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ER-α deficiency impairs BRCA1 mediated HRR and promote NHEJ. (A) Homologous recombination efficiency of MCF-7, MCF-7ShER-α, MCF-7ShBRCA1 and MCF-pEGFP ER-α cells, as analyzed by HR assay; (B, C) HR product formed upon recombination is quantified and normalized with respective backbone plasmids dl1 and dl2. (D) Homologous recombination efficiency of MDA-MB-231 and MDA-MB-231shBRCA1 cells as analyzed by HR assay, (E, F) normalized to backbone plasmids dl1 and dl2. (G, H) Homologous recombination efficiency of MCF-7 cells analyzed post estrogen deprivation and follow on activation using 17-β estradiol. (I, J) MCF-7 cells were treated with 5 μM Tamoxifen citrate for 12 hours to inhibit ER-α activity and HR efficiency was further scored using HR assay. (K-N) DRGFP based HR assay: MCF-7, MCF-ShER-α, and MDA-MB-231 cells stably expressing pDR-GFP, were transfected with I-SceI expression vector (pCBASceI) or mock <t>(pCAGGS-mCherry)</t> to generate a DSB within the Sce-GFP. (K) Immmunofluorescence and (L) Fluorescence-activated cell sorting (FACS) analysis carried out to quantify HR-repaired GFP+ cells. ER-α expressing cells generated higher percentage of GFP+ cells. Each value represents the mean ± SD of three independent experiments. For assessing NHEJ activity, DR-GFP-integrated MCF-7 and MDA-MB-231 cells were transfected with mock or I-Scel and 48 hours post transfection, genomic DNA was extracted for PCR amplification (Figure S5A), followed by I-SceI or I-SceI plus BcgI digestion, and the digested products were subjected to gel electrophoresis (M). Since HR repair will replace the I-SceI site with the BcgI site and the NHEJ repair will diminish both the enzyme sites, the ratio between uncut DNA and cut DNA after I-SceI digestion represents “HR + NHEJ repair” efficiency while this ratio between uncut DNA and cut DNA after I-SceI and BcgI digestion reflects only the NHEJ repair efficiency (N). (See Figure S4B for detailed protocol) (Error bars, mean ± SD, *P≤0.05 and **P≤0.005 and as ***P≤0.001, ****P≤0.0001, unpaired t-test).
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ER-α deficiency impairs BRCA1 mediated HRR and promote NHEJ. (A) Homologous recombination efficiency of MCF-7, MCF-7ShER-α, MCF-7ShBRCA1 and MCF-pEGFP ER-α cells, as analyzed by HR assay; (B, C) HR product formed upon recombination is quantified and normalized with respective backbone plasmids dl1 and dl2. (D) Homologous recombination efficiency of MDA-MB-231 and MDA-MB-231shBRCA1 cells as analyzed by HR assay, (E, F) normalized to backbone plasmids dl1 and dl2. (G, H) Homologous recombination efficiency of MCF-7 cells analyzed post estrogen deprivation and follow on activation using 17-β estradiol. (I, J) MCF-7 cells were treated with 5 μM Tamoxifen citrate for 12 hours to inhibit ER-α activity and HR efficiency was further scored using HR assay. (K-N) DRGFP based HR assay: MCF-7, MCF-ShER-α, and MDA-MB-231 cells stably expressing pDR-GFP, were transfected with I-SceI expression vector (pCBASceI) or mock (pCAGGS-mCherry) to generate a DSB within the Sce-GFP. (K) Immmunofluorescence and (L) Fluorescence-activated cell sorting (FACS) analysis carried out to quantify HR-repaired GFP+ cells. ER-α expressing cells generated higher percentage of GFP+ cells. Each value represents the mean ± SD of three independent experiments. For assessing NHEJ activity, DR-GFP-integrated MCF-7 and MDA-MB-231 cells were transfected with mock or I-Scel and 48 hours post transfection, genomic DNA was extracted for PCR amplification (Figure S5A), followed by I-SceI or I-SceI plus BcgI digestion, and the digested products were subjected to gel electrophoresis (M). Since HR repair will replace the I-SceI site with the BcgI site and the NHEJ repair will diminish both the enzyme sites, the ratio between uncut DNA and cut DNA after I-SceI digestion represents “HR + NHEJ repair” efficiency while this ratio between uncut DNA and cut DNA after I-SceI and BcgI digestion reflects only the NHEJ repair efficiency (N). (See Figure S4B for detailed protocol) (Error bars, mean ± SD, *P≤0.05 and **P≤0.005 and as ***P≤0.001, ****P≤0.0001, unpaired t-test).

Journal: American Journal of Cancer Research

Article Title: Modulation of BRCA1 mediated DNA damage repair by deregulated ER-α signaling in breast cancers

doi:

Figure Lengend Snippet: ER-α deficiency impairs BRCA1 mediated HRR and promote NHEJ. (A) Homologous recombination efficiency of MCF-7, MCF-7ShER-α, MCF-7ShBRCA1 and MCF-pEGFP ER-α cells, as analyzed by HR assay; (B, C) HR product formed upon recombination is quantified and normalized with respective backbone plasmids dl1 and dl2. (D) Homologous recombination efficiency of MDA-MB-231 and MDA-MB-231shBRCA1 cells as analyzed by HR assay, (E, F) normalized to backbone plasmids dl1 and dl2. (G, H) Homologous recombination efficiency of MCF-7 cells analyzed post estrogen deprivation and follow on activation using 17-β estradiol. (I, J) MCF-7 cells were treated with 5 μM Tamoxifen citrate for 12 hours to inhibit ER-α activity and HR efficiency was further scored using HR assay. (K-N) DRGFP based HR assay: MCF-7, MCF-ShER-α, and MDA-MB-231 cells stably expressing pDR-GFP, were transfected with I-SceI expression vector (pCBASceI) or mock (pCAGGS-mCherry) to generate a DSB within the Sce-GFP. (K) Immmunofluorescence and (L) Fluorescence-activated cell sorting (FACS) analysis carried out to quantify HR-repaired GFP+ cells. ER-α expressing cells generated higher percentage of GFP+ cells. Each value represents the mean ± SD of three independent experiments. For assessing NHEJ activity, DR-GFP-integrated MCF-7 and MDA-MB-231 cells were transfected with mock or I-Scel and 48 hours post transfection, genomic DNA was extracted for PCR amplification (Figure S5A), followed by I-SceI or I-SceI plus BcgI digestion, and the digested products were subjected to gel electrophoresis (M). Since HR repair will replace the I-SceI site with the BcgI site and the NHEJ repair will diminish both the enzyme sites, the ratio between uncut DNA and cut DNA after I-SceI digestion represents “HR + NHEJ repair” efficiency while this ratio between uncut DNA and cut DNA after I-SceI and BcgI digestion reflects only the NHEJ repair efficiency (N). (See Figure S4B for detailed protocol) (Error bars, mean ± SD, *P≤0.05 and **P≤0.005 and as ***P≤0.001, ****P≤0.0001, unpaired t-test).

Article Snippet: The backbone vectors pgkPURO and pCAGGSS-mCherry (#41583, addgene) were respectively used for mock transfections.

Techniques: Homologous Recombination, Activation Assay, Activity Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Fluorescence, FACS, Generated, Amplification, Nucleic Acid Electrophoresis