Fig 2: BMI1 indirectly inhibits Runx1 and Pten expression with histone deacetylation. (A) The heat map of poor diagnosis genes in six MDS CD34+ cells of two refractory anaemia (RA), two RA with excess blasts I (RAEB-1) and two RAEB-2 compared to the normal control on the cDNA microarray. (B) The relative transcription of Runx1 and Pten tested by Q-PCR in K562. The transcript level in K562 was set to 1. (C and D) The histone H3 acetylation (H3ac) and histone H4 acetylation (H4ac) at Runx1 and Pten promoter regions tested by chromatin immunoprecipitation (ChIP) and Q-PCR assay in K562, n = 3. The histone modification in 10% input DNA was set to 1. IgG was as negative control.

Fig 3: BMI1 directly suppresses Zmym3 and activates c-fos pathway. (A) Electrophoresis strips of Zmym3 promoter region in BMI1 and H2A with ubiquitination ChIP-PCR products. (B) The relative transcript level of Zmym3 in Bmi1-transfected K562 cells. The expression level in K562 was set to 1. (C) The relative transcript expression of c-fos in Bmi1-transfected K562. The expression level in K562 was set to 1. (D) The relative histone H3K27 acetylation (H3K27ac) and H3 acetylation (H3ac) level of c-fos promoter region, n = 3. The histone modification in 10% input DNA was set to 1. IgG was as negative control.

Fig 4: BMI1 enhances the malignancy of leukaemic cells (A) Western blotting of BMI1 in subclones of transfected U937 and K562 cells. Numbers are the folds compared to the first line control. SC1 and SC2 represent two subclones of Bmi1 transfected U937 and K562 cells. (B) The viability of cell was examined by vi-cell XR cell viability analyser after cells were cultured without foetal bovine serum for 72 hrs, n = 3. (C) Colony forming assay of K562 and Bmi1-transfected K562. Cells were cultured in MethoCult®H4230 with 5*104cells/mL for 7 days and then replated two cycles. Numbers are colonies with diameter larger than 0.3 mm, n = 3. The colony number of Bmi1-transfected K562 was greater than control with statistical significance, P < 0.05. The colony number means the total number of colonies/number of cell input after two cycles replating. (D) The apoptosis ratio of K562 after 48 hrs of 5 µM arsenic trioxide treatment tested by annexin V and propidium iodide in flow cytometry.

Fig 5: Bmi1 transcript expression in myeloid progression (A–E). The Bmi1 transcript expression levels were all performed by quantitative PCR analysis (Q-PCR). GPADH was as control. Relative expression was calculated by the comparative Ct method. (A) The relative Bmi1 transcript expression in myelodysplastic syndromes (MDS) and non-MDS cytopaenia bone marrow mononuclear cells (BMMCs). “NR MDS” means MDS not in remission. “R MDS” means MDS in remission. (B) The relative BMI1 expression in two MDS groups separated by the ratio of blast cells in MDS bone marrow smear. (C) The transcription of Bmi1 in non-MDS cytopaenia, MDS, MDS transformed acute myeloid leukaemia [MDS-acute myeloid leukaemia (AML)] and de novo AML (dAML) CD34+ cells. (D) The correlation of BMI1 transcription in MDS CD34+ cells and international prognostic scoring system score was evaluated by linear regression. The patient with one arrow progressed to refractory anaemia (RA) with excess blasts (RAEB) from RA and the patients with double arrows transformed to AML from RAEB. (E) The relative Bmi1 transcription in chronic myeloid leukaemia in chronic phase (CML-CP), CML in blast phase (CML-BP) and matched dAML BMMCs. (A, B, E) Quartile figure is used to present the data, and bar represents the median of the data. “*” indicates statistically significant P < 0.05; and “**” means without statistically significant.