Fig 2: Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs.A, specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2, Zbtb1, Cbfa2t2, or Cbfa2t3 was introduced into the Cas9-expressing (GFP+) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP+hNGFR+ cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B, an experimental scheme for the deletion of Lmo2, Zbtb1, Cbfa2t3, and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1-deficient LPs is shown. C, retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP+) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP+hNGFR+ sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D, the percentage of CD25+ cells among GFP+hNGFR+ sgRNA transduced cells (C) is shown with standard deviation (SD). E, ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP+hNGFR+ sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F, the percentage of CD25+ cells among GFP+hNGFR+ sgRNA transduced cells (E) is shown with SD. Data are representative of two (A) or three (C and E) independent experiments. Data represent the mean values of three independent biological replicates (D and F). ∗∗p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.

Fig 3: In vitro foetal haemoglobin (HbF) induction with decitabine. (A) Representative dose‐response curves of decitabine and hydroxyurea in two healthy donors. Data points for 3 µM decitabine and 30 µM hydroxyurea were excluded due to high cytotoxicity. Mean ± SD of technical triplicates are shown. (B) Baseline F‐cells, maximum F‐cells at 1 µM decitabine, and EC50 (half maximal effective concentration) of four healthy donors. (C) HbF protein levels by HPLC of two donors. (D) Immunoblot showing the expression of DNMT1 and BCL11A in erythroblasts at 48 h after decitabine treatment.

Fig 4: BCL11A overexpression exacerbated UVB-induced cell damage by inhibiting autophagy.a, b Representative immunohistochemistry images show ATG5 and LC3 expression in mice skin with BCL11A overexpressing post-UVB irradiation. Scale: 20 μm. c, d Western blot and quantitative grayscale analysis of BCL11A, LC3І/Ⅱ and ATG5 was conducted in HaCaT cells overexpressing BCL11A. Grayscale normalized to β-action of NC cells. e CCK-8 assay evaluated the viability of HaCaT cells with BCL11A overexpression following UVB exposure. f Flow cytometry measured apoptosis rate of HaCaT cells with BCL11A overexpression after 24 h UVB exposure. g Immunofluorescence assessed γH2A.X in HaCaT cells with BCL11A overexpression and 30 J m−2 UVB exposure (n = 5). Scale: 20 μm. h ROS levels were detected after HaCaT overexpression of BCL11A combined with UVB irradiation. i The representative immunohistochemical images of γH2A.X in the dorsal skin tissues of BCL11A overexpression mice were obtained after UVB treatment (n = 5). Scale bars: 20 µm. Data are presented as mean ± SD. One-way ANOVA, ns no significance, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.