Journal: PLOS One

Article Title: GATA4-targeted compounds induce apoptosis and diminish viability of hepatoblastoma cells

doi: 10.1371/journal.pone.0342565

Figure Lengend Snippet: COS-1 cells were transfected for 6 hours after which the small molecules were added in culture media for 24 hours. (a) The cells transfected with GATA4 binding sites containing reporter plasmid together with GATA4 expression plasmid or (b) three NKX2-5 binding sites containing reporter together with both GATA4 and NKX2-5 expression plasmids (G + N). Empty plasmid (pMT2) was used as control. The cell toxicity of GATA4 targeting compounds was assayed by MTT assay in COS-1 (c) and hiPSC (d) cells after 24 h incubation. The Fig shows the average, + SEM and the result of each independent experiments, n ≥ 3, except for 3i-2014 at panel B n = 2. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. (a) GATA4 or (b) G + N (one-way ANOVA followed by the Tukey test or Welch ANOVA followed by the Games-Howell test).

Article Snippet: Membranes were incubated with anti-GATA4 antibody in 1:1000 dilution in 1% milk-TBST (sc-9053, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at +4◦C for overnight and goat anti-rabbit IgG secondary antibody in 1:10,000 dilution in 5% milk-TBST (#711-035-152, Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 1 h. Protein bands were detected utilizing Enhanced Chemiluminescence detection kit (RPN2232, Amersham, GE Healthcare, Barrington, IL, USA) and analyzed with Image Lab Software 6.0 (Bio-rad).

Techniques: Transfection, Binding Assay, Plasmid Preparation, Expressing, Control, MTT Assay, Incubation

Journal: EMBO Reports

Article Title: Nanog mediated control of TBX3-GATA6 circuitry in primitive endoderm differentiation of mESCs

doi: 10.1038/s44319-026-00707-6

Figure Lengend Snippet: ( A ) qPCR analysis for transcript levels of the Gata4 and Sox17 in RCNβH (Ctrl), RCNβHt ( Nanog −/− ), and RCNβH-Gata6 −/− ESCs in the absence ( Gata6 −/− ), and presence ( Nanog/Gata6 dKO) of 4-OHT over a 4-day period. Data are presented as mean ± SD ( n = 3, biological replicates) Gata4 , P = 5.49 × 10 −6 (Row 1), P = 4.22 × 10 −6 (Row 2); Sox17 , P = 1.05 × 10 −5 (Row 1), P = 1.05 × 10 −5 (Row 2). ( B ) qPCR analysis for transcript levels of the Gata4 and Sox17 in Ctrl (RCNβH), Nanog −/− (RCNβHt), Nanog/Gata6 dKO, and Nanog/Gata6 dKO with exo- Gata6 ESCs. Data are presented as mean ± SD ( n = 3, biological replicates) Gata4 , P = 6.25 × 10 −6 (Row 2); Sox17 , P = 5.21 × 10 −5 (Row 1), P = 3.90 × 10 −6 (Row 2). ( C ) GATA6 levels at Gata6 promoter and enhancer regions in RCNβH (Ctrl.) and RCNβHt ( Nanog −/− ) ESCs, determined by ChIP-qPCR. Data are presented as mean ± SD ( n = 3, biological replicates). ( D ) qPCR analysis of transcript levels for Gata6 in Gata6 −/− (RCNβH-Gata6 −/− ), Nanog/Gata6 dKO (RCNβH-Gata6 −/− t), and dKO , exo-Gata6 (RCNβH-Gata6 −/− t with exogenous Gata6 expression) ESCs. Primers were designed based on coding, 5’-UTR, and 3’-UTR regions of the Gata6 gene. Data are presented as mean ± SD ( n = 3, biological replicates). ( E ) Confirmation of the interaction between Flag-TBX3 and GATA6 through Co-Immunoprecipitation (Co-IP) followed by western blot analysis. Lysates from RCNβH (Ctrl.) or RCNβHt ( Nanog −/− ) cells were used for IP with anti-Flag, while western blot analysis using anti-GATA6 and anti-GATA4. Anti-IgG served as control. ( F , G ) ChIP-qPCR analysis of GATA6 ( F ) and H3K27ac ( G ) levels at the promoter and enhancer regions of the Gata6, Gata4 , and Sox17 genes in RCNβH (Ctrl), RCNβHt ( Nanog −/− ), and RCNβH-Tbx3 −/− t ( Nanog/Tbx3 dKO) ESCs; Data are presented as mean ± SD ( n = 3, biological replicates) ( F ) Sox17 -DE, P = 4.55 × 10 −5 (Row 1); ( G ) Gata6 , P = 8.07 × 10 −5 (Row 1), P = 3.73 × 10 −5 (Row 2). ( H ) Dotplot showing indicated gene expression levels in Nanog low, middle and high clusters in E3.5 embryos. ( I ) Dotplot showing indicated gene expression levels in Nanog low and high clusters in E4.5 embryos. ( J ) A model for the regulation of PrE-differentiation by Nanog . The unpaired two-tailed Student’s t test was used for the statistical analysis in ( A – D , F , G ). Source data are available online for this figure. .

Article Snippet: Anti-GATA4 , Santa Cruz , SC25310.

Techniques: ChIP-qPCR, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Control, Gene Expression, Two Tailed Test