Fig 2: Increase in proportion of hemoglobin-containing, benzidine-positive cells in K562 and K562(BCL11A-XL) clone 12 cells treated with different fetal hemoglobin inducers. K562 (A) and K562(BCL11A-XL) clone 12 cells (B) were cultured in the presence of 30 nmol/L MTH (?), 175 µmol/L HU (?), 1.5 mmol/L BA (?), 25 µmol/L 1-OctylT (¦), 150 nmol/L RAPA (?), and 30 µmol/L RSV (•), and the proportion of benzidine-positive cells was determined. Data represent averages ± SD of four independent experiments. The differences between inducer-treated and untreated K562 cells were highly significant (p < 0.01) for all inducers, when data relative to 6 and 7 days of induction were considered. On the contrary, only K562(BCL11A-XL) clone 12 cells treated with MTH exhibited significant differences (p < 0.05) with respect to untreated cells.

Fig 3: BCL11A overexpression leads to thickening of the airways and abnormal organoid formation. a Schematic representing strategy to explore the role of BCL11A in vivo and ex vivo. Left Panel: Adenovirus-Cre was nasally administered to BCL11Aovx mice and the lungs were analysed after eight months. Right panel: for the tracheosphere organoid model, basal cells from the trachea of either BCL11Aovx or BCL11Acko mice were FACS sorted, embedded in matrigel and analysed after 15 days. Three independent mice were used for each experiment. b Images of airways from control and BCL11Aovx. Arrows indicate small hyperchromatic cells with irregular nuclei. c Quantificaiton of airway epithelial layer hyperplasia from two control and BCL11Aovx mice. d Bright field images of organoids from Bcl11acko and BCL11Aovx mice treated with vehicle or tamoxifen. e Sectioned organoids from BCL11Aovx mice stained with haematoxylin and eosin, BCL11A, Sox2, Krt5, Trp63 and Krt8. Arrows indicate positive staining. Scale bar indicates 50 µm

Fig 4: BCL11A regulates both SETD8 and SKIL gene expression. SETD8 (a), SKIL (b), TBX2 (c), BCL11A (d), SOX2 (e) gene expression in NSCLC cell lines. f Sorting strategy for RNA extraction from WT or BCL11AOVX mice treated with tamoxifen. SETD8 (g), SKIL (h), TBX2 (i), BCL11A (j), SOX2 (k) gene expression in basal stem cells. SETD8 (l), SKIL (m), TBX2 (n), BCL11A (o), SOX2 (p) gene expression in epithelial Epcam+ve cells. The whiskers in all box plots represent minimum and maximum points. The box extends from 25th to 75th percentile and the line represents the median. Data presented as mean ± s.d. (LUSC n = 5 and LUAD n = 6; WT n = 3 and BCL11AOVX n = 3). Student’s t-test performed, *p < 0.05, **p < 0.005, and ***p < 0.001

Fig 5: Production and characterization of K562(BCL11A-XL) cells. (A) Scheme describing the strategy. K562 cells were transfected with the pCDNA3.1-BCL11A-XL vector containing BLC11A-XL cDNA under the control of the cytomegalovirus promoter. (B, C) Quantitative analysis of BCL11A-XL expression in 12 K562(BCL11A-XL) clones, determined by real-time quantitative polymerase chain reaction (B) and Western blotting (C). The data represent the average ± SD of three independent analyses. Fold expression is relative to K562(BCL11A-XL) clone 10. A representative result of Western blotting is shown on the left in (C) and was performed using protein extracts isolated from K562 and K562(BCL11A-XL) clone 12, as indicated. The BCL11A-XL protein is arrowed. As an internal control, the Western blotting filters were stripped, and binding reaction was performed using an antibody recognizing p70.