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: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

doi: 10.3390/v15122304

Figure Lengend Snippet: Figure 1. SARS-COV-2 N protein interacts with UBC9. (A) pCDNA3.1 (3XFlag Tag) with N protein- coding genes and pEGFP-N1 (GFP Tag) with UBC9-coding genes were co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. (B) pDsRED-mono-N1 (RFP Tag) with SARS-COV-2 N protein-coding genes and pEGFP-N1 (GFP Tag) with UBC9 or UBC9C93A coding genes were expressed or co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cells were stained with DAPI before confocal microscopy imaging. Endogenous UBC9 in HEK293T cells was also detected by FITC-conjugated Goat Anti-Mouse IgG(H + L), which recognizes anti-UBC9 mouse antibodies. Scale bars: 10 µm. (C) pET28a (HIS6 tag, Kan+) with SARS-COV-2 N protein coding genes and pGEX-6P-1 (GST tag, Amp+) with UBC9 or UBC9C93A coding genes were co-expressed in BL21 (DE3) E. coli. cell lysates were subjected to Co-IP after IPTG (1 mM) induction for 6 h. (D) pCDNA3.1 (3XFlag Tag) with SARS-COV-2 N protein-coding genes was over-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat AntiMouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Transfection, Co-Immunoprecipitation Assay, Staining, Confocal Microscopy, Imaging

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

doi: 10.3390/v15122304

Figure Lengend Snippet: Figure 2. Human beta coronavirus N protein interacts with UBC9. (A) pET28a-N (HIS6 tag, Kan+) containing five human beta coronavirus N protein-coding genes and pGEX-UBC9 (GST tag, Amp+) harboring the UBC9 coding gene were co-expressed in BL21 (DE3) E. coli Cell lysates were prepared and subjected to Co-IP following IPTG induction (1 mM) for 6 h. (B) pEGFP-N1 (GFP Tag) with three human beta coronavirus N protein-coding genes and pCDNA3.1 (3XFlag Tag) with the UBC9 coding gene were co-expressed in HEK293T cells. After a 24- h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat AntiMouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Co-Immunoprecipitation Assay, Transfection

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

doi: 10.3390/v15122304

Figure Lengend Snippet: Figure 3. SARS-COV-2 N protein enhanced the molecular interaction between UBC9 and MAVS. (A) pEGFP-N1 (GFP Tag) with SARS-COV-2 N protein or UBC9 coding genes and pCDNA3.1 (3XFlag Tag) with MAVS coding gene were co-expressed in HEK293T cells. After a 24-h lipofec- tamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. (B) Relative Ubc9 band intensity before or after IP: Flag in (A) were calculated by using ImageJ in triplet replicates. ns: no significant difference, *** p < 0.001. (C) Schematic diagram of wild-type SARS- CoV-2 N protein and truncated mutants. (D) pEGFP-N1 (GFP Tag) with UBC9 coding genes and pCDNA3.1 (3XFlag Tag) with SARS-COV-2 N protein or truncation coding gene were co-expressed in HEK293T cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat AntiMouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

doi: 10.3390/v15122304

Figure Lengend Snippet: Figure 4. The expression of UBC9 in HEK293T, Vero, Vero E6, Huh7, and HRT18 cells was detected by Western blotting.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat AntiMouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Expressing, Western Blot

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

doi: 10.3390/v15122304

Figure Lengend Snippet: Figure 5. The interaction between the SARS-CoV-2 N protein and UBC9 inhibits MAVS ubiquitination by enhancing its SUMOylation. (A,B) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA, or K63-His6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 highly expressed) or Vero E6 cells (endogenous UBC9 underexpressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat AntiMouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

doi: 10.3390/v15122304

Figure Lengend Snippet: Figure 6. The interaction of N protein and UBC9 plays a crucial role in regulating the SUMOylation and ubiquitination modifications of MAVS. (A) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA or SUMO3KR-HA, K63-His6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 was highly expressed). After a 24-h lipofec- tamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. (B) pEGFP-N1 (GFP Tag) with SARS-COV-2 N coding genes or pCDNA3.1 with MAVS-Flag, SUMO3- HA, UBC9 or UBC9C93A, K63-His6 coding genes were co-expressed in Vero E6 cells (endogenous UBC9 underexpressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays. (C) pEGFP-N1 (GFP Tag) with SARS-COV-2 N, N44–419 or N174–419 coding genes or pCDNA3.1 with MAVS-Flag, SUMO3-HA, and K63-His6 coding genes were co-expressed in HEK293T cells (endogenous UBC9 highly expressed). After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Co-IP assays.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat AntiMouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay

Journal: Viruses

Article Title: The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

doi: 10.3390/v15122304

Figure Lengend Snippet: Figure 7. UBC9 plays a critical role in the process of impaired IFN I response caused by the in-teraction between the N protein and MAVS during virus infection. (A,B) pCDNA3.1 with SARS-CoV-2 N protein-coding genes was gradually increased in HEK293T cells or VERO E6 cells. After a 24-h lipofectamine transfection followed by 8 h of SeV stimulation, cell lysates were collected for Western blotting. (C,D) The relative band intensity of Western blotting results (a relative band quantification of phosphorylation protein to total protein) in (A,B) were calculated by using ImageJ in triplet replicates. ns: no significant difference, p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: Subsequently, the transfected cells were exposed to Sev for an additional 8 h, followed by PBS washing and fixation with 4% paraformaldehyde in PBS for a duration of 20 min. Endogenous UBC9 in HEK293T cells was also detected by FITC–conjugated Goat AntiMouse IgG(H + L) (Proteintech, Cat No. SA00003-1), which recognizes anti-UBC9 mouse antibody (Proteintech, Cat No. 60201-1-Ig).

Techniques: Virus, Infection, Transfection, Western Blot, Phospho-proteomics