Journal: EMBO Reports

Article Title: VGLL4 modulates Paneth cells and sustains intestinal homeostasis

doi: 10.1038/s44319-026-00699-3

Figure Lengend Snippet: ( A ) IF staining of small intestinal samples from Control and ISC-specific knockout (ISC-KO) mice at 1 week after TAM injection. Top 2 panels: Staining for Ki67 (gray), GFP (green), RFP (purple) and DAPI (blue). Bottom 2 panels: Staining for Lysozyme (gray), GFP (green), RFP (purple) and DAPI (blue). Scale bars, 10 μm. ( B ) Quantification of Ki67 + cells and Lysozyme + cells per crypt in ( A ) ( n = 4). Unpaired Student’s t test. ns P = 0.5512. ** P = 0.0281. ( C ) qRT-PCR analysis of Gfi1 and Sox9 mRNA levels in Vgll4 fl/fl and Vgll4 IEC-KO mice ileum samples ( n = 5). Unpaired Student’s t test. *** P = 0.0008. ns P = 0.1057. ( D ) Immunoblot analysis of GFI1, SOX9, ATOH1, and VGLL4 in ileum samples of Vgll4 fl/fl and Vgll4 IEC-KO mice. Samples were derived from the same experiment and blots were processed in parallel. ( E ) Quantification of protein levels in ( D ) ( n = 5). Unpaired Student’s t test. GFI1: ** P = 0.0018. ATOH1: ns P = 0.6427. SOX9: ns P = 0.4877. ( F ) IF staining of GFI1 (red) in Vgll4 fl/fl and Vgll4 IEC-KO mice small intestinal organoids ( n = 5). DAPI, blue. Scale bars, 30 μm. ( G ) Co-immunoprecipitation assay results between ATOH1 and TEAD4 in HEK293T cells. ( H ) Co-immunoprecipitation assay results between ATOH1 and TEAD4 in HEK293T cells with DNase. ( I ) Co-immunoprecipitation assay results between ATOH1 and different mutations of TEAD4 in HEK293T cells. ( J ) Co-immunoprecipitation assay results between ATOH1 and VGLL4 in HEK293T cells with or without TEAD4. ( K ) Immunoblot analysis of GST pull-down assay between purified ATOH1-ΔN, VGLL4, and TEAD4 proteins. Results are shown as mean + SD. ns no significance. .

Article Snippet: HEK293T , ATCC , ACS-4500.

Techniques: Staining, Control, Knock-Out, Injection, Quantitative RT-PCR, Western Blot, Derivative Assay, Co-Immunoprecipitation Assay, Pull Down Assay, Purification

Journal: EMBO Reports

Article Title: VGLL4 modulates Paneth cells and sustains intestinal homeostasis

doi: 10.1038/s44319-026-00699-3

Figure Lengend Snippet: ( A ) qRT-PCR analysis of Math1 from Vgll4 fl/fl and Vgll4 IEC-KO mice ileum samples ( n = 5). Unpaired Student’s t test. ns P = 0.7009. ( B ) Co-immunoprecipitation assay results between ATOH1 and VGLL4 in HEK293T cells. ( C ) Schematic illustration of TEAD4 and its short forms. ( D ) Co-immunoprecipitation assay results between ATOH1 and TEAD4 short forms in HEK293T cells. The black arrow indicates IgG heavy chain. ( E ) Co-immunoprecipitation assay results between ATOH1 and TEAD4-N and TEA domain short forms in HEK293T cells. ( F ) Schematic illustration of ATOH1 and its short forms. ( G ) Co-immunoprecipitation assay results between ATOH1 short forms and TEAD4 in HEK293T cells. ( H ) Coomassie brilliant blue staining of the GST-pulldown assay between ATOH1-ΔN, VGLL4, and TEAD4. ( I ) FLAG antibody pulldown assay results between VGLL4-TEAD4-ATOH1 complex in HEK293T cells overexpressing FLAG-VGLL4, MYC-ATOH1 and HA-TEAD4. ( J ) ChIP-qPCR analysis of TEAD4 enrichment at the GFI1 promoter in HEK293T cells overexpressing FLAG-TEAD4. Unpaired Student’s t test. **** P < 0.0001. Three biological replicates per group. ( K ) ChIP-qPCR analysis of TEAD4 at the GFI1 promoter in 293T cells with or without VGLL4 overexpression. Unpaired Student’s t test. **** P < 0.0001. Left *** P = 0.0007. Right *** P = 0.0003. Three biological replicates per group. ( L ) ChIP-qPCR analysis of ATOH1 at the GFI1 promoter in HEK293T cells with or without VGLL4 overexpression. Unpaired Student’s t test. Left *** P = 0.0003. Right *** P = 0.0007. Three biological replicates per group. ( M ) A model of GFI1 luciferase construction strategy. Red arrow: TBS, TEAD4 binding sequence. Black arrow: ABS, ATOH1 binding sequence. RR: regulatory region. ( N ) GFI1 Luc reporter activity in HCoEpiC cells transfected with ATOH1, VGLL4, and TEAD4. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. ( O ) GFI1 Luc reporter activity in HCoEpiC cells transfected with ATOH1, TEAD4 wild-type or mutant form, VGLL4 wild-type or mutant form. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. Results are shown as mean + SD. ns no significance.

Article Snippet: HEK293T , ATCC , ACS-4500.

Techniques: Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, GST Pulldown Assay, ChIP-qPCR, Over Expression, Luciferase, Binding Assay, Sequencing, Activity Assay, Transfection, Mutagenesis

Journal: EMBO Reports

Article Title: VGLL4 modulates Paneth cells and sustains intestinal homeostasis

doi: 10.1038/s44319-026-00699-3

Figure Lengend Snippet: ( A ) Heatmap analysis of defensin expression with RNA-seq data ( n = 4). ( B ) ChIP-qPCR analysis of TCF4 enrichment at the DEFA5 promoter in SW620 cells. Unpaired Student’s t test. *** P = 0.0008. Three biological replicates per group. ( C ) ChIP-qPCR analysis of TEAD4 enrichment at the DEFA5 promoter in SW620 cells. Unpaired Student’s t test. *** P = 0.0003. Three biological replicates per group. ( D ) Luciferase reporter activity driven by DEFA5 promoter in HEK293T cells transfected with indicated plasmids. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. ( E ) Luciferase reporter activity driven by DEFA5 promoter in HEK293T cells transfected with TCF4, VGLL4 and TEAD4 wild-type or TEAD4-TEA short form. One-way ANOVA. **** P < 0.0001. *** P = 0.0004. Three biological replicates per group. ( F ) Luciferase reporter activity driven by DEFA5 promoter in HEK293T cells transfected with TCF4, TEAD4 and VGLL4 wild-type or VGLL4-HF4A short form. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. ( G ) Luciferase reporter activity driven by DEFA5 promoter in HCoEpiC cells transfected with indicated plasmids. One-way ANOVA. **** P < 0.0001. ** P = 0.0025. Three biological replicates per group. ( H ) Luciferase reporter activity driven by DEFA5 promoter in HCoEpiC cells transfected with TCF4, TEAD4 wild-type or TEAD4-TEA short form, and VGLL4 wild-type or VGLL4-HF4A short form. One-way ANOVA. **** P < 0.0001. Three biological replicates per group. Results are shown as mean + SD.

Article Snippet: HEK293T , ATCC , ACS-4500.

Techniques: Expressing, RNA Sequencing, ChIP-qPCR, Luciferase, Activity Assay, Transfection

Journal: Journal of proteome research

Article Title: Interactome of Site-Specifically Acetylated Linker Histone H1.

doi: 10.1021/acs.jproteome.1c00396

Figure Lengend Snippet: Figure 1. Modification sites of H1.2. Scheme of H1.2 PTMs after IP enrichment from HEK 293T cells and identification by LC−MS/MS. Positions investigated within this study are indicated in bold (K17 and K64). Reprinted with adaptations from ref 15.

Article Snippet: To determine the stability of the acetylated H1.2-conjugates in human cell lysates, 4.7 μM H1.2 or H1.2 KxAc was incubated in HEK 293T cell lysates (5 mg/mL) at 4 or 25 °C for up to 24 h. Samples were analyzed by western blot with anti-H1 (Santa Cruz) and anti-AcK (Cell Signaling) antibodies (Figure S6).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Journal of proteome research

Article Title: Interactome of Site-Specifically Acetylated Linker Histone H1.

doi: 10.1021/acs.jproteome.1c00396

Figure Lengend Snippet: Figure 2. Generation of site-specifically acetylated H1.2. (A) AcK is incorporated into H1.2 during gene expression in E. coli in response to an amber stop codon (TAG/UAG) at positions K17 and K64 (mono-acetylation) or both positions within one protein (di-acetylation). (B) Purified H1.2 and H1.2 KxAc variants resolved by SDS-PAGE and Coomassie staining. (C) Chromatosome assembly with H1.2 KxAc variants; crDNA indicates competitor DNA, Chr. refers to mono-chromatosomes, and Nucl. indicates mono-nucleosomes. (D) Stability assay of acetylated histones in cell lysate. Proteins were incubated in HEK 293T cell lysate as indicated. Samples were analyzed by western blot, showing constant levels of acetylated protein, indicating the stability of the modification over time at different temperatures.

Article Snippet: To determine the stability of the acetylated H1.2-conjugates in human cell lysates, 4.7 μM H1.2 or H1.2 KxAc was incubated in HEK 293T cell lysates (5 mg/mL) at 4 or 25 °C for up to 24 h. Samples were analyzed by western blot with anti-H1 (Santa Cruz) and anti-AcK (Cell Signaling) antibodies (Figure S6).

Techniques: Gene Expression, SDS Page, Staining, Stability Assay, Incubation, Western Blot

Journal: Journal of proteome research

Article Title: Interactome of Site-Specifically Acetylated Linker Histone H1.

doi: 10.1021/acs.jproteome.1c00396

Figure Lengend Snippet: Figure 3. Identification of H1.2 KxAc-specific interactions. A heat map representing the hierarchical clustering of statistically significant interactors following ANOVA analysis (FDR = 0.01, s0 = 2, n = 3). Interacting proteins are shown in rows, and columns represent bait proteins. AP-MS experiments were carried out in biological triplicate with HEK 293T cell lysates. Empty beads (i.e., no bait protein) served as a control. Clusters of proteins with similar interaction behavior are marked with different colors (left). Profile plots of clusters indicating specific binding patterns are shown on the right.

Article Snippet: To determine the stability of the acetylated H1.2-conjugates in human cell lysates, 4.7 μM H1.2 or H1.2 KxAc was incubated in HEK 293T cell lysates (5 mg/mL) at 4 or 25 °C for up to 24 h. Samples were analyzed by western blot with anti-H1 (Santa Cruz) and anti-AcK (Cell Signaling) antibodies (Figure S6).

Techniques: Protein-Protein interactions, Control, Binding Assay

Journal: Sensors and actuators. B, Chemical

Article Title: Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

doi: 10.1016/j.snb.2013.08.012

Figure Lengend Snippet: Characterization of the engineered HEK-β2AR-GFP cells. (a-c) Representative fluorescence microscopic images of the engineered cells after overnight culture: (a) epifluorescence image; (b) TIRF image before any treatment; (c) TIRF image after treatment with 1μM isoproterenol for 1hr. For (a-c) the image scale bar is 50 μm. (d-f) The DMR dose responses of isoproterenol in cells after overnight culturing to form monolayer: (d) the parental HEK293 cells; (e) the engineered cells; (f) comparison of the dose-dependent responses of isoproterenol in both types of cells, wherein for the parental cells the DMR amplitude at 50min post stimulation was plotted as a function of isoproterenol dose, and for the engineered cells the DMR amplitudes at both 5min and 50min post stimulation were plotted. For (d-f), the data represents mean±s.d. (n =4).

Article Snippet: Briefly, HEK293 cells were transfected with human pCMV-β 2 AR-GFP plasmid (OriGene Technologies, Inc., Rockville, MD, USA) using Lipofectamine™ LTX and Plus Reagent (Invitrogen) in a 6-well cell culture plate.

Techniques: Fluorescence

Journal: Sensors and actuators. B, Chemical

Article Title: Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

doi: 10.1016/j.snb.2013.08.012

Figure Lengend Snippet: DMR characterization of HEK-β2AR-GFP cell adhesion. (a, b) The DMR of the engineered cells adherent onto different surfaces under ambient condition: (a) real-time; (b) the DMR amplitudes at 5hr after cell seeding. (c,d) The DMR of the engineered cells adherent onto different surfaces under physiological condition: (c) real-time; (d) the DMR amplitudes at 4hr after cell seeding. The medium only was used as the negative control. For all, the total number of cells added were the same (18,000 cells per well). The data represents mean±s.d. (n =12).

Article Snippet: Briefly, HEK293 cells were transfected with human pCMV-β 2 AR-GFP plasmid (OriGene Technologies, Inc., Rockville, MD, USA) using Lipofectamine™ LTX and Plus Reagent (Invitrogen) in a 6-well cell culture plate.

Techniques: Negative Control

Journal: Sensors and actuators. B, Chemical

Article Title: Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

doi: 10.1016/j.snb.2013.08.012

Figure Lengend Snippet: DMR characterization of HEK-β2AR-GFP cell adhesion in the presence of different inhibitors under ambient condition. (a-f) The DMR of the engineered cells adherent onto different sensor surfaces: tissue culture treated (a, b), fibronectin coated (c,d), and collagen IV coated (e,f). (a,c,e) real-time DMR; (b,d,f) the DMR amplitudes at 5hr after cell seeding as a function of inhibitor dose. For all, the total number of cells added were the same; that is, 18,000 cells per well. For (a,c,e), the inhibitor dose was fixed to be 10μM, 10μM, 10μM and 1mM for nocodazole, vinblastine, cytochalasin D, and RGD peptide, respectively. The data represents mean±s.d. (n=12 for a, c, and e; n =4 for b, d, and f).

Article Snippet: Briefly, HEK293 cells were transfected with human pCMV-β 2 AR-GFP plasmid (OriGene Technologies, Inc., Rockville, MD, USA) using Lipofectamine™ LTX and Plus Reagent (Invitrogen) in a 6-well cell culture plate.

Techniques:

Journal: Sensors and actuators. B, Chemical

Article Title: Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

doi: 10.1016/j.snb.2013.08.012

Figure Lengend Snippet: DMR characterization of HEK-β2AR-GFP cell adhesion in the presence of different inhibitors under physiological condition. (a-f) The DMR of the engineered cells adherent onto different sensor surfaces: tissue culture treated (a, b), fibronectin coated (c,d), and collagen IV coated (e, f). (a, c, e) real-time DMR; (b, d, f) the DMR amplitudes at 4hr after cell seeding as a function of inhibitor dose. For all, the total number of cells added were the same; that is, 18,000 cells per well. For (a, c, e), the inhibitor dose was fixed to be 10μM, 10μM, 10μM and 1mM for nocodazole, vinblastine, cytochalasin D, and RGD peptide, respectively. The data represents mean±s.d. (n=12 for a, c, and e; n =4 for b, d, and f).

Article Snippet: Briefly, HEK293 cells were transfected with human pCMV-β 2 AR-GFP plasmid (OriGene Technologies, Inc., Rockville, MD, USA) using Lipofectamine™ LTX and Plus Reagent (Invitrogen) in a 6-well cell culture plate.

Techniques:

Journal: Sensors and actuators. B, Chemical

Article Title: Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

doi: 10.1016/j.snb.2013.08.012

Figure Lengend Snippet: TIRF images of HEK-β2AR-GFP cells on the fibronectin coated surface under ambient condition. The images were taken 2hrs after cell adhesion onto the fibronectin-coated biosensor surface in the absence and presence of an inhibitor. (a) no any inhibitor; (b) 10μM cytochalasin D; (c) 1mM RGD peptide. The inhibitors were present throughout the cell adhesion process. Image scale bar: 50 μm.

Article Snippet: Briefly, HEK293 cells were transfected with human pCMV-β 2 AR-GFP plasmid (OriGene Technologies, Inc., Rockville, MD, USA) using Lipofectamine™ LTX and Plus Reagent (Invitrogen) in a 6-well cell culture plate.

Techniques: