Fig. 2: CNOT3 suppress differentiation of primary HSPCs.

A–F Cord blood-derived human CD34+ hematopoietic stem/progenitor cells (CB-CD34+ cells) were transduced with lentiviruses expressing either a scramble (control) shRNA or CNOT3-targeting shRNAs (KD33 and KD-37). Cells were selected for puromycin resistance and assayed 3 days post-transduction. A Representative immunoblots showing efficient depletion of CNOT3. ACTIN serves as loading control. B Cell proliferation in liquid culture supplemented with cytokines. C Colony formation. Cells were plated on methylcellulose supplemented with cytokines. D Percentage of apoptotic cells by flow cytometry analysis for Annexin-V positivity. E Quantitative summary of flow cytometry analysis of myeloid differentiation markers CD11b, CD14, and CD13. F Representative H&E images of morphological evaluation of CB-CD34+ cells upon CNOT3 depletion. All graphs (A–E) show data as mean ±  s.e.m. n = 3 independent experiments, p values calculated by two-tailed student’s t test. G–I CB-CD34+ cells were transduced with lentiviruses carrying either an empty vector backbone or cDNA expressing CNOT3. Reporter gene YFP is present within the lentiviral vector Cells were sorted based on YFP positivity 3 days post-transduction. G Cell proliferation in liquid culture supplemented with cytokines. H Colony formation. Cells were plated on methylcellulose supplemented with cytokines. I Quantitative summary of flow cytometry analysis of myeloid differentiation markers CD11b, CD14, and CD13. All graphs G–I showing data as mean ± s.e.m, n = 3 independent experiments, p values calculated by two-tailed student’s t test. Source data are provided as Source Data files for figures (A–E, G–I).