DLC1 is an E2-responsive gene. (A) DLC1 mRNA is increased after oestrogen stimulation. MCF-7 cells were treated with different concentrations of E2 for 16 h and analysed by northern blotting. (B) Effect of actinomycin D on the ability of E2 to upregulate DLC1 mRNA in MCF-7 cells. Cells were collected at different time points after treatment with E2 and/or actinomycin D (10 μg/ml) and DLC1 mRNA was analysed by northern blotting. (C) Upregulation of DLC1 promoter activity in MCF-7 and Ishikawa cells treated with E2 for 24 h. Cells were transfected with ERE-luc for 24 h, treated with E2 for 24 h and ERE-luc activity was measured. (D) Upregulation of DLC1 protein in MCF-7 and Ishikawa cells treated with E2 for 24 h.

DLC1 overexpression leads to E2 hypersensitivity. (A) Dox-mediated upregulation of T7-DLC1 expression in MCF-7/DLC1 clone. (B) DLC1 overexpression potentiates Bcl-2 expression by E2. MCF-7/DLC1 cells were treated with or without Dox and stimulated with E2 for 24 h. (C) DLC1 knockdown reduces the expression of progesterone receptor (PR) and cathepsin D. Cells were treated with control or DLC1specific siRNA for 48 h and stimulated with E2 for 24 h. Then, lysates were immunoblotted with the indicated antibodies. (D) DLC1 overexpression potentiated E2-mediated stimulation of ERE-luc activity in MCF-7 and Ishikawa cells. MCF-7 and Ishikawa cells were transfected with ERE-luc, treated with E2 for 24 h and ERE-luc activity was measured. (E) DLC1 upregulation potentiates growth stimulation by E2. MCF-7/DLC1 cells were treated with or without Dox and stimulated with E2 for 5 days. (F) DLC1 upregulation promoted anchorage-independent colony formation. MCF-7/DLC1 cells were plated in soft agar, treated or not treated with Dox and stimulated with E2 for 21 days. (G) DLC1 expression level influences the growth of MCF-7 cells. Cells were treated with control or DLC1 siRNA for 48 h, and treated with E2 for 72 h. (H) DLC1-mediated potentiation of ER transactivation depends on DLC1 status in MCF-7 cells. Cells were treated with control or DLC1 siRNA for 48 h. Cells were transfected with ERE-luc and treated with E2 for 24 h, after which ERE-luc activity was measured.

DLC1 and oestrogen receptor localization and chromatin studies. (A–C) Recruitment of ER and DLC1 to the pS2 promoter chromatin. MCF-7/DLC1 cells were treated or not treated with Dox for 24 h, stimulated with E2 for 1 h and ChIP was performed with anti-ER or anti-T7 antibodies. IP, immunoprecipitation. (A) DLC1 enhances ER recruitment. Upper panel: PCR analysis of the 304-bp pS2 promoter fragment associated with ER. Lower panel: PCR analysis of the input DNA. Relative recruitment of ER or T7-DLC1 on the target promoter is shown as fold change. (B) Oestrogen induces recruitment of DLC1 to the target chromatin. PCR analysis of the 304-bp pS2 promoter fragment associated with T7-DLC1 (upper panel); PCR analysis of the input DNA is shown below. (C) DLC1 recruitment to the pS2 promoter chromatin depends on the presence of ER (upper panel). PCR analysis of the 304-bp pS2 promoter fragment associated with T7-DLC1; PCR analysis of the input DNA is shown below. (D) Ishikawa cells grown on coverslips and treated with control or DLC1 siRNA for 48 h. The coverslips were incubated with antibodies against DLC1 and ER and then incubated with secondary antibodies conjugated with Alexa-546 (red) and Alexa-488 (green), respectively. Arrows indicate the cells with exclusively cytoplasmic staining of ER. (E) ER interacts with DIC in vivo. Cell lysates from MCF-7 stimulated with E2 for 1 h were immunoprecipitated with anti-DIC monoclonal antibody and blotted with anti-DLC1, anti-ER or anti-DIC antibodies. WB, western blot. (F) Role of DLC1 in ER–DLC1 interaction with DIC. Lysates from MCF7 cells were treated with control or DLC1 siRNA and were immunoprecipitated with anti-DIC monoclonal antibody and blotted with DLC1, ER and DIC antibodies. The right panel shows the extent of DLC1 knockdown by siRNA.

Signalling pathways in oestrogen regulation of DLC1. (A) Increased expression of DLC1 mRNA and protein in MCF-7/DA-Pak1 cells. Cells were induced with Dox, and DLC1 mRNA and protein levels were assessed. (B) Decreased expression of DLC1 mRNA and protein in MCF-7/DN-Pak1 cells. Cells were induced for 8 h with Dox and DLC1 mRNA and protein levels were assessed. (C) Downregulation of DLC1 mRNA in MCF-7 cells treated with siRNA-Pak1 for 24 h. (D) Modulation of DLC1 promoter activity in MCF-7 cells by the expression of Pak1 or DN-Pak1 for 24 h. WT, wild type.

DLC1 interacts with oestrogen receptor in vitro and in vivo. (A) Interaction of DLC1 with ER in vitro. GST pull-down assay shows the association of GST–DLC1 with in vitro-translated ³⁵S-labelled ER. (B) Interaction of DLC1 with the AB domain of ER. GST pull-down assay shows the association of GST–ER deletion constructs with the in vitro-translated ³⁵S-labelled DLC1. (C) Mapping the ER-binding region in DLC1. GST pull-down assay shows the association of GST–DLC1 deletion constructs with the in vitro-translated ³⁵S-labelled ER. (D) DLC1 and ER interact in vivo. Cell lysates were immunoprecipitated with anti-ER monoclonal antibody and blotted with anti-DLC1 or anti-ER antibodies. (E) DLC1 overexpression increases DLC1–ER interaction. MCF-7/DLC1 cells were treated or not treated with Dox and were stimulated with E2 for 1 h. Cell lysates were immunoprecipitated with anti-ER monoclonal antibody and blotted with anti-T7 or anti-ER antibodies. (F) ERE-luc assay shows that the deletion of the binding site of ER on DLC1 blocked transactivation.