Figure 1
Expression of oestrogen receptor alpha (ERα) and BMI1 in human mammary epithelial cells. (a) Plot of microarray data [18] showing BMI1 expression in ERα-positive and ERα-negative breast tumours. BMI1 is significantly overexpressed in ERα-positive tumours (p < 0.001). (b) Colony formation assay. Human mammary epithelial cells (HMECs) transduced with either glucuronidase (GUS; a negative control gene) alone, ERα alone or ERα and BMI1 were fixed after growth for 10 days in the presence of oestrogen and then stained with crystal violet. The surface area covered with cells in the fixed plates was used to estimate growth. (c) Western blot for ERα and β-tubulin in MCF7 control cells or in passage 3 (P3, left) and passage 4 (P4, right) HMECs transduced with BMI1 alone or with ERα and BMI1 together. Endogenous ERα expression is progressively lost with passage. (d) Western blot for ERα, BMI1 and β-tubulin in passage 6 (P6) HMECs transduced with BMI1 alone or with ERα and BMI1 together. Before being harvested, cells were treated for 24 hours with 1 nM oestrogen (E) or 1 μM fulvestrant (F). Endogenous ERα expression is no longer detectable. Exogenous ERα is destabilised by fulvestrant. (e) Immunofluorescent staining of HMECs infected with ERα and BMI1 viruses for ERα (lower left panel) and BMI1 (upper left panel). 4',6-Diamidino-2-phenylindole (DAPI; right panels) was used to counterstain nuclei. ERα and BMI1 are both nuclear. (f) Western blot for TERT, ERα, BMI1, MYC, p14ARF, p16CDKN2A and β-tubulin in HMECs infected with control virus (GUS, lane 1) or ERα, BMI1, TERT and MYC lentiviruses. HMECs from three different patients are shown in lanes 2 to 4. The transgenes are expressed, and BMI1 suppresses p14ARF and p16CDKN2A expression. (g) Immunofluorescent staining of HMECs for keratin 14 (K14, green) and keratin 18 (K18, red). DAPI (blue) was used to counterstain nuclei. At passage 1, HMECs infected with control virus and plated at clonal density formed mixed colonies with central K18-positive luminal cells and peripheral K14-positive myoepithelial cells (GUS P1). Passaging of these cultures led to progressive loss of luminal cells (GUS P4). HMECs infected with ERα and BMI1 viruses maintained K18 expression at passage 4, but individual cells were positive for both K14 and K18 (ERα BMI1 P4).