Fig 2: Western blotting validation of crucial targets of SCU against HCC. (A) Expression of crucial targets as histone H1-4 (H1-4) and PTPRC on SCU-treated HepG2 cells for 48 h. Graphical representations of protein expression were shown and the proteins were normalized with an internal control β-actin. (B) Results obtained from three independent experiments were expressed as mean ± standard deviation (SD) compared with the control group. ***P<0.001. PTPRC, protein tyrosine phosphatase receptor type C; SCU, scutellarein; HCC, hepatocellular carcinoma; H1-4, histone H1-4.

Fig 3: Deficiency of H1.2 or H1.4 is rescued by inhibition of GATA-2.(a–c) Deficiency in GATA-2 but not GATA-1 rescues H1.2 and H1.4 deficiency. Populations are batches of PLB-985. scr. (a), H1.4 (b) and H1.2 (c) knockout lines were transduced with an sgRNA against GATA1 or GATA2 or with a scr. sgRNA as control and analyzed for ROS production in response to PMA at d4 of differentiation. (d) Deficiency in GATA-2 rescues CD11b expression of H1.2 and H1.4-deficient PLB-985. CD11b expression of the indicated genotypes was measured at d4 of differentiation. (e) Treatment of PLB-985 with GATA-2 but not GATA-1 inhibitor rescues expression of CD11b, especially in lines deficient in H1.2 and H1.4. (f–k) ROS production of PLB-985 in response to PMA is affected upon GATA-2, but not GATA-1 inhibition during differentiation. (f–k) PLB-985 ROS production was measured at d7 of differentiation, indicated genotypes were treated with indicated inhibitors or solvent before the onset of differentiation and at d4. (a–k) Shown is the mean -/+ SEM of 3 independent experiments.

Fig 4: H1 effects on PLB-985 differentiation are subtype-specific.(a, b) Expression of the surface marker CD11b at d3 (a) or d7 (b) of differentiation. Depicted is the percentage of CD11b-positive cells out of all living singlets for wt, scrambled (scr.) and two knockout clones per H1 subtype. (c, d) Growth curves of PLB-985 during differentiation (c) and cell counts at d7 of differentiation (d) show restricted growth of H1.1, H1.3 and H1.5-deficient cells and enhanced growth of H1.2 and H1.4-deficient clones. (a–d) Depicted are five independent experiments, error bars are SEM. (e, f) Viability, as determined by DAPI negative cells, at d8 (e) and d12 (f) of differentiation of 3 independent experiments. Error bars are SEM.

Fig 5: RNA-seq reveals H1 subtype-specific effects on differentiation and an eosinophilic signature of undifferentiated H1.2/H1.4 knockout lines.(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.Figure 4—source data 1.RNA-seq expression tables.The individual sheets display expression values of indicated conditions (Cont. is wild type and scr. combined, for the respective H1 subtypes values of 2 clones are combined).