American Society of Hematology

Figure 2.

$CITED2 is dispensable for human mast cell differentiation, survival, and proliferation. (A) Single-cell RNA-sequencing data visualized using the UMAP embedding. The gene expression level of CITED2 is plotted in the hematopoietic progenitor landscape. (B) Editing efficiency shown as percentage of InDels and percentage of frameshift mutations for CITED2 sgRNA1 of live cells sorted 5 days after electroporation. Five independent experiments were performed. (C) Flow cytometry analysis of mast cells 12 days after electroporation with CITED2 sgRNA1 in CD34+ cells. Representative of 5 independent experiments. Live single cells are shown. (D) The fraction of mast cells 11 to 12 days after electroporation. The results show the frequency of c-Kithi FcεRI+ mast cells (among live singlets) in the CITED2 sgRNA1 sample normalized to the corresponding NC sgRNA sample. The cell culture medium was supplemented with IL-3 during the second week in 2 of the 5 experiments. Two-tailed 1 sample t test, hypothetical value is 1. The experiments highlighted with red dots were analyzed with regards to editing efficiency in panel E. (E) Editing efficiency of percentage of InDels and percentage of frameshift for CITED2 sgRNA1 of c-Kithi FcεRI+ mast cells and non-mast cells within individual samples sorted 12 days after electroporation. Two independent experiments were performed. (F) Representative flow cytometry plots showing the frequency of mast cells derived from sorted FcεRI– progenitors and MCPs 12 days after electroporation with CITED2 sgRNA1. (G) The fraction of mast cells after electroporation and culture of sorted FcεRI– progenitors and MCPs. The results show the frequency of c-Kithi FcεRI+ mast cells (among live singlets) in the CITED2 sgRNA1 sample normalized to the corresponding NC sgRNA sample. The plots show 3 independent experiments in which CITED2 was knocked out in sorted FcεRI– progenitors and 2 independent experiments in which CITED2 was knocked out in sorted MCPs. Two-tailed 1 sample t test, hypothetical value is 1. (H) Editing efficiency shown as percentage of InDel and percentage of frameshift of CITED2 sgRNA1 in c-Kithi FcεRI+ mast cells derived from FcεRI- progenitors (upper panel) and MCPs (lower panel). (I) Flow cytometry plots and histograms showing the survival and the proliferation of LAD2 and HMC-1 electroporated with NC sgRNA and CITED2 sgRNA1, respectively. Representative of 2 independent experiments. Control (gray) refers to all live cells analyzed after staining with CellTrace Far Red and incubating the cells in the fridge. Live single cells are shown. (J) Editing efficiency shown as percentage of InDel and percentage of frameshift in LAD2 and HMC-1 in 2 independent experiments. The NC sgRNA condition refers to nontargeting sgRNA. ns, nonsignificant.$

CITED2 is dispensable for human mast cell differentiation, survival, and proliferation. (A) Single-cell RNA-sequencing data visualized using the UMAP embedding. The gene expression level of CITED2 is plotted in the hematopoietic progenitor landscape. (B) Editing efficiency shown as percentage of InDels and percentage of frameshift mutations for CITED2 sgRNA1 of live cells sorted 5 days after electroporation. Five independent experiments were performed. (C) Flow cytometry analysis of mast cells 12 days after electroporation with CITED2 sgRNA1 in CD34⁺ cells. Representative of 5 independent experiments. Live single cells are shown. (D) The fraction of mast cells 11 to 12 days after electroporation. The results show the frequency of c-Kit^hi FcεRI⁺ mast cells (among live singlets) in the CITED2 sgRNA1 sample normalized to the corresponding NC sgRNA sample. The cell culture medium was supplemented with IL-3 during the second week in 2 of the 5 experiments. Two-tailed 1 sample t test, hypothetical value is 1. The experiments highlighted with red dots were analyzed with regards to editing efficiency in panel E. (E) Editing efficiency of percentage of InDels and percentage of frameshift for CITED2 sgRNA1 of c-Kit^hi FcεRI⁺ mast cells and non-mast cells within individual samples sorted 12 days after electroporation. Two independent experiments were performed. (F) Representative flow cytometry plots showing the frequency of mast cells derived from sorted FcεRI^– progenitors and MCPs 12 days after electroporation with CITED2 sgRNA1. (G) The fraction of mast cells after electroporation and culture of sorted FcεRI^– progenitors and MCPs. The results show the frequency of c-Kit^hi FcεRI⁺ mast cells (among live singlets) in the CITED2 sgRNA1 sample normalized to the corresponding NC sgRNA sample. The plots show 3 independent experiments in which CITED2 was knocked out in sorted FcεRI^– progenitors and 2 independent experiments in which CITED2 was knocked out in sorted MCPs. Two-tailed 1 sample t test, hypothetical value is 1. (H) Editing efficiency shown as percentage of InDel and percentage of frameshift of CITED2 sgRNA1 in c-Kit^hi FcεRI⁺ mast cells derived from FcεRI^- progenitors (upper panel) and MCPs (lower panel). (I) Flow cytometry plots and histograms showing the survival and the proliferation of LAD2 and HMC-1 electroporated with NC sgRNA and CITED2 sgRNA1, respectively. Representative of 2 independent experiments. Control (gray) refers to all live cells analyzed after staining with CellTrace Far Red and incubating the cells in the fridge. Live single cells are shown. (J) Editing efficiency shown as percentage of InDel and percentage of frameshift in LAD2 and HMC-1 in 2 independent experiments. The NC sgRNA condition refers to nontargeting sgRNA. ns, nonsignificant.

This Feature Is Available To Subscribers Only