Fig 2: Sp-1 cooperates with CHIR to sustain mESC self-renewal and pluripotency.A, AP staining in 46C mESCs cultured with serum-containing medium supplemented with the indicated molecules for 8 days. CS, CHIR plus Sp-1. Scale bar, 100 µM. B, immunostaining analysis of Oct4 expression in mESCs treated with or without CS for five passages. Scale bar, 100 µM. C, qRT–PCR analysis of Oct4, Nanog, Klf4, and Tfcp2l1 expression levels in mESCs maintained in serum-containing medium supplemented with or without CS. Data represent mean ± SD (N = 3 biological replicates). *p < 0.05, **p < 0.01 versus CHIR. D, immunostaining analysis of Gata6, Myosin, and Tuj1 expression in EB-derived cells. Scale bar, 100 µM. E, immunostaining of female mESCs and mEpiSCs for H3K27me3 and Oct4. mEpiSCs exhibit a nuclear body indicative of the inactive X chromosome. Scale bar, 100 µM. F, generation of chimeric mice after the injection of blastocysts with CS-treated C57BL/6 mESCs.

Fig 3: Bioinformatics analyses of GATA6-associated genes. (A) Relative gene expression analysis of GATA6 in head and neck squamous cell carcinoma was conducted through LinkedOmics (https://www.linkedomics.org/login.php/) using HiSeq RNA Platform (17). (B) Gene enrichment analysis was conducted by WebGestalt (http://www.webgestalt.org/) using Gene Set Enrichment Analysis tool and Kyoto Encyclopedia of Genes and Genomes functional database (FDR <0.25, adjusted P-value <0.05). And association between GATA6 and ECM-receptors revealed the highest association between FN1 and GATA6. GATA6, GATA binding protein 6; ECM, extracellular matrix; FN1, fibronectin 1.

Fig 4: Single-cell transcriptional analysis of GATA6 mutant hiPSC-CMs during differentiation.(A) tSNE of single-cell RNA-Seq of day 4 GATA6 mutant hiPSC-CMs identified four clusters. Labels reflect marker gene expression: SC, Stem cell; CPC, cardiac progenitor cells; E-CPC, cardiac progenitors enriched with endoderm markers; ECM, endodermal-like cells enriched for extracellular matrix proteins. (B) Examples of marker gene expression in clustered hiPSC-derived cells. CPC cells express mesodermal factors (MESP1, EOMES) as well as cardiac transcription factors (GATA6, GATA4, ISL1, NKX2.5, HAND1, and HAND2). SCs expressed OCT4 (POU5F1). E-CPCs expressed GATA6, SOX17, HNF4A, and FOXA2. (C) tSNE clustering of single-cell RNA-Seq of day 8 GATA6 mutant hiPSC-CMs identified five clusters. Labels reflect marker gene expression: CM, cardiomyocytes; CM (‘RA’), cardiomyocytes with increased RA-signaling pathway genes; SC, Stem Cell; CM (‘Endo’), cardiomyocytes with enrichment in endothelial genes; FB; fibroblast-like cells (D) Examples of marker gene expression in clustered cells. SC expressed OCT4 (POU5F1). CMs expressed sarcomere protein genes (MYH6, MYH7), SMYD1, a CM-specific histone methyl-transferase, and HAND2, a second heart field transcription factor. CMs (RA) also expressed retinoic acid pathway genes (SOX6, HOXB1). CMs (Endo) have upregulated endothelial cell gene expression (MEF2C, BMPER), while FB cells expressed ECM markers (IGFBP7).

Fig 5: GATA6 is highly expressed in OSCC tissues and cells. (A) TNMplot data was employed to analyze the expression of GATA6 in OSCC tissues and adjacent tissues. (B) The GSE dataset GSE146483 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146483) consisting of 11 samples (8 OSCC and 3 human normal oral mucosal epithelial keratinocytes) was downloaded from the GEO database and analyzed using GEO2R. GATA6 expression was detected in HOK and OSCC cell lines using (C) western blot analysis and (D) reverse transcription-quantitative PCR. **P<0.01, ***P<0.001 vs. HOK. GATA6, GATA binding protein 6; OSCC, oral squamous cell carcinoma.