(a) Concordantly and discordantly regulated genes when comparing PLB-985 differentiation (wt and scr., d0 to d7) and human neutrophil differentiation (Blueprint, neutrophilic myelocyte to segmented neutrophil of bone marrow) using discordance scores and tmod. Most modules are concordantly regulated. (b) Volcano plot of all differentially expressed genes of control cells (wt and scr.) between d7and d0, darker gray depicts p<0.05 and logFC >2. A selection of strongly upregulated genes is labelled and neutrophil markers are highlighted in red. Expression of granule proteins, such as MPO, neutrophil elastase (ELANE), proteinase 3 (PRTN3) and cathepsin G (CTSG) decreases during differentiation. (c) Principal component analysis (PCA) of control and mutant PLB-985 during differentiation shows clustering according to experimental replicates and genotypes and separates samples according to differentiation state. Lines indicate the center-of-mass trajectory for ‘normal’ (wild type and scr. cells), ‘hyper’ (H1 clones with enhanced differentiation) and ‘hypo’ (H1 clones with reduced differentiation) samples. While lines deficient in H1.1, H1.3 and H1.5 already at d3 cluster closely to wt and scr. at d7, indicating faster and more efficient differentiation, H1.2 and H1.4-deficient clones at d7 still only cluster with wt and scr. at d3, indicating a stalled development.(d) Scatter dot plot depicting differentially expressed genes of H1.2 knockouts vs control cells at d0 of differentiation (x axis) and H1.4 knockouts vs control cells at d0 of differentiation (y axis), color (alpha) indicates p-value of differential expression between H1.2 and H1.4 at d0. Neutrophil genes such as neutrophil elastase (ELANE), proteinase 3 (PRTN3), cathepsin G (CTSG), azurocidin (AZU1) and MPO are strongly downregulated in both conditions when compared to wt and scr., whereas eosinophil genes such as galectin 10 (CLC), interleukin-5 receptor alpha (IL5RA), GATA-1 (GATA1), GATA-2 (GATA2) or eosinophil peroxidase (EPX) are upregulated. (e) Gene set enrichment analysis of indicated samples at d0 of differentiation shows enhanced expression of eosinophil and GATA-1 gene sets (see Materials and methods) in H1.2 and H1.4-deficient lines, but not in other knockout conditions.