Figure 6
Regulation of ALDH1A1 expression by tamoxifen-activated ERα36. (A) Putative sites in ERα36 involved in interaction with 4-OHT. All aa residues close to 4-OHT in less than 4 Å are shown by lines. The analysis was performed with Discovery Studio 2.0 (Accelrys Software Inc.). (B) Binding of 4-OHT to purified GST-ERα36 fusion protein. GST-ERα36 was immobilized to an SPR sensor chip by GST capturing and 4-OHT was introduced as the soluble-phase analyte. The sensorgrams reached equilibrium and rapidly returned to baseline, demonstrating quick interaction kinetics between GST-ERα36 and 4-OHT. The KD was estimated as 11.6 ± 1.0 μM using the Biacore Evaluation Software. (C) Nuclear localization of ERα36 (green) in MDA-MB 436 cells after treatment with 4-OHT (1 μM) for 20 and 40 min. Heochst (blue) was used for nuclear staining. Scale bar, 20 μm. (D) Western blot of ERα36 in the cytoplasm or nuclei of MDA-MB 436-ALDH1high cells after 4-OHT or E2 treatment. Lamin-B1 was used as a nuclear protein control, β-actin as a cytoplasm protein control. C, cytoplasm; N, nuclei. (E) HS578 ERE-luciferase assays showing the transcriptional ability of ERα36 activated by E2 or 4-OHT. ERα36 or ERα66 was transfected into HS578 cells along with an ERE-luciferase element. The transcriptional activity was measured. Error bars represent SEM from mean of triplicate samples. (F) Two potential ERE-binding sites in the ALDH1A1 promoter as analyzed by Transcription Element Search System. (G) ChIP/PCR analysis of MDA-MB-436 cell lysates showing endogenous ERα36 bound to ALDH1A1 promoter after treatment with E2 (1 nM) or 4-OHT (1 μM). An unrelated mouse IgG was used as an immunoprecipitation control. *P < 0.05. (H) Luciferase activity of the reporter fused to a wild-type ALDH1A1 promoter observed in MDA-MB-436/shControl cells treated with E2 (1 nM) or 4-OHT (1 μM). DMSO was used as a vehicle control. *P < 0.01. (I) Abolished transcriptional activity (shown by relative luc activity) of ALDH1A1 promoter with mutant ERE sites in MDA-MB 436/shControl cells. Results are presented as mean ± SEM. Statistical significance was determined by two-tailed Student's t test. #P < 0.05.
