Project description:To identify genes regulated by SMAD3, we have employed whole transcriptome microarray expression profiling to identify genes downstream of SMAD3. Total RNAs from SMAD3-overexpression (7721-SMAD3 vs 7721-VEC) or SMAD3-knockdown (LM3-shSMAD3 vs LM3-CON) cell lines were measured.
Project description:In order to identify genes co-bound by SOX4 and SMAD3 in the context of breast cancer, different breast cell lines (HMLEs, MDA-MB-231 or HCC-1954) were used. Due to low endogenous expression levels for SOX4 in HMLEs in untreated conditions, doxycycline-dependent SOX4 overexpression was obtained by transducing HMLE cells with pIINDUCER21-SOX4 vector..Cells were plated and SOX4 and SMAD3 chromatine-immunoprecipitation was performed in untreated conditions, TGF-beta (2.5ng/ml), doxycycline (0.5ug/ml) or both as indicated. Genome-wide binding sites for SOX4 and SMAD3 was identified in HMLEs, MAD-MB-231 and HCC-1954 cells.
Project description:TGF? activates a signal transduction cascade that results in the transcription of genes, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify SMAD3 binding targets and the molecular pathways affected by the TGF?1/SMAD3 signal transduction on a genome-wide scale. Cultured A549 cells at 30-50% confluence were treated with 10 uM SIS3 in dimethyl sulfoxide (DMSO), or DMSO (vehicle-only) 30 min prior to TGF?1 treatment. Cells were treated with 2 ng/mL recombinant TGF?1 for 0, 2, 12, and 24 h.