Figure 6.
Restoration of defective granulopoiesis in COPZ1 mutated HSPCs by COPZ2 overexpression or HIF1α stabilizer IOX2. (A) Schematic of the in vitro COPZ2 rescue experiment in COPZ1-TR CD34+ cord blood (CB)-HSPCs. Created with BioRender.com. (B) Cell number for mature neutrophils (CD45+CD15+CD66b+CD11b+CD16+) derived from 5 healthy donors on day 14 of LCD, after introduction of COPZ1-TR or AAVS1 edited control, and overexpression of an RFP empty vector, COPZ1 WT, or COPZ2 WT, as assessed by flow cytometry. Data shown as means of total cell counts (large symbols) from 5 donors (color coded), with each donor's experiment performed in technical duplicates (small symbols). Statistical significance: ∗∗P < .01; ns, not significant. (C) Representative May-Grünwald-Giemsa–stained images of indicated samples on day 14 of LCD; magnification ×60. (D) CFU assay of COPZ1-TR and AAVS1 control-edited CD34+ CB-HSPCs, transduced with lentiviral particles containing an RFP empty vector, COPZ1 WTRFP, or COPZ2 WTRFP (n = 3; color coded), on day 14 of LCD. CFU types included granulocytes, erythrocytes, monocytes, megakaryocytes (GEMM); granulocytes, monocytes (GM); granulocytes (G); monocytes (M); and burst-forming unit erythroid (BFU-E). Data represent means (large symbols) from 3 independent experiments in duplicates (small symbols). Statistical significance ∗P < .05. (E) Cell counts of CD45+CD15+CD66b+CD11b+CD16+ neutrophils of COPZ1-TR and AAVS1 edited control CD34+ CB-HSPCs treated with 10 μM IOX2 or DMSO. Cell counts are derived from 3 donors on day 14 of LCD, as assessed by flow cytometry. Data show the mean counts from every experiment (large symbols), performed in technical duplicates (small symbols). Statistical significance, ∗∗P < .01; ∗∗∗P < .001. (F) Representative May-Grünwald-Giemsa–stained images of indicated samples on day 14 of LCD. Original magnification ×60. (G-H) CFU assay of HSPCs treated with DMSO or IOX2 (10 μM). (G) COPZ1-TR and AAVS1 control-edited CD34+ CB-HSPCs (n = 3, in technical duplicates). (H) COPZ1-MS and WT control CD34+ CB-HSPCs (n = 3, in technical duplicates). (I-J) Quantification of mpo:gfp+ neutrophils after treatment with DMSO (control) or 10 μM IOX2 in wild-type and Copz1-TR zebrafish embryos (I) and wild-type and hax1 morpholino-induced KD zebrafish embryos (J). Each of the small dots represents the number of cells of an individual embryo. Each big dot represents the mean of 1 of 3 independent experiments. (#) represents the number of stained cells. Statistical significance, ∗∗∗∗P < .0001.

Restoration of defective granulopoiesis in COPZ1 mutated HSPCs by COPZ2 overexpression or HIF1α stabilizer IOX2. (A) Schematic of the in vitro COPZ2 rescue experiment in COPZ1-TR CD34+ cord blood (CB)-HSPCs. Created with BioRender.com. (B) Cell number for mature neutrophils (CD45+CD15+CD66b+CD11b+CD16+) derived from 5 healthy donors on day 14 of LCD, after introduction of COPZ1-TR or AAVS1 edited control, and overexpression of an RFP empty vector, COPZ1 WT, or COPZ2 WT, as assessed by flow cytometry. Data shown as means of total cell counts (large symbols) from 5 donors (color coded), with each donor's experiment performed in technical duplicates (small symbols). Statistical significance: ∗∗P < .01; ns, not significant. (C) Representative May-Grünwald-Giemsa–stained images of indicated samples on day 14 of LCD; magnification ×60. (D) CFU assay of COPZ1-TR and AAVS1 control-edited CD34+ CB-HSPCs, transduced with lentiviral particles containing an RFP empty vector, COPZ1 WTRFP, or COPZ2 WTRFP (n = 3; color coded), on day 14 of LCD. CFU types included granulocytes, erythrocytes, monocytes, megakaryocytes (GEMM); granulocytes, monocytes (GM); granulocytes (G); monocytes (M); and burst-forming unit erythroid (BFU-E). Data represent means (large symbols) from 3 independent experiments in duplicates (small symbols). Statistical significance ∗P < .05. (E) Cell counts of CD45+CD15+CD66b+CD11b+CD16+ neutrophils of COPZ1-TR and AAVS1 edited control CD34+ CB-HSPCs treated with 10 μM IOX2 or DMSO. Cell counts are derived from 3 donors on day 14 of LCD, as assessed by flow cytometry. Data show the mean counts from every experiment (large symbols), performed in technical duplicates (small symbols). Statistical significance, ∗∗P < .01; ∗∗∗P < .001. (F) Representative May-Grünwald-Giemsa–stained images of indicated samples on day 14 of LCD. Original magnification ×60. (G-H) CFU assay of HSPCs treated with DMSO or IOX2 (10 μM). (G) COPZ1-TR and AAVS1 control-edited CD34+ CB-HSPCs (n = 3, in technical duplicates). (H) COPZ1-MS and WT control CD34+ CB-HSPCs (n = 3, in technical duplicates). (I-J) Quantification of mpo:gfp+ neutrophils after treatment with DMSO (control) or 10 μM IOX2 in wild-type and Copz1-TR zebrafish embryos (I) and wild-type and hax1 morpholino-induced KD zebrafish embryos (J). Each of the small dots represents the number of cells of an individual embryo. Each big dot represents the mean of 1 of 3 independent experiments. (#) represents the number of stained cells. Statistical significance, ∗∗∗∗P < .0001.

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