Figure 5: NEDD9 is a TGFβ/Smad and MRTF-SRF target.

(a) Western blot using the indicated antibodies of untreated (−) and TGFβ (2.5 ng ml⁻¹)-treated (+) MDA-MB-231 M1 cultures transfected with siRNA pools against SRF, Smad2 (S2), Smad3 (S3) Smad2 and 3 (S2+3) and Smad4 (S4). (b) qRT–PCR analysis of NEDD9 in same conditions as in (a). Error bars represent ±s.d. of three experiments. (c) qRT–PCR of the indicated transcripts in HCC1954 (left panel) and MDA-MB-231 (right panel) M1 mammospheres treated with TGFβ (2.5 ng ml⁻¹), 5 μM CD or 0.5 μM LB as indicated. Expression was normalized against beta-2-microglobulin and expressed as arbitrary units. Error bars represent ±s.d. of at least three independent experiments. (d) ChIP assay on MDA-MB-231 M1 mammospheres, untreated (−) or treated with TGFβ (2.5 ng ml⁻¹) (Tβ) for 1 h to detect binding of endogenous SRF and Smad2/3 to R1, R2 and R3. KRT19SBE region was used as an internal positive control for Smad2/3 binding. Results are expressed as TGFβ-induced fold change in binding from untreated control cultures. Error bars represent ±s.d. of three independent experiments. (e) ChIP assay as described in (d) to detect binding of endogenous MRTF-A and MRTF-B. (f) UCSC Genome Browser display showing regions R1, R2 and R3 that were selected to design primers to detect binding of Smad, SRF, MRTF-A and MRTF-B by ChIP-qPCR assays. Error bars represent ±s.d. of three independent experiments. Also showing ChIP-sequencing enrichment of Smad2/3 binding in M1 mammosphere cultures treated with TGFβ (2.5 ng ml⁻¹) for 1 h across the NEDD9 genomic region. Black boxes correspond to called binding events obtained using both MACS and SWEMBL algorithms.