Supplementary MaterialsSupplementary Document. AR-C155858 2and and and and and and and and and and and and expression can be associated with a poorer clinical outcome. In summary, these data reflect correlations between higher-level as opposed to lower-level BRCA1 gene expression and greater tumor-based aneuploidy and a poorer clinical prognosis. One possible KLHL22 antibody explanation for this relationship is usually that, as suggested in reporter (U2OS-DR) were used following a previously described methodology (51). Cell Culture. AR-C155858 All cells were cultivated at 37 C in a humidified incubator in an atmosphere made up of 10% CO2. U2OS cells were produced in DMEM supplemented with 10% FBS. Breast malignancy cell lines were cultured according to the guidelines provided by American Type Culture Collection or the suppliers. RNA Interference. The following siRNA or shRNA sequences were used in this study: siBRCA1-1: AGAUAGUUCUACCAGUAAA siBRCA1-2: GAAUCCUAGAGAUACUGAA siPARP1-1: CCAAAGGAAUUCCGAGAAA siPARP1-2: CCGAGAAAUCUCUUACCUCAA siPARP1-3: ACGGUGAUCGGUAGCAACAAA siTP53BP1: GGACUCCAGUGUUGUCAUU shLuciferase: GTGCGCTGCTGGTGCCAAC shBRCA1-1: AGAATCCTAGAGATACTGAA shBRCA1-2: TATAAGACCTCTGGCATGAAT shPARP1: AAGGTGGTTGACAGAGATTCT Nontargeting siRNA pools from Dharmacon were used as siRNA controls, and shRNA targeting luciferase was used as an shRNA control in all experiments. siRNA transfections were performed using HiPerFect (Qiagen) or Lipofectamine RNAiMax (Invitrogen) according to the manufacturers instructions. Chromosome Analysis. U2OS cells were exposed to the indicated siRNA or drugs or were transfected with an indicated cDNA for 48 h and then exposed to 150 rads of IR. At 5 h after IR, 30 ng/mL colcemid was added to each culture, and cells were incubated for an additional 3 h, collected, and then prepared for an analysis of metaphase spreads. Spreads were stained with DAPI. Immunofluorescence. Immunofluorescence following irradiation was performed as described previously (31, 52). Data Availability. All of the data supporting the findings of this study are available within the paper and SI Appendix. Supplementary Material Supplementary FileClick here to view.(2.5M, pdf) Acknowledgments We thank Dr. Richard Baer for generously providing the anti-CtIP antibody and Dr. Sharon Cantor for providing the anti-BACH1 antibodies. This work was supported by Grant R01 CA136512 from the National Malignancy Institute; grants from the Breast Cancer AR-C155858 Research Foundation, the Susan G. Komen Foundation, the BRCA Foundation, and the Gray Foundation (to D.M.L.); as well as a National Malignancy Institute SPORE (Specialized Programs of Research Excellence) grant for breast malignancy research to the Dana-Farber/Harvard Cancer Center. AR-C155858 The results presented herein are based in part on data generated by AR-C155858 the TCGA Research Network ( Footnotes Competing interest statement: D.M.L. serves as a consultant to Constellation Pharma, the Novartis Institute for Biomedical Research, and NextechInvest. He’s also a known person in the Exterior Advisory Planks from the Rutgers Tumor Middle, MIT Tumor Middle, and Sidney Kimmel Johns Hopkins Tumor Center. The various other writers declare no contending interests. This informative article supporting ://www information online at