brca1 cdna pcdna3 mammalian expression vector  (Thermo Fisher)

Journal: International Journal of Molecular Epidemiology and Genetics

Article Title: Effects of 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate on oxidative stress and breast carcinogenesis

doi:

Figure Lengend Snippet: The percentage of inhibition activity of SOD induced by 3300 del A-1061 Ter BRCA1 frameshift mutation in HCC1937 cell line.

Article Snippet: BRCA1 ) consisting of the full-length BRCA1 cDNA within the pcDNA3 mammalian expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) and 3300 del A-1061 Ter BRCA1 expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) were used in this study.

Techniques: Inhibition, Activity Assay, Mutagenesis

Journal: International Journal of Molecular Epidemiology and Genetics

Article Title: Effects of 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate on oxidative stress and breast carcinogenesis

doi:

Figure Lengend Snippet: The percentage of inhibition activity of SOD induced by 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate conc. 0.5, 1.0, 1.5 and 2.0 mg/mL in SKBR3 cell line

Article Snippet: BRCA1 ) consisting of the full-length BRCA1 cDNA within the pcDNA3 mammalian expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) and 3300 del A-1061 Ter BRCA1 expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) were used in this study.

Techniques: Inhibition, Activity Assay, Mutagenesis

Journal: Frontiers in Endocrinology

Article Title: Identification of BRCA1 As a Potential Biomarker for Insulin-Like Growth Factor-1 Receptor Targeted Therapy in Breast Cancer

doi: 10.3389/fendo.2017.00148

Figure Lengend Snippet: Insulin-like growth factor-1 receptor (IGF1R) gene expression in wild-type- and mutant-BRCA1-containing breast cancer cells. (A) Confluent cultures of wild-type BRCA1-expressing MCF7, MCF10A, HB2, and MDA-MB-231, and mutant BRCA1-expressing HCC1937 cells, were harvested and total protein and RNA was extracted. The bar graphs represent the IGF1R and BRCA1 mRNA levels in the various cell lines, as measured by RT-qPCR. An arbitrary value of 1 in the y -axis was given to the mRNA levels in HCC1937 cells. Bars represent mean ± SEM of three independent experiments (* p < 0.05 versus HCC1937; ** p < 0.01 versus HCC1937). Equal amounts of protein (50 μg) were separated by 6 and 10% SDS-PAGE, transferred to nitrocellulose filters and blotted with anti-BRCA1 or anti-total IGF1R antibodies, respectively. The positions of the ~220-kDa BRCA1, ~97-kDa IGF1R β-subunit, 42-kDa β-actin, and 100-kDa Cbl bands are indicated. (B) Effect of BRCA1 expression on endogenous IGF1R levels. HCC1937 cells were seeded in 10-cm plates at a density of 1 × 10 6 cells per plate. After 24 h, cells were transiently transfected with 10 μg of the pcDNA3-BRCA1 expression vector, or empty vector, using the jetPRIME reagent. After 48 h, cells were harvested, and levels of BRCA1 and endogenous IGF1R were assessed by Western blotting. Tubulin was used as a loading control.

Article Snippet: To generate wild-type BRCA1-expressing HCC1937 cells, naïve HCC1937 cells were transiently transfected with 10 μg of a pcDNA3-BRCA1 expression vector, or empty pcDNA3 vector (Invitrogen, Carlsbad, CA, USA), using the jetPRIME ® reagent (Polyplus Transfection, Illkirch, France).

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, SDS Page, Transfection, Plasmid Preparation, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Identification of BRCA1 As a Potential Biomarker for Insulin-Like Growth Factor-1 Receptor Targeted Therapy in Breast Cancer

doi: 10.3389/fendo.2017.00148

Figure Lengend Snippet: Effect of BRCA1 silencing on the anti-proliferative activity of MK-0646. (A) MCF7, MCF10A, and HB2 cells were seeded in 10-cm plates at a density of 1 × 10 6 cells per plate. After 24 h, cells were infected with 3 μg of the lentivirus vector pGIPZ encoding BRCA1 shRNA or empty vector (NS). Levels of BRCA1 mRNA and protein in untransfected cells, empty vector-transfected cells, and a number of selected clones were measured by RT-qPCR (bar graphs) and Western blots, respectively. Figures above the bars denote arbitrary units of absorbance. Equal loading was confirmed by Cbl or tubulin measurement. Bars represent mean ± SEM of three independent experiments (** p < 0.01 versus respective control). (B) Untransfected MCF7 cells, empty vector-transfected cells (NS), and MCF7-BRCA1 knockdown cells (C5 and C8 clones) were plated in 96-well plates at a density of 3 × 10 3 cells/well. The cells were serum starved for 24 h, after which the medium was changed to serum-free media, including or lacking MK-0646 antibody (MK, 10 μg/ml). Cell proliferation was examined by XTT assays. Bars represent mean ± SEM of three independent experiments (* p < 0.05 versus respective control). (C) Wild-type BRCA1-containing MCF7 and MDA-MB-231 cells, and mutant BRCA1-containing HCC1937 cells, were seeded in 96-well plates at a density of 3 × 10 3 cells/well. The effect of MK-0646 on cell proliferation was assessed as described above. Bars represent mean ± SEM of three independent experiments (* p < 0.05 versus control).

Article Snippet: To generate wild-type BRCA1-expressing HCC1937 cells, naïve HCC1937 cells were transiently transfected with 10 μg of a pcDNA3-BRCA1 expression vector, or empty pcDNA3 vector (Invitrogen, Carlsbad, CA, USA), using the jetPRIME ® reagent (Polyplus Transfection, Illkirch, France).

Techniques: Activity Assay, Infection, Plasmid Preparation, shRNA, Transfection, Clone Assay, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: Frontiers in Endocrinology

Article Title: Identification of BRCA1 As a Potential Biomarker for Insulin-Like Growth Factor-1 Receptor Targeted Therapy in Breast Cancer

doi: 10.3389/fendo.2017.00148

Figure Lengend Snippet: Effect of BRCA1 status on the synergistic activity of MK-0646. MCF7 and HCC1937 cells were plated in 12-well plates at a density of 25 × 10 3 cells/well. After 24 h, the medium was changed to 5% charcoal-treated fetal bovine serum, including or lacking MK-0646 (MK, 10 μg/ml) with (open bars) or without (solid bars) etoposide (2 μM). Control cells were incubated for the same period of time in the absence of the antibody. Cell proliferation was examined by XTT assays. A value of 100% was given to the viability of untreated cells. Bars represent mean ± SEM of three independent experiments (* p < 0.05 versus no etoposide).

Article Snippet: To generate wild-type BRCA1-expressing HCC1937 cells, naïve HCC1937 cells were transiently transfected with 10 μg of a pcDNA3-BRCA1 expression vector, or empty pcDNA3 vector (Invitrogen, Carlsbad, CA, USA), using the jetPRIME ® reagent (Polyplus Transfection, Illkirch, France).

Techniques: Activity Assay, Incubation