tryptophan and leucine to a density of 5107 cells/ml. Serial 10-fold dilutions have been generated and four ul of each dilution was spotted on synthetic total media lacking theNature. Author manuscript; out there in PMC 2019 January 18.Mirman et al.Pagenutrients tryptophan, leucine, adenine, histidine and containing 3-aminotriazole (3-AT) as indicated. Plates were then incubated for 5 days at 30 just before imaging. Representative of three experiments.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptExtended Information Figure six. Localization of CST and Pol to DSBsa, Quantification of HA-Stn1 localization to FOKI-induced DSBs as in Fig. 3e. Suggests (center bars) and SDs (error bars) from 4-6 independent Gene Inhibitors products experiments (80 induced nuclei for every single situation in each and every experiment) are shown. b, IF for endogenous Pol in FOKI-LacINature. Author manuscript; readily available in PMC 2019 January 18.Mirman et al.PageU2OS cells in S phase and just after RO3306 treatment (G2). Dotted line: outline with the nucleus. Representative of two experiments. c, Examples of HA-Stn1 and Pol localization at FOKIinduced DSBs in G2-arrested FOKI-LacI U2OS cells (as in Fig. 3f). Representative of 3 experiments. d, Quantification of co-localization of Pol with FOKI-induced DSBs (as in Fig. 3f). Means (center bars) and SDs (error bars) from 3 independent experiments (80 induced nuclei for every single situation in each experiment) are shown. All statistical evaluation as in Fig. 1.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptExtended Data Figure 7. Effect of Stn1 knockdown around the intensity of IR-induced RPA fociQuantification of myc-RPA32 intensity per nucleus inside the experiments shown in Fig. 3g-h. Medians (center bars and numbers below) obtained from 4 independent experiments with 20 nuclei for every single experimental condition in every experiment. Every single symbol represents one particular nucleus. Statistical evaluation as in Fig. 1.Nature. Author manuscript; readily available in PMC 2019 January 18.Mirman et al.PageAuthor Manuscript Author Manuscript Author ManuscriptExtended Data Figure 8. Impact of CST and Pol on PARPi treatment of BRCA1-deficient cellsAuthor Manuscripta-f, Immunoblots around the MEFs used in Fig. 4a-e to confirm the absence of deleted proteins and efficacy of your shRNAs. Reduction in Stn1 expression is used as a proxy for the efficacy of your Ctc1 shRNA considering the fact that no antibody to mouse Ctc1 is readily available. Each immunoblot is representative of three experiments. g, Immunoblots for BRCA1 and Stn1 within the cells utilized in Fig. 4f. Representative of two experiments. h-j, Manage experiment to assess that cells analyzed in Fig. 4f progressed by means of S phase in the course of PARPi therapy. h, Experimental.
