Fig 2: GATA1 Is a Potentially Interacted Protein with FAM122A and the Gene Regulation Profiling upon FAM122A Knockout(A and B) The potential interacted proteins with FAM122A were analyzed by in vitro binding assay and MS analysis. Schematics showed the process of the experiments (A). Coomassie brilliant blue-stained gel of proteins bound to GST and GST-FAM122A are shown, followed by identification with tandem mass spectrometry (B). The indicated bands referred to GST-FAM122A, GATA1, and the known FAM122A interacting proteins, PP2A-Aα, -Bα, and -Cα (left panel). The indicated proteins were analyzed by scaffold 4 proteome software and their information is presented (right panel). Peptides and coverage indicate the numbers of unique peptides and the percentage of sequence coverage for identified proteins.(C and D) FAM122A KO and NC K562 cells were subjected to RNA sequencing and bioinformatics analysis. Differentially expressed genes were analyzed by gene ontology (C), and the most upregulated genes in FAM122A KO K562 cells were enriched as indicated in molecular functions, cellular components and biological processes. Heatmap presents the enriched candidate and upregulated genes involved in erythroid maturation and function as indicated with red words (D).(E) The representative candidate genes regulated by FAM122A were confirmed by qPCR (n = 3). Data indicate means with bar as SD in an independent experiment.

Fig 3: GATA1 Mediates FAM122A Deletion-Enhanced Erythroid DifferentiationK562 cells with stably expressing shNC and shFAM122A were further infected with lentiviruses expressing two pairs of GATA1 shRNAs, together with control shRNA as controls (shcontrol). The indicated cells were treated with or without 50 μM hemin for 48 h.(A) The specific knockdown effect of GATA1 was confirmed by western blot (n = 3).(B) HbG protein expression as an indicator for erythroid differentiation was examined by western blot (left panel) and its relative expression level was calculated by normalizing against shNC/shcontrol cells (right panel) (n = 3).(C) Representative images of benzidine staining are shown in the indicated cells (upper panel) and the percentages of DAB+ cells were calculated and shown by observing five random fields (bottom panel) (n = 3).(D) Expressions of various globin genes were examined by qPCR. Data were normalized as relative fold changes against shNC/shcontrol cells without hemin treatment and displayed as means ± SD in an independent experiment (n = 3).

Fig 4: RAP‐536 up‐regulates Gata1 and its target gene signature in erythroid precursors of Hbbth3/+ β‐thalassaemic mice. A, Heat map for RNA sequencing analysis of sorted erythroid precursors from Hbbth3/+ mice depicting differentially regulated genes 16 h after treatment with a single dose of RAP‐536 (30 mg/kg, i.p) or vehicle. B, Main transcription factors up‐regulated in erythroid precursors by RAP‐536 treatment. FDR—false discovery rate. C, Gene set enrichment analysis to identify those differentially regulated in erythroid precursors by RAP‐536 treatment, showing plots for GATA‐1, HSF and NFE2. Bar graph indicates pathways that were significantly activated by GATA‐1 in erythroid precursors by RAP‐536 treatment. D, Representative heat map with each column representing genes up‐regulated (red) or down‐regulated (green) in erythroid precursors of an individual mouse by treatment with RAP‐536 or vehicle (VEH). E, Pathways affected by RAP‐536 treatment and some key genes involved. F, Linkage analysis model depicting RAP‐536‐induced changes in gene expression (all up‐regulated) in erythroid precursors

Fig 5: Schematic model for CPM action in regulating megakaryopoiesis and thrombopoiesis. CPM induces IL-6 and IL1R1 expressions, causing the activation of PI3K/Akt and MEK/ERK (MAPK) signaling pathways, followed by the activation of downstream hematopoietic TFs, including GATA1, TAL1, PBX1 and EGR1, which stimulate MK differentiation and platelet production.