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. 2019 Dec 11;10:5661. doi: 10.1038/s41467-019-13530-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a MDA-MB-436 cells engineered to express mCherry (mCh), HA-BRCA1 full-length/wild-type (FL), or the HA-BRCA1 BRCT mutations (M) c.5251 C > T-(M#1), c.5263insC-(M#2), c.5277insTA-(M#3), c.5445 G > A-(M#4), that are endogenously present in HCC1395, HCC1937, MDA-MB-436 and SNU-251 BRCA1 BRCT mutant cell lines, respectively; and were assessed for HA mRNA by qRT-PCR. HA expression normalized to FL expressing cells is shown (above). HA protein expression was assessed from the same cells by Western blotting (below). b MDA-MB-436 cells engineered to express mCh, BRCA1-FL, BRCA1-I14-(I14), and BRCA1-I15-(I15) were assessed as described in a. See Supplementary Fig. 4b. c Cells from panel b were subject to HA-immunoprecipitation with the indicated antibodies. d mCh, BRCA1-FL, BRCA1–14, BRCA1-I15, and BRCA1 c.5445 G > A (SNU-251 mutation) expressing MDA-MB-436 cells were seeded at decreasing densities in the presence of 20 nM rucaparib, 20 nM olaparib or 20 ng/ml cisplatin and assessed for colony formation. ***p < 0.001 compared to mCh cells. Below, representative plates. For panels a−d, data are the mean ± SD of n = 3 biological replicates. Statistical significance was assessed by unpaired, two-tailed t tests. e mCh, BRCA1-FL, BRCA1-I15, and BRCA1 c.5445 G > A expressing MDA-MB-436 tumor xenografts were harvested and assessed for HA expression by Western blotting. f Mice harboring mCh, BRCA1-FL, BRCA1-I15, and BRCA1 c.5445 G > A expressing MDA-MB-436 tumor xenografts were treated with vehicle (black line) or 200 mg/kg rucaparib bi-daily (red line) for 2 × 5 days with a 2-day interval. Mean ± SD. Tumor volumes are shown for five tumors implanted in five separate mice per cell line per treatment from the same experiment. A linear mixed effects model tested the difference in slopes of log- tumor volume, and p values shown are tests of differences in growth rates between vehicle and rucaparib-treated tumors. Moreover, differences-in-slopes (i.e. vehicle to PARPi differences) were compared using Tukey’s correction for multiple comparisons: BRCA1 5445 G > A vs. BRCA1-I15 (p = 0.02), BRCA1 5445 G > A vs. BRCA1-FL (p < 0.001), BRCA1-I15 vs. mCh (p = 0.04), and mCh vs. BRCA1-FL (p = 0.001), 5445 G > A vs. mCh (p = 0.99) or BRCA1-I15 vs. BRCA1-FL (p = 0.57). See Supplementary Data 1 file for more details.