Transcription profiling by array of human SRC-2-depleted MCF-7 human breast cancer cells and MCF-7 cells exposed to PKA activating agents to identify genes that may be regulated through PKA-induced downregulation of SRC-2

ABSTRACT: We have characterized the global program of transcription in SRC-2-depleted MCF-7 breast cancer cells using short-hairpin RNA technology, and in MCF-7 cells exposed to PKA activating agents. In order to identify genes that may be regulated through PKA-induced downregulation of SRC-2, overlapping transcriptional targets in response to the respective treatments were characterized. MCF-7 human breast cancer cells transduced with shRNA targeting SRC-2 (SRC-2 shRNA) or infected with control shRNA vector (Ctr shRNA) were seeded in 9,2 cm petri dishes, 2,0 x106 cells in each dishes, in DMEM supplemented with 5% charcoaled stripped FBS and 10 nM 17b-estradiol. After 48 hours a selection of the Ctr shRNA cells were treated with cAMP analog (8-CPT-cAMP, 150 µM) and cAMP elevating agents (Forskolin (10 µM) and IBMX (50 µM)) The cells were then incubated for another 24 hour in the cell incubator. Cells were harvested by washing them twice with 8 ml PBS (37 oC), before adding 1,5 ml 0,25 % trypzine. The trypzine reaction was stopped after 5 minutes by adding 2,5 ml DMEM to the cells. Trypzine and cell medium was removed by centrifugating the cell suspensions at 1000 rpm for 3 minutes. The cells were then washed with 400 µl PBS, before addition of 350 µl RLT buffer to the cell pellets. The cells were then homogenized by vortexing for 1 minute and then freezed in -80 oC.

ORGANISM(S): Homo sapiens

SUBMITTER: Helland Thomas 

PROVIDER: S-ECPF-MTAB-1662 | biostudies-other |

REPOSITORIES: biostudies-other