Deregulated signaling pathways downstream of COPZ1 mutations. (A) Volcano plot of negative log10 transformed adjusted (adj.) P values against log2 fold change (log2FC) values, showing DEGs between COPZ1-TR vs AAVS1 CTRL HSPCs. Red dots represent the DEGs with log2FC >1 or less than −1 and adj. P value <.05. Genes with −1 <log2FC <1 and adj. P value <.05 are shown in blue; genes with log2FC >1 or less than −1 and adj. P value >.05 are shown as green dots; gray dots are genes with −1 < log2FC <1 and adj. P value >.05. Horizontal dashed line marks gene expression with adj. P value of .05; vertical dashed lines represent log2FC of 1 (right line) and log2FC of −1 (left line). The names of the 20 top DEGs are displayed. (B-E) Supervised clustering of significantly upregulated or downregulated genes (adj. P < .05; |log2FC| > 1 [B,E] or adj. P < .05; |log2FC| > .5 [C-D]) from “Hallmark Interferon Alpha Response” (B), “JAK/STAT Signaling Pathway” (C), “Hallmark Hypoxia” (D), and “Hallmark Oxidative Phosphorylation” (E) gene sets of gene set enrichment analysis (GSEA). Gene expression heat maps were created using regularized logarithm transformation of read counts as an input. (F) mRNA expression of JAK/STAT signaling-related genes CSF3R, PIM1, and CEBPE. The data are normalized to ACTB and COPZ1-TR group is compared with AAVS1 control-edited group using the 2^–ΔΔCt method. Data are plotted as means (large symbols) from 3 independent experiments (color coded per experiment) performed in technical triplicates (small symbols). Statistical significance: ∗∗P < .01. (G) Mean fluorescence intensity (MFI) of surface G-CSF3R expression in COPZ1-TR and AAVS1 control-edited cells, measured by flow cytometry at day 4 of LCD. Data are presented as the mean ± standard deviation (SD) from 3 independent experiments, with each experiment color coded accordingly. Statistical significance: ∗P < .05. (H) mRNA expression of PDK4 and the hypoxia-related genes ALDOC and BNIP3 in 2 COPZ1-TR HSPCs samples at day 4 of LCD. The data are normalized to ACTB and compared with the specific gene expression in AAVS1 control-edited cells using the 2^–ΔΔCt method. (I) Supervised clustering of DEGs (|log2FC|>1 and adj. P < .05) in COPZ1-MS HSPCs compared with COPZ1-WT HSPCs. (J) Pathway analysis of COPZ1-MS vs COPZ1-WT RNA-seq data using GSEA (preranked fgsea) with gene sets from GO Biological Process in iDEP v96. The figure displays top upregulated and downregulated gene sets. Normalized enrichment score (NES) of the gene sets are plotted and bar colors represent adj. P values. GO, gene ontology.