Journal: Oncotarget

Article Title: CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

doi: 10.18632/oncotarget.7711

Figure Lengend Snippet: Female nu/nu mice (N=24) were chronically fed (16 weeks) with HFD or CD. A. Mice body weight follow up during the experiment. B. Cholesterol, C. Glucose, and D. Triglycerides serum levels determined after mice euthanasia. Histograms show media and SD values of one representative experiment from two biological replicates. Significance was analyzed by t-test (*, p < 0.05; **, p < 0.01). E. H&E staining from liver from animals fed with CD or HFD. Magnifications x400. F. Mammary glands from mice fed with HFD or CD were whole mounted and stained with aluminum carmine red solution. G. Branches, lateral buds and TEB from mammary ducts were quantified. Plots show media and SD values of one representative experiment (N=2) with seven replicates each (*, p value < 0.05). H. H&E staining and IHC using anti-CtBP1 and anti-Cyclin D1 specific antibodies were performed in mammary tissue from mice fed with CD or HFD. HFD Group 1: mammary glands from HFD fed mice that show normal ducts; HFD Group 2: mammary glands from HFD fed mice that show prominent duct pattern. Magnifications are x40. I. RNA from mammary tissue was isolated and Cyclin D1 and E-cadherin expression were determined by RT-qPCR. Data were normalized to β-actin and CD (*, p value < 0.05).

Article Snippet: MDA-MB-231 pcDNA3 cells and pcDNA3 CtBP1 cells were generated by transient transfection using 6 μg of plasmid and polyethylenimine methodology (PEI - PolySciences INC) with PEI:DNA ratio 2:1. pcDNA3 plasmid was from Invitrogen. pcDNA3 CtBP1 plasmid was kindly provided by Dr. Richard H. Goodman (Vollum Institute, Oregon Health & Sciences University Portland). pGIPZ shRNA Scramble plasmid was from Open Biosystems. shRNA CtBP1 plasmids mix was from Santa Cruz Biotechnology Inc. LM38-LP cell line, derived from a murine mammary papillary adenocarcinoma [ ], were grown in DMEM/F12 medium with non-essential amino acids and 2 μM L-glutamine (Gibco), supplemented with 10% FBS (Internegocios) and 80 mg/ml gentamicin at 37°C.

Techniques: Staining, Isolation, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

doi: 10.18632/oncotarget.7711

Figure Lengend Snippet: A. CtBP1 expression was determined by WB in the indicated cell lines. Band quantifications performed by Image J software are indicated. Data were normalized to β-actin and NIH 3T3 cells. B. LM38-LP cells were exposed to CDS or HFDS during 6 days, and mammosphere formation assay was performed. Number of mammospheres was quantified. C. Left panel: Diameter of mammospheres (*, p value < 0.05). Right panel: mammospheres images. D. Mammospheres derived from LM38-LP were exposed to CDS or HFDS during 6 days and secondary mammosphere formation assay was performed. Number and diameter of mammosphere were determined. E. Clonogenic assay was performed in LM38-LP cells exposed to CDS or HFDS during 6 days. Histograms show media and SD values from three replicates (*, p value < 0.05; **, p value < 0.01). Magnifications x40.

Article Snippet: MDA-MB-231 pcDNA3 cells and pcDNA3 CtBP1 cells were generated by transient transfection using 6 μg of plasmid and polyethylenimine methodology (PEI - PolySciences INC) with PEI:DNA ratio 2:1. pcDNA3 plasmid was from Invitrogen. pcDNA3 CtBP1 plasmid was kindly provided by Dr. Richard H. Goodman (Vollum Institute, Oregon Health & Sciences University Portland). pGIPZ shRNA Scramble plasmid was from Open Biosystems. shRNA CtBP1 plasmids mix was from Santa Cruz Biotechnology Inc. LM38-LP cell line, derived from a murine mammary papillary adenocarcinoma [ ], were grown in DMEM/F12 medium with non-essential amino acids and 2 μM L-glutamine (Gibco), supplemented with 10% FBS (Internegocios) and 80 mg/ml gentamicin at 37°C.

Techniques: Expressing, Software, Tube Formation Assay, Derivative Assay, Clonogenic Assay

Journal: Oncotarget

Article Title: CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

doi: 10.18632/oncotarget.7711

Figure Lengend Snippet: A. CtBP1 expression was determined in MCF7, T-47D, MDA-MB-231, MDA-MB-453, MDA-MB-468 and BT-474 cells by RT-qPCR. B. CtBP1 expression was determined in CtBP1-diminished expression stable transfected MDA-MB-231 cells and control cells by WB ( left ) and RT-qPCR ( right ). Numbers below bands indicate quantification using Image J software. Data were normalized to lamin A/C for WB or β-actin for RT-qPCR and control cells (*, p value < 0.05). C. CtBP1 expression was determined by WB and RT-qPCR as indicated above in MDA-MB-231 transiently transfected with CtBP1 overexpression or control vector. D. Stable ( left ) or transient ( right ) transfected MDA-MB-231 cells were exposed to the indicated percentages of FBS during 72 h and cell viability was determined by MTS assay. Media and SD values of one representative experiment (N=2) from three replicates are shown (*, p value < 0.05). E. Stable transfected MDA-MB-231 cells were grown with media without FBS during 72 h and cell cycle analysis was performed. Media and SD values from two biological replicates are shown. F-G. Cyclin D1 expression was determined in the indicated cells by RT-qPCR. Data were normalized to β-actin and control cells (*, p value < 0.05).

Article Snippet: MDA-MB-231 pcDNA3 cells and pcDNA3 CtBP1 cells were generated by transient transfection using 6 μg of plasmid and polyethylenimine methodology (PEI - PolySciences INC) with PEI:DNA ratio 2:1. pcDNA3 plasmid was from Invitrogen. pcDNA3 CtBP1 plasmid was kindly provided by Dr. Richard H. Goodman (Vollum Institute, Oregon Health & Sciences University Portland). pGIPZ shRNA Scramble plasmid was from Open Biosystems. shRNA CtBP1 plasmids mix was from Santa Cruz Biotechnology Inc. LM38-LP cell line, derived from a murine mammary papillary adenocarcinoma [ ], were grown in DMEM/F12 medium with non-essential amino acids and 2 μM L-glutamine (Gibco), supplemented with 10% FBS (Internegocios) and 80 mg/ml gentamicin at 37°C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Software, Over Expression, Plasmid Preparation, MTS Assay, Cell Cycle Assay

Journal: Oncotarget

Article Title: CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

doi: 10.18632/oncotarget.7711

Figure Lengend Snippet: A. Tumor growth from CD or HFD orthotopic xenografts inoculated with shRNA scramble or shRNA CtBP1 cell lines. Curves indicate media and SD values of one representative experiment with 6 mice. B. CtBP1 RT-qPCR from HFD or CD xenograft samples. Data were normalized to β-actin and control. C. CtBP1 IHC from tumor xenografts. Magnification x400. D. RT-qPCR from xenograft tumors described above. Specific primers for the indicated genes were used. Data were normalized to β-actin and control. (*, p < 0.05; **, p < 0.01).

Article Snippet: MDA-MB-231 pcDNA3 cells and pcDNA3 CtBP1 cells were generated by transient transfection using 6 μg of plasmid and polyethylenimine methodology (PEI - PolySciences INC) with PEI:DNA ratio 2:1. pcDNA3 plasmid was from Invitrogen. pcDNA3 CtBP1 plasmid was kindly provided by Dr. Richard H. Goodman (Vollum Institute, Oregon Health & Sciences University Portland). pGIPZ shRNA Scramble plasmid was from Open Biosystems. shRNA CtBP1 plasmids mix was from Santa Cruz Biotechnology Inc. LM38-LP cell line, derived from a murine mammary papillary adenocarcinoma [ ], were grown in DMEM/F12 medium with non-essential amino acids and 2 μM L-glutamine (Gibco), supplemented with 10% FBS (Internegocios) and 80 mg/ml gentamicin at 37°C.

Techniques: shRNA, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

doi: 10.18632/oncotarget.7711

Figure Lengend Snippet: List of differentially expressed miRNAs

Article Snippet: MDA-MB-231 pcDNA3 cells and pcDNA3 CtBP1 cells were generated by transient transfection using 6 μg of plasmid and polyethylenimine methodology (PEI - PolySciences INC) with PEI:DNA ratio 2:1. pcDNA3 plasmid was from Invitrogen. pcDNA3 CtBP1 plasmid was kindly provided by Dr. Richard H. Goodman (Vollum Institute, Oregon Health & Sciences University Portland). pGIPZ shRNA Scramble plasmid was from Open Biosystems. shRNA CtBP1 plasmids mix was from Santa Cruz Biotechnology Inc. LM38-LP cell line, derived from a murine mammary papillary adenocarcinoma [ ], were grown in DMEM/F12 medium with non-essential amino acids and 2 μM L-glutamine (Gibco), supplemented with 10% FBS (Internegocios) and 80 mg/ml gentamicin at 37°C.

Techniques: shRNA

Journal: Oncotarget

Article Title: CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

doi: 10.18632/oncotarget.7711

Figure Lengend Snippet: GeneChip miRNA 4.0 Affymetrix was hybridized to total RNA from xenografts with CtBP1 knockdown or control grown in mice fed with HFD. Predicted target genes for the A. CtBP1 downregulated miRNAs; or B. CtBP1 upregulated miRNAs were determined using miRecords data base. Pie charts show GO analysis of predicted target genes using GO Panther. Four top processes over-represented were drilled down to smaller categories.

Article Snippet: MDA-MB-231 pcDNA3 cells and pcDNA3 CtBP1 cells were generated by transient transfection using 6 μg of plasmid and polyethylenimine methodology (PEI - PolySciences INC) with PEI:DNA ratio 2:1. pcDNA3 plasmid was from Invitrogen. pcDNA3 CtBP1 plasmid was kindly provided by Dr. Richard H. Goodman (Vollum Institute, Oregon Health & Sciences University Portland). pGIPZ shRNA Scramble plasmid was from Open Biosystems. shRNA CtBP1 plasmids mix was from Santa Cruz Biotechnology Inc. LM38-LP cell line, derived from a murine mammary papillary adenocarcinoma [ ], were grown in DMEM/F12 medium with non-essential amino acids and 2 μM L-glutamine (Gibco), supplemented with 10% FBS (Internegocios) and 80 mg/ml gentamicin at 37°C.

Techniques: Knockdown, Control

Journal: Oncotarget

Article Title: CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

doi: 10.18632/oncotarget.7711

Figure Lengend Snippet: HFD induces NADH activating CtBP1 expression in the mammary gland which, in turn, induce prominent epithelia ducts and trigger TEB formation. After tumor growth initiates, high CtBP1 expression might provide a worse prognosis to the patients modulating the expression of genes and miRNAs that are involved in cell proliferation, progenitor cells phenotype, EMT and mammary development in breast tumors.

Article Snippet: MDA-MB-231 pcDNA3 cells and pcDNA3 CtBP1 cells were generated by transient transfection using 6 μg of plasmid and polyethylenimine methodology (PEI - PolySciences INC) with PEI:DNA ratio 2:1. pcDNA3 plasmid was from Invitrogen. pcDNA3 CtBP1 plasmid was kindly provided by Dr. Richard H. Goodman (Vollum Institute, Oregon Health & Sciences University Portland). pGIPZ shRNA Scramble plasmid was from Open Biosystems. shRNA CtBP1 plasmids mix was from Santa Cruz Biotechnology Inc. LM38-LP cell line, derived from a murine mammary papillary adenocarcinoma [ ], were grown in DMEM/F12 medium with non-essential amino acids and 2 μM L-glutamine (Gibco), supplemented with 10% FBS (Internegocios) and 80 mg/ml gentamicin at 37°C.

Techniques: Expressing