Fig 1: GATA4 binds to the conserved GATA-binding sites on the HAND2 gene. (A) Enhancer region of the HAND2 gene contains two conserved GATA-binding sites (underlined sequence, transcription factor binding sequence). (B) EMSA results revealed that the MU GATA4 protein exhibited decreased DNA-binding affinity. Lane 1, labeled probe; lane 2, protein from HeLa cells transfected with GATA4 (WT) + labeled probe; lane 3, protein from HeLa cells transfected with GATA4 (WT) + unlabeled competitor probe + labeled probe; lane 4, protein from HeLa cells transfected with GATA4 (MU) + labeled probe; lane 5, protein from HeLa cells transfected with GATA4 (WT) + labeled probe + anti-GATA4. The specific DNA/protein complexes are indicated by arrows. A supershift, indicated by the red arrow, revealed that the GATA4 antibody could specifically bind with whole cell lysate, which was transfected with the GATA4 plasmid. EMSA, electrophoretic mobility shift assay; GATA4, GATA-binding factor 4; HAND2, heart and neural crest derivatives expressed 2; MU, mutant; WT, wild-type.
Fig 2: Ablation of Mps1 in gonocytes and spermatogonia led to the depletion of germ cells.a qPCR analysis of Mps1 expression level of liver, kidney, spleen, brain, and testis of wild-type mice at 2 month of age. n = 3; Student’s t test; **P < 0.01, ***P < 0.001. b qPCR analysis of Mps1 expression level of wildtype mouse testes from P3 to P60. n = 3; Student’s t test; n.s. no significance, *P < 0.05, **P < 0.01. c Images of control and Mps1dKO testes at P0, at P10, and during adulthood. Scale bar = 1 mm. d H&E staining of control and Mps1dKO testes at P0, at P10, and during adulthood. Scale bar = 50 µm. e Immunofluorescence staining of DDX4 and GATA4 in control and Mps1dKO testes at P0. Scale bar = 50 µm. f Immunofluorescence staining of DDX4 and GATA4 in control and Mps1dKO testes at P10. Scale bar = 50 µm.
Fig 3: Cataloguing in utero epithelial maturation and crypt development(A) UMAP plot visualizing epithelial compartment populations (i) and epithelial cell distribution based on location (ii) and developmental time course (iii).(B) Dot plot of epithelial cluster markers, with color indicating average expression within cluster and dot size indicating percentage of cells within cluster expression the gene.(C) Selected population abundance changes over developmental time course shown as bar plots. Wilcox rank test, p-value < 0.05 ∗; p-value < 0.01 ∗∗; p-value < 0.001∗∗∗; n.s = not significant. For location-specific clusters, only location-matched samples were considered. Error bars represent standard error of the mean (SEM).(D) Violin plots showing expression of selected time-course varying genes in distal and proximal stem cells.(E) UMAP overlay visualizing expression of GATA4 in epithelial cells (i) and representative images of SI sections from 10, 17 and 22PCW embryonic tissue stained for GATA4 by immunohistochemistry (IHC) (n = 3 for each individual image shown repeated on samples +-1pcw PCW, 10x/20x magnification scale bar=360/180 μm) (ii)(F) Representative images of SI sections from 10 and 17 PCW embryonic tissue stained for Transferrin (TF) by IHC (n = 3 on samples +-1pcw to example image, 20x/100x magnification scale bar=180/40μm)(G) Representative images of colonic sections from 10, 12 and 17 PCW embryonic tissue and adult colonic tissue stained for LGR5 expression by single molecule in-situ hybridization (sm-ISH) (n=3 on samples +-1pcw in fetal images, 20x/100x magnification scale bar=180/40μm)(H) UMAP overlay of ASCL2 TF module AUC score over developmental time course in epithelial cells.(I) Representative images of colonic sections from 10 and 15 PCW embryonic tissue stained for BEST4 by IHC (n=3 on samples +-1pcw to example image, 20x/40x magnification scale bar=180/90μm respectively).
Fig 4: Subcellular localization of GATA4, as determined by immunofluorescence in HeLa cells. For each construct, anti-FLAG (red) and DAPI (blue) staining was presented individually and merged. WT and MUT GATA4 were localized exclusively to the nuclei with normal nuclear distribution. GATA4, GATA-binding factor 4; MUT, mutant; WT, wild-type.
Fig 5: Expression of WT and MUT GATA4 in HeLa cells. (A) Reverse transcription-quantitative PCR results revealed that the mRNA expression levels of GATA4 were decreased in the mutant group when compared with the wild-type group. ****P<0.0001 vs. GATA4-WT. (B) Western blot analysis detected the protein expression levels of GATA4 in the MUT and WT groups. Anti-GATA4 antibody was used as the primary antibody and anti-GAPDH was used as the internal control. The c.1306C>T mutation significantly reduced GATA4 protein expression. GATA4, GATA-binding factor 4; MUT, mutant; WT, wild-type.
Supplier Page from Abcam for Anti-GATA4 antibody