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. Author manuscript; available in PMC: 2016 Nov 28.
Published in final edited form as: Cancer Discov. 2012 Aug 22;2(11):1036–1047. doi: 10.1158/2159-8290.CD-11-0348

Figure 1.

Figure 1

BRCA downregulation and γH2AX staining following PI3K inhibition in vitro. A, fluorescence-activated cell-sorting (FACS) analysis showing staining of γH2AX during the 3 phases of the cell cycle (G1, S, G2) in MDA-MB-468 cells transfected with control, BRCA1, or PIK3CA siRNAs and treated with either dimethyl sulfoxide (DMSO) or 1 μmol/L olaparib. B, immunofluorescence of γH2AX in MDA-MB-468 cells transfected with control, BRCA1, or PIK3CA siRNAs and treated with either DMSO or 1 μmol/L of olaparib. Percentages of positive nuclei (10 foci or more per nucleus being considered positive) from 10 fields per sample are indicated. C, Western blot analysis of cell lysates (7 days posttransfection) from MDA-MB-468 cells transfected with control or PIK3CA siRNAs using the indicated antibodies. Tubulin was used as loading control. D, Western blot analysis of lysates from MDA-MB-468 treated with BKM120 (750 nmol/L) for 7 days using the indicated antibodies. Total ERK (tERK) was used as a loading control. E, quantitative real-time PCR (qRT-PCR) measuring both BRCA1 and BRCA2 mRNA levels in MDA-MB-468 treated with BKM120. Measurements were normalized to 18S mRNA levels and expressed as fold change compared with controls (log2 scale). Data are shown as mean ± SE of 3 independent replicates for each condition.