Journal: Advanced Science

Article Title: The TET3/GATA6 Axis Drives Lipid Metabolism and Therapeutic Vulnerabilities in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202501774

Figure Lengend Snippet: TET3 transcriptionally represses GATA6 through histone deacetylation. A) Western blot analysis of GATA6 protein levels in wild‐type (WT) and TET3 knockout (KO) PANC‐1 cells. B) UMAP visualization showing the expression distribution of TET3 and GATA6 in epithelial cells from scRNA‐seq of 25 PDAC patients ( GSE242230 ). C) Violin plots showing the expression levels of GATA6 in type 1 and type 2 ductal cells previously identified in PDAC patients (CRA001160; n = 24). D) RT‐qPCR analysis of GATA6 mRNA in TET3 knockout PANC‐1 cells transduced with doxycycline‐inducible wild‐type TET3 (TET3 wt , unfilled bars) or catalytically inactive mutant TET3 (TET3 mut , striped bars), treated with (purple) or without (gray) doxycycline (1 µg mL −1 ) (n = 3). E) ChIP‐qPCR assay of H3K27ac levels in WT and KO PANC‐1 cells (n = 3). Primers targeted regions upstream or downstream of the GATA6 transcription start site, as indicated in Figure , Supporting Information. F) RT‐qPCR measuring GATA6 mRNA expression in wild‐type PANC‐1 cells treated with SAHA at 0, 5, or 10 µM for 24 or 48 h (n = 3). G) Western blot analysis of GATA6 protein levels in PANC‐1 cells treated with SAHA (0, 5, 10 µM) for 24 h. H) ChIP‐qPCR assay of V5 in PANC‐1 cells ectopically expressing V5‐tagged TET3 (n = 3). qPCR primers are the same as those used in (E). I) Immunoprecipitation of V5‐tagged TET3 in PANC‐1 cells, followed by immunoblotting for HDAC1 and HDAC2 using an anti‐V5 antibody. Data represent mean ± SD. Statistical significance was determined by two‐tailed Wilcoxon test (C), one‐way ANOVA (D, F) or two‐tailed unpaired t ‐test (E, H). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: GATA6 expression vector (Addgene, #120445) was packaged into a lentivirus transduction system and used to infect cancer cells.

Techniques: Western Blot, Knock-Out, Expressing, Quantitative RT-PCR, Transduction, Mutagenesis, ChIP-qPCR, Immunoprecipitation, Two Tailed Test

Journal: Advanced Science

Article Title: The TET3/GATA6 Axis Drives Lipid Metabolism and Therapeutic Vulnerabilities in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202501774

Figure Lengend Snippet: GATA6 suppresses lipogenic gene expression and limits tumor growth. A) Western blot showing knockout efficiency of GATA6 in TET3 knockout (KO) PANC‐1 cells. B) RT‐qPCR analysis of lipid metabolic gene expression in PANC‐1 cells with wild‐type (WT), KO, and TET3 / GATA6 double knockout (KO‐sgGATA6) (n = 3). C) Western blot showing overexpression efficiency of GATA6 (GATA6 OE ) in wild‐type PANC‐1 cells. D) RT‐qPCR analysis of lipid metabolic gene expression in PANC‐1 cells with constitutive GATA6 overexpression (GATA6 OE ) (n = 3). E) Representative images of subcutaneous xenograft tumors (left) and tumor weight at 8 weeks post‐implantation (right) for the indicated cell lines. Nude mice were transplanted with WT (n = 5), KO (n = 5), or KO‐sgGATA6 PANC‐1 cells (n = 5). F) Cell viability of wild‐type PANC‐1 cells treated for 24 or 48 h with gemcitabine (1 µM), gemcitabine + SAHA (5 µM), gemcitabine + Erastin (1 µM), or a triple combination of gemcitabine, SAHA, and Erastin (n = 3). G) Representative images of xenografts (left) and tumor weight at 7 weeks post‐implantation (right) following treatment beginning at week 4 with the indicated agents. Nude mice were transplanted with wild‐type PANC‐1 cells. Data represent mean ± SD. Statistical significance was determined by one‐way ANOVA (B, E, F, G) or two‐tailed unpaired t ‐test (D). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: GATA6 expression vector (Addgene, #120445) was packaged into a lentivirus transduction system and used to infect cancer cells.

Techniques: Gene Expression, Western Blot, Knock-Out, Quantitative RT-PCR, Double Knockout, Over Expression, Two Tailed Test

Journal: Advanced Science

Article Title: The TET3/GATA6 Axis Drives Lipid Metabolism and Therapeutic Vulnerabilities in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202501774

Figure Lengend Snippet: TET3 promotes invasive PDAC through activation of TGF‐β signaling pathway. A) mRNA expression levels of TET3 in normal pancreatic tissues (n = 7), IPMA tissues (n = 6), and invasive PDAC tissues (n = 3) from GSE19650 (n = 16). B) Representative images and quantification of transwell invasion and migration assays in wild‐type (WT) and TET3 knockout (KO) PANC‐1 cells (n = 3). Scale bars = 200 µm. C) Western blot analysis of epithelial‐mesenchymal transition (EMT) markers E‐cadherin, N‐cadherin, and vimentin in WT and KO PANC‐1 cells. D) Western blot analysis of TGF‐β pathway proteins in WT, KO, and TET3 / GATA6 double knockout (KO‐sgGATA6) PANC‐1 cells. E) Western blot analysis of TGF‐β signaling proteins in wild‐type PANC‐1 cells constitutively overexpressing GATA6 (GATA6 OE ). F) Representative images and quantification of transwell invasion and migration assays in WT and KO in CFPAC‐1 cells (n = 3). G) Survival analysis of TCGA‐PAAD patients stratified by SMAD4 expression (high: top 50%, n = 89; low: bottom 50%, n = 89) and further subdivided by TET3 expression (high: top 25%, n = 22; low: bottom 25%, n = 22). Data are shown as mean ± SD. Statistical significance was assessed by one‐way ANOVA (A), two‐tailed unpaired t ‐test (B, F), or log‐rank Mantel‐Cox test (G). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: GATA6 expression vector (Addgene, #120445) was packaged into a lentivirus transduction system and used to infect cancer cells.

Techniques: Activation Assay, Expressing, Migration, Knock-Out, Western Blot, Double Knockout, Two Tailed Test

Journal: Advanced Science

Article Title: The TET3/GATA6 Axis Drives Lipid Metabolism and Therapeutic Vulnerabilities in Pancreatic Ductal Adenocarcinoma

doi: 10.1002/advs.202501774

Figure Lengend Snippet: Schematic representation of the TET3/GATA6 axis in regulating lipogenic metabolism and promoting tumor growth and invasion in pancreatic cancer.

Article Snippet: GATA6 expression vector (Addgene, #120445) was packaged into a lentivirus transduction system and used to infect cancer cells.

Techniques:

Journal: bioRxiv

Article Title: A Test of the Pioneer Factor Hypothesis

doi: 10.1101/2021.08.17.456650

Figure Lengend Snippet: (a) Schematic of experimental design to infect K562 cells with FoxA1- or Hnf4a-lentivirus and then perform functional assays on dox-induced cells. In CUT&Tag, a protein A-protein G fusion (pA/G) increases the binding spectrum for Fc-binding and allows Tn5 recruitment to antibody-labeled TF binding sites. In ATAC-seq, Tn5 homes to any accessible site. And in RNA-seq, polyA RNA is captured and sequenced. (b) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FoxA1-Hnf4a compared to the number actually activated. Both liver-( P < 10 −38 ) and intestinal-enrichment ( P < 10 −13 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. (c) Genome browser view of a representative liver-specific locus ( ALB ) in FoxA1-Hnf4a clonal line that shows uninduced and induced accessibility, FoxA1 binding, and Hnf4a binding. (d) Meta plot showing uninduced and induced accessibility at all FoxA1-Hnf4a co-bound sites within 50 kb of each FoxA1-Hnf4a activated liver-specific gene (n = 53).

Article Snippet: We used PCR to add V5 epitope tags to the 3’ end of FoxA1 (Addgene #120438) and Hnf4a (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Functional Assay, Binding Assay, Labeling, RNA Sequencing

Journal: bioRxiv

Article Title: A Test of the Pioneer Factor Hypothesis

doi: 10.1101/2021.08.17.456650

Figure Lengend Snippet: (a) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FoxA1 compared to the number actually activated. Liver-enrichment ( P < 10 −4 ) is significant. There are 242 total liver-enriched genes. (b) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by Hnf4a compared to the number actually activated. Liver-( P < 10 −8 ) and intestine-enrichment ( P < 10 −15 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. (c) 242 liver genes characterized as activated by Foxa1, Hnf4a, both, or neither. (d) 122 intestine genes characterized as activated by FoxA1, Hnf4a, both, or neither.

Article Snippet: We used PCR to add V5 epitope tags to the 3’ end of FoxA1 (Addgene #120438) and Hnf4a (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques:

Journal: bioRxiv

Article Title: A Test of the Pioneer Factor Hypothesis

doi: 10.1101/2021.08.17.456650

Figure Lengend Snippet: (a) Genome browser view of a representative liver-specific locus ( Arg1 ) in FoxA1 clonal line showing uninduced and induced accessibility and FoxA1 binding. (b) Genome browser view of a representative liver-specific locus ( ApoC3 ) in Hnf4a clonal line showing uninduced and induced accessibility and Hnf4a binding. (c) Meta plot of uninduced and induced accessibility at all FoxA1 binding sites within 50 kb of each FoxA1-activated liver-specific genes (n = 59). (d) Meta plot of uninduced and induced accessibility at all Hnf4a binding sites within 50 kb of each Hnf4a-activated liver-specific genes (n = 76). (e) FoxA1 or Hnf4a motif count at FoxA1 or Hnf4a binding sites within 50 kb of each FoxA1- or Hnf4a-activated liver-specific genes, respectively. Motifs were called with FIMO using 1e-3 a p-value threshold. For each boxplot, the center line represents the median, the box represents the first to third quartiles, and the whiskers represent any points within 1.5 times the interquartile range.

Article Snippet: We used PCR to add V5 epitope tags to the 3’ end of FoxA1 (Addgene #120438) and Hnf4a (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: A Test of the Pioneer Factor Hypothesis

doi: 10.1101/2021.08.17.456650

Figure Lengend Snippet: (a) Venn diagram of all liver genes categorized as either activated by FoxA1, Hnf4a, FoxA1-Hnf4a, some combination, or by none of the three cocktails. (b) Genome browser view of a representative liver-specific locus ( AMDHD1 ) showing examples of a co-bound site that is “FoxA1 Pioneered” (FP), “Hnf4a Pioneered” (HP), and “Collaboratively Co-bound” (CC). The first two tracks are FoxA1 and Hnf4a binding in the FoxA1-Hnf4a co-expression clone and the last two tracks are FoxA1 and Hnf4a binding in their individual expression clones. (c) List of the 31 liver genes that are only activated by FoxA1-Hnf4a co-expression. The columns indicate how many co-bound FP, HP, or CC peaks exist within 100 kb of the gene. (d) Venn diagram of all genome-wide co-bound peaks categorized as either bound by FoxA1 individually (FP), Hnf4a individually (HP), by both, or by neither (CC). (e) Overlap of FP, HP, and CC sites from (D) with ChromHMM annotations showing the fraction of each co-binding site type in each chromatin region.

Article Snippet: We used PCR to add V5 epitope tags to the 3’ end of FoxA1 (Addgene #120438) and Hnf4a (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay, Expressing, Clone Assay, Genome Wide

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Reflux Linked GATA Factor Fulcrum Dictates Lineage Commitment Through GPRC5B During the Esophageal Dysplastic Transition

doi: 10.1016/j.jcmgh.2025.101552

Figure Lengend Snippet: EAC-associated transcription factors and GERD history. ( A ) Waterfall plot (oncoplot) of copy number variation of metaplasia-associated transcription factors from Duggan et al in The Cancer Genome Atlas data of patients with EAC with (+GERD) and without (-GERD) reported GERD history. This visualization highlights the prevalence and variation of key transcription factor alterations across patient cohort. ( B ) Box plot showing the relationship between GATA4 mRNA expression (y-axis) relative to copy number variation of GATA4 (x-axis) in tumors from 87 patients with EAC ( blue ) versus 95 patients with esophageal squamous cell carcinoma ( red ). ( C ) Box plot showing the relationship between GATA6 mRNA expression (y-axis) relative to copy number variation of GATA6 (x-axis) in tumors from 87 patients with EAC ( blue ) versus 95 patients with esophageal squamous cell carcinoma ( red ). ( D ) Box plot showing the relationship between TRPS1 mRNA expression (y-axis) relative to copy number variation of TRPS1 (x-axis) in tumors from 87 patients with EAC ( blue ) versus 95 patients with esophageal squamous cell carcinoma ( red ). ( E ) Gene ontology (GO) enrichment analysis of cancer genome atlas EAC RNAseq gene expression data segregated according to GERD status, showing enrichment of squamous cell–associated GO terms in GERD(-) and intestine cell–associated GO terms in GERD(+) EAC cases. ( F ) Heatmap illustration of squamous cell– and metaplastic/columnar cell–associated gene expression levels in cancer genome atlas EAC cases with and without reported GERD. GERD enriched genes signature included a metaplastic columnar signature positive for marker ANPEP. Cases with no reported GERD history were enriched for squamous cell associated marker genes TP63, KRT15, and demarcated previous reported SOX15 high population of EACs. ( G ) Western blot analysis of TRPS1, GATA4, and GATA6 levels in various esophageal cell lines, providing insights into the protein expression dynamics of these transcription factors in the context of EAC cell lines. ( H ) TRPS1 levels in esophageal cell lines assessed by RT-qPCR. ( I ) GATA4 levels in esophageal cell lines assessed by RT-qPCR. ( J ) GATA6 levels in esophageal cell lines assessed by RT-qPCR. ( K ) TRPS1 Knockdown in SKGT4 cell line using 2 different shRNA compared with shGFP (NTC), assessed by RT-qPCR (N = 3). The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± standard deviation (SD). ∗∗∗ P < .001. ( L ) Graphical representation of mechanistic question, what is the coregulatory network of atypical (TRPS1) and classical (GATA6) GATA factor action in the BE-HGD-EAC cells. ( M ) Venn diagram showing the overlap between TRPS1 up-regulated genes and GATA6 down-regulated genes ( top ), and the overlap between TRPS1 down-regulated genes and GATA factors that are up-regulated ( bottom ). Accompanying heatmap representation highlights up-regulated genes ( red ) and down-regulated genes ( blue ), indicating coregulation between TRPS1 and GATA6, particularly in cytokines and the transforming growth factor-β pathway, along with changes in squamous markers on loss of TRPS1 and GATA6. NTC, not targeting control.

Article Snippet: GATA4 overexpression plasmid (Addgene #120444) added was used for GATA4 expression EF1a_GATA4_P2A_Hygro.

Techniques: Expressing, Gene Expression, Marker, Western Blot, Quantitative RT-PCR, Knockdown, shRNA, Standard Deviation, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Reflux Linked GATA Factor Fulcrum Dictates Lineage Commitment Through GPRC5B During the Esophageal Dysplastic Transition

doi: 10.1016/j.jcmgh.2025.101552

Figure Lengend Snippet: TRPS1 suppresses GATA6, and consequently interleukin (IL)8 levels, in esophageal cells. ( A ) RT-qPCR and Western blot in SKGT4 cell lines showing increased GATA6 expression on TRPS1 knockdown compared with shGFP (NTC). The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗ P < .001 (N = 3). ( B ) RT-qPCR and Western blot in GOhTRT cell lines showing increased GATA6 expression on TRPS1 knockdown. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (n = 3). ( C ) RT-qPCR and Western blot showing TRPS1 overexpression suppresses GATA4 in GOhTRT cell line. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( D ) RT-qPCR and Western blot showing TRPS1 overexpression suppresses GATA4 expression in FLO1 cell line. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗ P < .05 (N = 3). ( E ) RT-qPCR showing TRPS1 overexpression suppresses GATA6 in BE-ASC. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( F ) RT-qPCR analysis of EAC amplified genes from TCGA database (VEGFA, NFKIBE, AGO2, FGF19) on TRPS1 overexpression in FLO1 cells. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗ P < .05 (N = 3). ( H-K ) RT-qPCR showing IL8 expression following TRPS1 suppression H , TRPS1 overexpression (I), GATA6 suppression ( J ), and GATA6 overexpression ( K ). The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗ P < .05, ∗∗∗ P < .001 (N = 3). ( L ) A proposed model illustrates the regulatory dynamics between TRPS1 and GATA6 in modulating TP63 and IL8 levels. TRPS1 acts as a GATA6 suppressor. When TRPS1 is degraded GATA6 level rises, leading to decrease the expression of TP63 and promote the expression of IL8 fostering an inflammatory environment.

Article Snippet: GATA4 overexpression plasmid (Addgene #120444) added was used for GATA4 expression EF1a_GATA4_P2A_Hygro.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Over Expression, Amplification

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Reflux Linked GATA Factor Fulcrum Dictates Lineage Commitment Through GPRC5B During the Esophageal Dysplastic Transition

doi: 10.1016/j.jcmgh.2025.101552

Figure Lengend Snippet: GATA factor fulcrum gene targets and spatially resolved dysplastic gene signature. ( A ) Schematic drawing of the GATA factor family cotargeted genes. Gene overlaps from 3 RNAseq dataset were used. Genes induced in response to TRPS1 knockdown (TRPS1 suppressed) and genes suppressed in response to the GATA4/6 knockdown (classical GATA factor activated). ( B ) Heat map of the identified target overlaps, from A , between the 3 RNAseq Data sets: TRPS1 knockdown, GATA4 knockout, and GATA6 knockout. ( C ) Violin plot of proliferation markers TOP2A, RRM2, CKD1, and KI67, showing progressively increasing expression from BE through LGD to HGD clusters. ( D ) Sankey plot of top up-regulated genes in BE, LGD, and HGD identified by spatial gene expression RNA sequencing (Visium, 10X Genomics). Columns represent disease stages. The left column represents BE, middle column represents LGD, and right column represent HGD. The edges represent the relationship between genes expression and the stages. Genes expression, top 10 up-regulated in BE ( green ), top 9 up-regulated in LGD ( blue ), and top 10 up-regulated in HGD ( purple ). The plot emphasizes the distinct expression patterns that characterize each disease stage.

Article Snippet: GATA4 overexpression plasmid (Addgene #120444) added was used for GATA4 expression EF1a_GATA4_P2A_Hygro.

Techniques: Knockdown, Knock-Out, Expressing, Gene Expression, RNA Sequencing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Reflux Linked GATA Factor Fulcrum Dictates Lineage Commitment Through GPRC5B During the Esophageal Dysplastic Transition

doi: 10.1016/j.jcmgh.2025.101552

Figure Lengend Snippet: Spatial transcriptomic analysis of TRPS1, GATA factors, and GPRC5B in esophageal tissues. Spatial transcriptomic profiling reveals regional expression patterns of TRPS1 and GATA factors and GPRC5B. ( i ) Hematoxylin and eosin staining of the tissue section of 4 different patients, illustrating the overall histologic architecture and pathologic annotation. ( ii ) Unsupervised k-means clustering, performed in giotto software, of transcriptomic profiles from the spatially resolved tissue section. Clustered gene expression patterns, revealing distinct spatial domains within each patient samples. ( iii ) Expression of GPRC5B, shown in the spatial transcriptomic map, indicates specific regions of enhanced expression, suggesting a potential role in disease progression. ( iv ) Spatial distribution of GATA6 expression across the tissue section, highlighting areas associated with development and differentiation of BE, HGD, and EAC, as indicated by the intensity of expression. ( v ) Expression of GATA4, depicted in the spatial transcriptomic data, illustrating its localized presence in regions relevant HGD. ( vi ) TRPS1 expression mapping, showing distinct spatial localization that may correlate with specific squamous tissue.

Article Snippet: GATA4 overexpression plasmid (Addgene #120444) added was used for GATA4 expression EF1a_GATA4_P2A_Hygro.

Techniques: Expressing, Staining, Software, Gene Expression, Biomarker Discovery

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Reflux Linked GATA Factor Fulcrum Dictates Lineage Commitment Through GPRC5B During the Esophageal Dysplastic Transition

doi: 10.1016/j.jcmgh.2025.101552

Figure Lengend Snippet: GATA factor-mediated regulation of GPRC5B expression in esophageal cells. ( A ) Verification of GATA4 suppression by CRISPR/Cas9 system in OE33 and FLO1 cell lines. ( B ) Verification of GATA6 suppression by CRISPR/Cas9 system in SKGT4, GOhTRT, and OE33 cell lines. ( C ) Verification of GATA4 expression following overexpression in FLO-1, OE33, SKGT4, and GOhTRT cell lines. ( D ) Verification of GATA6 expression following overexpression in FLO-1, OE33, SKGT4, and GOhTRT cell lines. ( E, F ) qRT-PCR and immunoblotting in SKGT4 cell lines showing increase GPRC5B levels on TRPS1 knockdown compared with shGFP (NTC). The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( G, H ) qRT-PCR and immunoblotting in GOhTRT cell line showing increase GPRC5B levels on TRPS1 knockdown compared with shGFP (NTC). The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( I, J ) qRT-PCR and immunoblotting in SKGT4 cell line showing decreased GPRC5B levels on TRPS1 overexpression compared with EV. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗ P < .05 (N = 3). ( K ) qRT-PCR in OE33 cell lines showing decrease GPRC5B levels on TRPS1 overexpression compared with EV. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( L ) qRT-PCR in FLO1 cell line showing decrease GPRC5B levels on TRPS1 overexpression compared with EV. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗ P < .05 (N = 3). ( M, N ) qRT-PCR and immunoblotting in SKGT4 cell line showing decreased GPRC5B levels on GATA6 knock out compared with sgNTC (NTC). The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( O, P ) qRT-PCR and immunoblotting in OE33 cell line showing decrease GPRC5B levels on GATA4/GATA6 knockout compared with NTC. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( Q, R ) qRT-PCR and immunoblotting in FLO-1 cell line showing decrease GPRC5B levels on GATA4 knockout compared with NTC. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( S, T ) qRT-PCR and immunoblotting in SKGT4 cell line showing decreased GPRC5B levels on GATA4 or GATA6 overexpression compared with EV. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( U, V ) qRT-PCR and immunoblotting in GohTRT cell line showing decreased GPRC5B levels on GATA4 or GATA6 overexpression compared with EV. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( W, X ) qRT-PCR and immunoblotting in OE33 cell line showing decreased GPRC5B levels on GATA4 or GATA6 overexpression compared with EV. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). ( Y, Z ) qRT-PCR and immunoblotting in FLO-1 cell line showing decreased GPRC5B levels on GATA4 or GATA6 overexpression compared with EV. The data were analyzed by 2-tailed unpaired Student t test and are represented by means ± SD. ∗∗∗ P < .001 (N = 3). EV, empty vector; KO, knockout. NTC, not targeting control.

Article Snippet: GATA4 overexpression plasmid (Addgene #120444) added was used for GATA4 expression EF1a_GATA4_P2A_Hygro.

Techniques: Expressing, CRISPR, Over Expression, Quantitative RT-PCR, Western Blot, Knockdown, Knock-Out, Plasmid Preparation, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Reflux Linked GATA Factor Fulcrum Dictates Lineage Commitment Through GPRC5B During the Esophageal Dysplastic Transition

doi: 10.1016/j.jcmgh.2025.101552

Figure Lengend Snippet: GPRC5B-mediated cellular reprogramming of BE-ASCs. ( A ) Percentage of cellular confluence following GPRC5B knockdown in SKGT4 cells compared with shGFP control-transfected cells, assessed over 9 days. ∗∗∗ P < .001 (N = 3). ( B ) Percentage of cellular confluence following GPRC5B knockdown in OE33 cells compared with shGFP control-transfected cells, assessed over 9 days. ∗∗∗ P < .001 (N = 3). ( C ) Percentage of cellular confluence following plasmid-mediated GPRC5B overexpression in SKGT4 cells compared with EV control-transfected cells. ∗∗∗ P < .001 (n = 3). ( D ) Percentage of cellular confluence following plasmid-mediated GPRC5B overexpression in OE33 cells compared with EV control-transfected cells. ∗∗∗ P < .001 (N = 3). ( E ) Results from the colony-formation assay following stable expression of GPRC5B in SKGT4 or OE33 EAC cells compared with EV or shGFP-transfected cells. ∗∗∗ P < .001 (N = 3). ( F ) Results from the colony-formation assay following suppression of GPRC5B in SKGT4 or OE33 EAC cells compared with EV or shGFP-transfected cells. ∗∗∗ P < .001 (N = 3). ( G-I ) Verification of GPRC5B levels following overexpression of suppression of GPRC5B by shRNA or plasmid transfection, respectively. ( J ) RT-qPCR analysis of GPRC5B expression showing a 3-fold increase in undifferentiated HGD-ASC compared with undifferentiated BE-ASC. Data are presented as means ± SD. ∗∗∗ P < .001. ( K ) Heatmap illustrating intestinal- and gastric-associated gene markers derived from RNA-seq analysis of HGD-ASC and BE-ASC, highlighting distinct differences in gene expression profiles. ( L ) Immunofluorescence staining of TFF3 ( red ) and GPRC5B ( green ) in differentiated BE-ASC ( top ) and differentiated HGD-ASC ( bottom ), showing negative expression in BE-ASC and positive cell surface and nuclear staining in HGD-ASC. ( M ) Increased expression of GATA4 following a 3-hour pulsed exposure of BE-ASCs to acidic media (pH 4.5) measured by RT-PCR. ∗ P < .005 (N = 3). ( N ) Increased expression of GPRC5B following a 3-hour pulsed exposure of BE-ASCs to acidic media (pH 4.5) measured by RT-PCR. ∗ P < .005 (N = 3). ( O ) GPRC5B overexpression in BE-ASCs promotes cell growth compared with control EV transfected cells. ∗∗∗ P < .001 (N = 3). ( P ) GPRC5B suppression in HGD ASCs hinders cell growth compared with shGFP-transfected cells. ∗∗∗ P < .001.

Article Snippet: GATA4 overexpression plasmid (Addgene #120444) added was used for GATA4 expression EF1a_GATA4_P2A_Hygro.

Techniques: Knockdown, Control, Transfection, Plasmid Preparation, Over Expression, Colony Assay, Expressing, shRNA, Quantitative RT-PCR, Derivative Assay, RNA Sequencing, Gene Expression, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction