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. 2015 Jan 21;6(6):4345–4356. doi: 10.18632/oncotarget.2999

Figure 2. GATA1 recruits HDAC3/4 to down-regulate E-cadherin transcription.

Figure 2

(A) pGL2-E-cad-luc and pRL-TK plasmids were co-transfected with pcDNA-GATA1 or control plasmid into HEK-293 cells and MCF7 cells. Then cells treated with or without TSA for luciferase assay. (B) HEK-293 cells were transfected with pGL2-E-cad-luc plasmid together with HDAC constructs expressing HDAC1–6, respectively. **p < 0.01. (C–D) HEK-293 cells were transfected with pGL2-E-cad-luc, pcDNA-GATA1 and increasing amounts of HDAC3/4 as indicated for Luciferase Assays. Simultaneously, increasing amounts of TSA was added to HEK-293 cells transfected with GATA1 and HDAC3/4 for Luciferase Assays. (E) MCF-7 cells were transfected with Flag-HDAC3/4 expression plasmids. ChIP assay was carried out using anti-GATA1 or anti-Flag antibody, followed by PCR with primers amplifying the E-cadherin promoter region (–1001/–753, –720/–402, –388/–179). ChIP Re-IP, soluble chromatin, prepared from MCF-7 cells transfected with Flag-HDAC3/4, was firstly immunoprecipitated with antibody against GATA1, then reimmunoprecipitated with anti-Flag antibody. (F) In vitro translated Myc-GATA1 or Flag-HDAC3/4 was incubated with GST-HDAC3/4 or GST-GATA1 fusion proteins for GST pull-down assay. (G) HEK-293 cells were transfected with GFP-GATA1 and Flag-HDAC3 or GFP-GATA1 and Flag-HDAC4. Lysates were immunoprecipitated with Flag antibodies and immunoblotted with Flag and GFP antibodies.