Journal: Scientific reports

Article Title: Mechanism of KIT gene regulation by GATA1 lacking the N-terminal domain in Down syndrome-related myeloid disorders.

doi: 10.1038/s41598-022-25046-z

Figure Lengend Snippet: Figure 1. Overview of genome editing of GATA1 gene by CRISPR/Cas9 system. (a) Gene structure diagram of GATA1. The arrows on C1 and C2 indicate the target positions of the two guide RNAs. (b) Detection of GATA1 protein in K562-G1s clones obtained by GATA1 genome editing. Western blotting was performed with anti-GATA1 antibody (D24E4, Cell signaling technology), which could detect both full length GATA1 (45 kDa) and GATA1s (37 kDa) and monoclonal antibody against β-actin (Sigma-Aldrich). Images were obtained using ChemiDoc MP (Bio-Rad Laboratories). Mutations in all clones caused loss of full-length GATA1 production but allowed expression of GATA1s protein from the second translation initiation codon. (c) GSEA for the gene sets with differential expression between K562-WT cells and K562-G1s clones. NES, normalized enrichment score; NOM, nominal; FDR, false discovery rate. (d) Expression levels of four representative GATA1 target genes in six K562-WT single clones and nine K562-G1s clones by qRT-PCR. Data points were added with dots, and sample means were indicated by crosses. The expression levels of each gene are shown relative to the mean values of the K562-WT single clones, which is set at 1. The P-values of the Mann–Whitney U test were noted.

Article Snippet: Western blotting was performed with anti-GATA1 antibody (D24E4, Cell signaling technology), anti-GATA2 (AF2046, R&D) anti-c-Kit (SC-17806, Santa Cruz) and anti-β-actin antibody (Sigma-Aldrich).

Techniques: CRISPR, Clone Assay, Western Blot, Expressing, Quantitative Proteomics, Quantitative RT-PCR, MANN-WHITNEY

Journal: Scientific reports

Article Title: Mechanism of KIT gene regulation by GATA1 lacking the N-terminal domain in Down syndrome-related myeloid disorders.

doi: 10.1038/s41598-022-25046-z

Figure Lengend Snippet: Figure 2. Changes in KIT gene expression by GATA1 siRNAs. (a) qRT-PCR analysis was performed for K562-WT (left) and one of the K562-G1s clone, C1 #26 (right). The vertical axis represents a relative value with each value of non-target siRNA set at 1. Results are averages of three independent experiments. Error bars represent standard deviation. *P < 0.05, **P < 0.01 (two-side t-test). (b) Representative immunoblots of siRNA experiments with K562-WT (left) and G1s C1 #26 (right). Western blotting was performed with anti-GATA1 antibody (D24E4, Cell signaling technology), anti-GATA2 (AF2046, R&D) anti-c-Kit (SC-17806, Santa Cruz) and anti-β-actin antibody (Sigma-Aldrich). Images were obtained using ChemiDoc MP.

Article Snippet: Western blotting was performed with anti-GATA1 antibody (D24E4, Cell signaling technology), anti-GATA2 (AF2046, R&D) anti-c-Kit (SC-17806, Santa Cruz) and anti-β-actin antibody (Sigma-Aldrich).

Techniques: Gene Expression, Quantitative RT-PCR, Standard Deviation, Western Blot

Journal: Scientific reports

Article Title: Mechanism of KIT gene regulation by GATA1 lacking the N-terminal domain in Down syndrome-related myeloid disorders.

doi: 10.1038/s41598-022-25046-z

Figure Lengend Snippet: Figure 3. ChIP analysis of GATA1 and GATA2 in K562-WT and K562-G1s clones. (a) Overlap of peaks in GATA1 ChIP-seq of K562-WT and G1s C1 # 26. (b) Relative distribution of GATA1 ChIP-seq peak regions in the genome. (c) Results of de novo motif analysis of GATA1 ChIP-seq data. The top six motifs are shown. (d) Overlap of ChIP-seq peaks of GATA1 and GATA2 in K562-WT (left) and in G1s C1 #26 (right). Since both K562-WT and C1#26 had low yields of ChIP for GATA2, one library from each of the four experimental samples was prepared and sequenced. (e) Genome browser view at the KIT locus. The y axis shows read coverages normalized to RPGC (Read per genomic content). Light gray boxes indicate active distal regions (− 115 kb, − 109 kb, − 87 kb, − 72 kb and − 69 kb) marked with arrows, and the promoter region is highlighted in orange. The top shows profiles of K562-WT and the bottom shows profiles of the K562-G1s C1 #26. (f) Quantification of GATA1 and GATA2 binding upstream of KIT by ChIP-qPCR. (Left) data of GATA1 ChIP- qPCR, (right) data of GATA2 ChIP-qPCR. Results are averages of three independent experiments. Error bars represent standard deviation.

Article Snippet: Western blotting was performed with anti-GATA1 antibody (D24E4, Cell signaling technology), anti-GATA2 (AF2046, R&D) anti-c-Kit (SC-17806, Santa Cruz) and anti-β-actin antibody (Sigma-Aldrich).

Techniques: Clone Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, Standard Deviation

Journal: American Journal of Clinical Pathology

Article Title: Loss of Full-Length GATA1 Expression in Megakaryocytes Is a Sensitive and Specific Immunohistochemical Marker for the Diagnosis of Myeloid Proliferative Disorder Related to Down Syndrome

doi: 10.1093/ajcp/aqy001

Figure Lengend Snippet: Schematic diagrams illustrating the relationship between GATA1 mutations, resultant N-terminally truncated GATA1 (GATA1s), and the monoclonal antibody used for detection. A, Nonsense mutations in exons 2 and 3 force the use of an alternative start codon in exon 3 and results in GATA1s. B, A monoclonal antibody (D52H6) is raised against a peptide centered on amino acid residue 13 within the portion of full-length GATA1 (GATA1f) that is not present in GATA1s. A second monoclonal antibody (D24E4) is raised against a peptide centered on amino acid residue 184 of GATA1.

Article Snippet: Antibodies Two commercially available rabbit monoclonal antibodies (mAb) against GATA1 were purchased from Cell Signaling Technology (Danvers, MA): (1) mAb D52H6 (3535) is raised against a peptide centered on Glu13 of GATA1, and (2) mAb D24E4 (4589) is raised against a peptide centered on Ala184 of GATA1.

Techniques: Residue

Journal: American Journal of Clinical Pathology

Article Title: Loss of Full-Length GATA1 Expression in Megakaryocytes Is a Sensitive and Specific Immunohistochemical Marker for the Diagnosis of Myeloid Proliferative Disorder Related to Down Syndrome

doi: 10.1093/ajcp/aqy001

Figure Lengend Snippet: A Western blot with lysates from erythroleukemic cell lines (K562 and HEL) shows D52H6 recognizes full-length GATA1 (GATA1f) only, and D24E4 recognizes both forms. β-Actin reactivity serves as a protein loading control.

Article Snippet: Antibodies Two commercially available rabbit monoclonal antibodies (mAb) against GATA1 were purchased from Cell Signaling Technology (Danvers, MA): (1) mAb D52H6 (3535) is raised against a peptide centered on Glu13 of GATA1, and (2) mAb D24E4 (4589) is raised against a peptide centered on Ala184 of GATA1.

Techniques: Western Blot, Control

Journal: American Journal of Clinical Pathology

Article Title: Loss of Full-Length GATA1 Expression in Megakaryocytes Is a Sensitive and Specific Immunohistochemical Marker for the Diagnosis of Myeloid Proliferative Disorder Related to Down Syndrome

doi: 10.1093/ajcp/aqy001

Figure Lengend Snippet: Detection of truncated and full-length forms of GATA1 (using mAb D24E4) in myeloid proliferative disorders associated with Down syndrome (MPD-DS) and non–Down syndrome (DS) cases. Strong nuclear reactivity is detected erythroid and megakaryocytic elements in normal marrow (A, ×100) and neoplastic blasts of acute megakaryocytic leukemia non-DS (B, ×100) in a similar pattern as mAb D52H6. However, in contrast to mAb D52H6, the neoplastic megakaryocytes (arrows) in transient abnormal myelopoiesis (C, ×100) and myeloid leukemia associated with Down syndrome (ML-DS) (D, ×100), as well as neoplastic blasts in ML-DS (E, ×100), retained robust nuclear reactivity, consistent with expression of truncated GATA1 proteins.

Article Snippet: Antibodies Two commercially available rabbit monoclonal antibodies (mAb) against GATA1 were purchased from Cell Signaling Technology (Danvers, MA): (1) mAb D52H6 (3535) is raised against a peptide centered on Glu13 of GATA1, and (2) mAb D24E4 (4589) is raised against a peptide centered on Ala184 of GATA1.

Techniques: Expressing