Figure 2. Loss of Lmo2 leads to the differentiation arrest in pro-B(+) cells.

(A) An experimental scheme for the deletion of Lmo2 using the CRISPR/Cas9 system in pro-B cell lines is shown. (B) Retroviral vectors encoding sgRNA against luciferase (sgCont.) or LMO2 (sgLMO2) were introduced into Cas9-expressing (GFP⁺) pro-B(+) cells. Five (cultured on OP9-Mock for 5 days, left panels) or 10 days (cultured on OP9-Mock for 10 days, right panels) after co-cultured on OP9-Mock cells following the infection, pro-B cells were cultured again on OP9-Mock (Mock) or OP9-Dll4 (Dll4) stromal cells for 3 days. GFP⁺hNGFR⁺ sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression (left). The percentages and numbers of CD25⁺ cells among GFP⁺hNGFR⁺ sgRNA-transduced cells, cultured on OP9-Dll4, are shown with SD (right). The data represent the mean values of three independent biological replicates. Each value is indicated by a closed circle. **p<0.01 by two-sided Student’s t-test.

Figure 2—figure supplement 1. CRISPR/Cas9-mediated deletion of Lmo2 in BM progenitors.

(A) Experimental scheme for Lmo2 deletion in primary BM progenitor cells using the CRISPR/Cas9 system is shown. (B) Flow cytometric analysis of BM progenitors transduced with sgRNA and co-cultured with OP9-Dll1 for 4 days. Representative CD45/hNGFR (sgRNA) profiles in single cells, FSC/Lin profiles in CD45⁺hNGFR⁺ cells, and CD44/CD25 profiles in CD45⁺hNGFR⁺Lin^- cells are shown. Results are representative of three biological replicates. Percentages of CD25⁺ cells among the CD45⁺hNGFR⁺Lin^- cells are shown with SD (right). (C) Specific depletion of targeted LMO2 protein. Four days after sgRNA transduction in Figure 2A, lysates from retrovirus-infected GFP⁺hNGFR⁺ Pro-B(+) cells were subjected to immunoblotting for LMO2. Two independent experiments were performed with similar results.