Characterization of the transcriptional response to hypoxia in MDA-MB-231-shCAIX, MCF7-shCAIX and the respective control cells grown as multicellular spheroids under hypoxic or normoxic conditions

ABSTRACT: To interrogate the hypoxia response pathways affected by the depletion of CAIX in BC cells, we performed gene expression profiling using SurePrint G3 Human Gene Expression 60K microarrays in MDA-MB-231-shCAIX, MCF7-shCAIX and the respective control cells grown as multicellular spheroids under hypoxic or normoxic conditions. The human breast cancer cell lines MDA-MB-231 and MCF7 were purchased from the European Collection of Cell Cultures (ECACC, UK) and maintained as recommended by the manufacturer. To generate stable CAIX silenced cells, cell lines were transfected with a pool of three plasmids each encoding CAIX-specific shRNA (1 µg) and a negative control (NC) plasmids encoding scrambled shRNAs (1 µg) (Santa Cruz Biotechnology, TX, USA) using shRNA transfection medium and shRNA transfection reagent (Santa Cruz Biotechnology, USA) according to the manufacturers’ protocol. For selection of stably transfected cells, puromycin dihydrochloride (Santa Cruz Biotechnology, USA) was added to medium 48 h post-transfection (6 µg/ml for MCF7, 2 µg/ml for MDA-MB-231 cells). The transfected cells were plated at density 2e+3 cells per ml of serum-free DMEM/F12 medium supplemented with EGF (20ng/ml, R&D Systems, #236-EG-200), bFGF (10ng/ml, SantaCruz, #sc-4573), hydrocortisone (50 ng/ml, Sigma-Aldrich, #H0135-1MG) and 1B27 (Invitrogen, #17504001) and grown as multicellular spheroids for 5 days. To establish hypoxic conditions, the cells were cultured at 1% oxygen, 94% N2 and 5% CO2 at 37oC using humidified multi-gas incubator (Sanyo, Sanyo Electric Co.,Ltd.) for 48 hours. The normoxic control cells were incubated at 37oC with 5% CO2 in a humidified incubator (Panasonic, Panasonic Healthcare Co., Ltd.). The efficiency of CAIX silencing was assessed by qRT-PCR and immunofluorescence before the experiments.

ORGANISM(S): Homo sapiens

SUBMITTER: Pawel Zayakin 

PROVIDER: E-MTAB-5512 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress