creert2 sequence  (Addgene inc)

Journal: Molecular Oncology

Article Title: Hypoxia‐inducible factor‐2α directly promotes BCRP expression and mediates the resistance of ovarian cancer stem cells to adriamycin

doi: 10.1002/1878-0261.12419

Figure Lengend Snippet: HIF ‐2α directly activates the BCRP gene in ovarian cancer cells. (A) The evolutionarily conserved hypoxia‐response element ( HRE ) sequence ( CACGTG ), located between −483 and −478 nucleotides upstream of the transcription start site of the human BCRP gene. (B) Luciferase reporter activity was measured in transient co‐transfections with EPAS 1 ‐ cDNA or NC ‐ cDNA plasmids and BCRP promoter vectors ( GV 238‐ BCRP ‐ WT ) or vectors containing mutated HRE sequence binding sites ( GV 238‐ BCRP ‐mut) in 293T and OVCAR ‐3 cells. The luciferase activities in the cells co‐transfected with NC ‐ cDNA and GV 238‐ BCRP ‐ WT or GV 238‐ BCRP ‐mut plasmids were set as 1. Data are presented as the mean ± SD from three independent experiments. (C) Ch IP assays were performed to verify HIF ‐2α binding to the BCRP gene in OVCAR ‐3 and OVCAR ‐3 S cells cultured under hypoxic conditions (1% O 2 ) for 48 h. (D) Ch IP ‐ qPCR shows the enhanced binding HIF ‐2α on BCRP promoter in OVCAR ‐3 S cells. Antibody enrichment was quantified relative to the amount of input DNA . Antibody directed against IgG was used as a negative control. (E) The mRNA expression of BCRP was analyzed by qRT ‐ PCR in the OVCAR ‐3 and CAOV ‐3 cells transduced with EPAS 1 ‐ cDNA or NC ‐ cDNA lentivirus, and in the OVCAR ‐3 S and CAOV ‐3 S cells transduced with sh‐ EPAS 1 or sh‐ NC lentivirus under hypoxic conditions (1% O 2 ) for 48 h. BCRP expression levels in OVCAR ‐3 cells or CAOV ‐3 cells transduced with EPAS 1 ‐ cDNA lentivirus and in OVCAR ‐3 S cells or CAOV ‐3 S cells transduced with sh‐ NC lentivirus were set as 1. Data are presented as the mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01. Statistical significance was evaluated by Student's t ‐test.

Article Snippet: After 24 h, cells were transiently transfected with either 0.1 μg GV238‐basic vectors containing upstream promoter regions of BCRP (GV238‐BCRP‐WT), vectors containing mutated HRE sequence binding sites (GV238‐ BCRP ‐mut), or NC promoter plasmids and 0.01 μg Renilla luciferase plasmid and 0.2 μg EPAS1 ‐cDNA or NC‐cDNA plasmids (all from Shanghai Genechem Co., Ltd) using Lipofectamine 3000 transfection regent.

Techniques: Sequencing, Luciferase, Activity Assay, Transfection, Binding Assay, Cell Culture, Negative Control, Expressing, Quantitative RT-PCR, Transduction

Journal: Molecular Oncology

Article Title: Hypoxia‐inducible factor‐2α directly promotes BCRP expression and mediates the resistance of ovarian cancer stem cells to adriamycin

doi: 10.1002/1878-0261.12419

Figure Lengend Snippet: Silencing HIF ‐2α increases the response of OCSC s to ADR in vivo . (A) A diagram showing the time of tumor formation in BALB /c mice transplanted with the OVCAR ‐3 S cells (1 × 10 6 ) stably transfected sh‐ EPAS 1 lentiviral transduction particles or sh‐ NC as a control and after ADR or control DMSO intraperitoneal injection in vivo ( n = 5 mice in each group). (B) Growth curves of tumor volumes measured every other day in xenograft mice. Error bars indicate SD . *** P < 0.001. (C) GFP expression was detected in xenograft mice by small animal imaging. (D) The tumor weight was compared after sacrifice of xenograft mice 4 weeks after cell inoculation. Error bars indicate SD . * P < 0.05, *** P < 0.001. (E) Representative immunohistochemical staining for the expression of HIF ‐2α and BCRP proteins and statistical results for IOD in the xenograft tumors. Error bars indicate SD . * P < 0.05, ** P < 0.01, *** P < 0.001. The length of the scale bars is 20 μm. (F) Representative images for the expression of HIF ‐2α and BCRP proteins in the xenograft tumors by western blot and the relative protein expression of HIF ‐2α and BCRP normalized to those of β‐actin were analyzed. The expression levels of HIF ‐2α/ BCRP in the xenograft tumors derived from cells transduced with sh‐ NC and without ADR treatment were set as 1. Error bars indicate SD . * P < 0.05, ** P < 0.01, *** P < 0.001. (G) ADR accumulation in xenograft tumors derived from sh‐ EPAS 1 or sh‐ NC transduced OVCAR ‐3 S cells by mass spectrometry. The representative chromatograms were for ADR , internal standard ( IS ) in the sh‐ NC xenograft tumors with ADR treatment, and ADR , IS in the sh‐ EPAS 1 xenograft tumors with ADR treatment. RT , Retention Time. Error bars indicate SD . *** P < 0.001. Statistical significance was evaluated by one‐way anova .

Article Snippet: After 24 h, cells were transiently transfected with either 0.1 μg GV238‐basic vectors containing upstream promoter regions of BCRP (GV238‐BCRP‐WT), vectors containing mutated HRE sequence binding sites (GV238‐ BCRP ‐mut), or NC promoter plasmids and 0.01 μg Renilla luciferase plasmid and 0.2 μg EPAS1 ‐cDNA or NC‐cDNA plasmids (all from Shanghai Genechem Co., Ltd) using Lipofectamine 3000 transfection regent.

Techniques: In Vivo, Stable Transfection, Transfection, Transduction, Control, Injection, Expressing, Imaging, Immunohistochemical staining, Staining, Western Blot, Derivative Assay, Mass Spectrometry