dpp4 activity  (TargetMol)

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: DPP4 expression is upregulated in intestinal fibro-stenotic areas of CD patients. (A) Volcano plot showing differentially expressed genes (DEGs) between stenotic (B2) and non-stenotic (B1) CD tissues ( GSE66207 dataset). Red dots indicate DEGs with |log2FC| >1 and p < 0.05. (B, C) Hub genes identified using the MCODE and CytoHubba plugins, respectively. (D) RT-qPCR analysis of DPP4 mRNA levels in intestinal tissues. (E) Western blot analysis of DPP4 protein expression in colonic tissues from healthy controls, non-stenotic, and stenotic regions of CD patients. (F) Densitometric quantification of DPP4 protein normalised to GAPDH (corresponding to panel E). (G) Representative endoscopic images of healthy controls, non-stenotic, and stenotic intestinal regions in CD patients, illustrating macroscopic features of stricture. Corresponding immunohistochemical (IHC) images show DPP4 expression in colonic tissues from the same groups. Adjacent serial sections stained with Masson’s trichrome highlight fibrotic areas. (H) Quantification of IHC DPP4 staining (IOD/area) across groups. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: Elevated DPP4 expression contributes to fibrotic remodelling in a chronic colitis model. (A) Colon length quantification in control ( n = 6) and DSS-treated ( n = 6) mice. (B) Representative H&E-stained colon sections (left) and histologic inflammation scores (right). (C) Representative Masson’s trichrome-stained colon sections and quantification of collagen volume fraction. (D) Immunofluorescence images and quantification of α -SMA⁺ fibrotic thickness in the muscularis propria. (E) qRT-PCR analysis of Col1a1 mRNA levels in colon tissues. (F) qRT-PCR analysis of Col6a1 mRNA levels in colon tissues. (G) Western blot analysis of DPP4 protein expression in control and DSS-treated mice. (H) Representative IHC staining of DPP4 expression in control and DSS-treated colons, with semiquantitative analysis. (I) Schematic overview of the chronic DSS-induced colitis model and pharmacological intervention. Mice received three cycles of 1.5% DSS (7 days per cycle), each followed by a 14-day recovery phase. The DPP4 inhibitor sitagliptin was administered via oral gavage for 21 days during the final cycle of the experiment. (J) Colon length quantification in the DSS group ( n = 6) and DSS + DPP4i group ( n = 6). (K) Representative H&E-stained colon sections (left) and histologic inflammation scores (right). (L) Representative Masson’s trichrome-stained colon sections and quantification of collagen volume fraction. (M) Immunofluorescence images and quantification of α -SMA⁺ fibrotic thickness in the muscularis propria. ( N ) RT-qPCR analysis of Col1a1 mRNA levels in colon tissues. (O) RT-qPCR analysis of Col6a1 mRNA levels in colon tissues. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Expressing, Control, Staining, Immunofluorescence, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: Membrane-bound DPP4 in fibroblasts drives intestinal myofibroblast activation and migration. (A) Multiplex IHC staining of FFPE colonic sections from healthy controls, non-stenotic, and stenotic regions of CD patients. (B) Volcano plot showing DEGs in fibroblasts isolated from paired stenotic and normal CD tissues ( GSE90607 dataset, left), with DPP4 mRNA expression levels specifically highlighted (right). (C) Western blot analysis of membrane-bound DPP4 and α -SMA protein in primary HIMFs. (D) Western blot analysis and quantification of DPP4 and α -SMA expression in TGF-β–stimulated HIMFs with or without DPP4 inhibitor. (E) qRT-PCR analysis of COL1A1 mRNA expression in HIMFs. (F) qRT-PCR analysis of ACTA2 mRNA expression in HIMFs. (G) Immunofluorescence staining and quantification of Ki67⁺ proliferating HIMFs. (H) Representative images and quantification of HIMF migration in scratch wound healing assays. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Membrane, Activation Assay, Migration, Multiplex Assay, Immunohistochemistry, Isolation, Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: Soluble DPP4 (sDPP4) promotes intestinal myofibroblast activation, proliferation, and migration. (A) ELISA-based quantification of soluble DPP4 (sDPP4) levels in plasma from healthy controls ( n = 10), CD patients without stenosis ( n = 20), and with stenosis ( n = 20). (B) RT-qPCR analysis of COL1A1 mRNA expression in HIMFs treated with sDPP4 in the presence or absence of DPP4 inhibitor. (C) RT-qPCR analysis of ACTA2 mRNA expression in HIMFs treated with sDPP4 in the presence or absence of DPP4 inhibitor. (D) Western blot of α -SMA protein expression in HIMFs treated with sDPP4 in the presence or absence of DPP4 inhibitor. (E) Immunofluorescence staining and quantification of Ki67⁺ proliferating HIMFs treated with sDPP4 in the presence or absence of DPP4 inhibitor. (F) Scratch wound healing assay evaluating HIMF migration at 0 and 24 hours following sDPP4 stimulation, with or without DPP4 inhibitor. Wound closure was quantified using ImageJ. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Activation Assay, Migration, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Wound Healing Assay

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: DPP4 drives intestinal myofibroblast activation via the PI3K-AKT pathway. (A) Western blot analysis of canonical (SMAD3) and non-canonical (PI3K-AKT, ERK, JNK, and p38) signalling pathways in TGF- β -stimulated HIMFs, treated with or without DPP4 inhibitor (sitagliptin, 20 nM). (B) Western blot analysis of p -PI3K, PI3K, p -AKT, AKT, and α -SMA expression in HIMFs treated with TGF- β , in the presence or absence of DPP4 inhibitor (20 nM) and the PI3K activator 740Y- P (10 μM). (C) Western blot analysis of p -ERK, ERK, and α -SMA expression in HIMFs treated with TGF- β , in the presence or absence of DPP4 inhibitor (20 nM) and the MEK/ERK activator C16-PAF (1 μM). (D) Scratch wound healing assay of HIMF migration at 0 and 24 hours following TGF- β stimulation with or without DPP4 inhibitor (20 nM) and the PI3K activator 740Y- P (10 μM). Wound closure was quantified using ImageJ. Ki67 immunofluorescence staining and quantification of proliferating HIMFs are shown in the lower panels. (E) Scratch wound healing assay of HIMF migration at 0 and 24 hours following TGF- β stimulation with or without DPP4 inhibitor (20 nM) and the MEK/ERK activator C16-PAF (1 μM). Wound closure was quantified using ImageJ. Ki67 immunofluorescence staining and quantification are shown in the lower panels. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Activation Assay, Western Blot, Expressing, Wound Healing Assay, Migration, Immunofluorescence, Staining

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: Gut microbiota-derived DPP4 is enriched in stenotic CD and associated with fibrotic remodelling. (A) Schematic overview of the faecal metagenomic sequencing strategy in controls ( n = 10), CD without stenosis ( n = 20), and CD with stenosis ( n = 20). (B) Relative abundance of microbial Dpp4 genes across the three groups, as determined by metagenomic sequencing. (C) Species-level contributions of microbial Dpp4 genes in CD patients with stenosis, showing the top five contributing species ranked by relative abundance. (D) Differential abundance of microbial Dpp4 genes assigned to four Bacteroides species in faecal metagenomes from healthy controls, non-stenotic CD, and stenotic CD patients. (E) Total faecal DPP4 enzymatic activity was measured by Gly-Pro-pNA assay in controls, CD patients without stenosis, and those with stenosis. (F) Faecal DPP4 enzymatic activity was measured in control mice and those with DSS-induced chronic colitis. (G) Time-dependent increase in DPP4 activity measured in the culture supernatants of B. thetaiotaomicron grown under anaerobic conditions. (H–I) RT-qPCR analysis of COL1A1 (H) and ACTA2 (I) mRNA levels in HIMFs treated with increasing concentrations of recombinant btDPP4 (0–400 ng/mL). (J) Western blot analysis of α -SMA protein expression in HIMFs treated with 200 ng/mL btDPP4. (K) Ki67 immunofluorescence staining and quantification of proliferating HIMFs following treatment with 200 ng/mL btDPP4. (L) Scratch wound healing assay of HIMF migration at 0 and 24 hours following treatment with 200 ng/mL btDPP4. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Derivative Assay, Sequencing, Activity Assay, Control, Quantitative RT-PCR, Recombinant, Western Blot, Expressing, Immunofluorescence, Staining, Wound Healing Assay, Migration

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: Colonisation with engineered bacteria overexpressing DPP4 worsens fibrotic remodelling in DSS-induced chronic colitis. (A) Schematic illustration of engineered E. coli btDPP4 construction. (B) PCR validation of btDPP4 genomic integration in engineered E. coli strains. (C) Growth curves comparing E. coli btDPP4 and wild-type (WT) strains under standard conditions. (D) Secreted DPP4 enzymatic activity measured in the culture supernatants of E. coli btDPP4 and E. coli WT. (E) Experimental design of the chronic DSS colitis model with oral gavage of PBS, E. coli WT, or E. coli btDPP4. (F) Relative faecal E. coli load in mice treated with PBS, E. coli WT, or E. coli btDPP4. (G) Expression of btDPP4 gene in faeces from mice treated with PBS, E. coli WT, or E. coli btDPP4. (H) Faecal DPP4 enzymatic activity in mice treated with PBS, E. coli WT, or E. coli btDPP4. (I) Colon length was measured and compared across groups. (J) Representative H&E-stained colon sections and quantification of histological inflammation scores. (K) Representative Masson’s trichrome-stained sections and quantification of collagen volume fraction. (L) Immunofluorescence staining of α -SMA⁺ fibrotic areas and quantification of fibrotic thickness. (M–N) RT-qPCR analysis of Col1a1 (M) and Col6a1 ( N ) mRNA levels in mouse colon tissues. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Bacteria, Biomarker Discovery, Activity Assay, Expressing, Staining, Immunofluorescence, Quantitative RT-PCR

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: Microbial-derived DPP4 inhibitor Dau-d4 suppresses intestinal myofibroblast activation. (A-B) RT-qPCR analysis of COL1A1 and ACTA2 mRNA expression in HIMFs treated with increasing concentrations of recombinant microbial DPP4 (btDPP4, hereafter referred to as mDPP4), with or without the microbial DPP4 inhibitor Dau-d4 (0–200 nM). (C) Western blot analysis of α -SMA protein expression in HIMFs stimulated with mDPP4, with or without 100 nM Dau-d4. (D) Ki67 immunofluorescence staining and quantification of proliferating HIMFs stimulated with mDPP4, with or without 100 nM Dau-d4. (E) Scratch wound healing assay showing HIMF migration at 0 and 24 hours following treatment with mDPP4, in the presence or absence of 100 nM Dau-d4. (F) Western blot analysis of p -PI3K, PI3K, p -AKT, AKT, and α -SMA expression in HIMFs treated with vehicle control, mDPP4 (200 ng/mL), mDPP4 + Dau-d4 (100 nM), or mDPP4 + Dau-d4 + PI3K activator 740Y- P (10 μM) for 24 hours. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Derivative Assay, Activation Assay, Quantitative RT-PCR, Expressing, Recombinant, Western Blot, Immunofluorescence, Staining, Wound Healing Assay, Migration, Control

Journal: Gut Microbes

Article Title: Dual-source DPP4 drives intestinal fibrosis in Crohn’s disease: synergistic therapeutic targeting of host and microbiota pathways

doi: 10.1080/19490976.2025.2593119

Figure Lengend Snippet: Dual inhibition of bacterial- and host-derived DPP4 synergistically attenuates intestinal fibrosis in vivo. (A) Schematic of the experimental design showing late-phase intervention with the microbiota-derived DPP4 inhibitor Dau-d4 (10 mg/kg), alone or in combination with the host-derived DPP4 inhibitor sitagliptin, during the final cycle of DSS-induced chronic colitis. (B) DPP4 activity was measured in the murine faeces following treatment. (C) Colon length was measured and compared across groups. (D) Representative H&E-stained colon sections and quantification of histological inflammation scores. (E) Representative images of Masson’s trichrome staining and quantification of collagen volume fraction. (F) Immunofluorescence staining of α -SMA⁺ areas and quantification. (G-H) RT-qPCR analysis of Col1a1 and Col6a1 mRNA expression in murine colonic tissues. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: DPP4 activity in human and murine faecal samples was quantified using Gly-Pro- p -nitroanilide hydrochloride (Gly-Pro-pNA, TargetMol, USA) as the substrate.

Techniques: Inhibition, Derivative Assay, In Vivo, Activity Assay, Staining, Immunofluorescence, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Overview of the pooled CRISPR screen. The genome-scale Brunello library was introduced into Cas9 expressing Caco2 cells, followed by selection of transduced cells. After 10 days, pooled Caco2 cells were infected with HAstV1 for 24h, followed by staining using anti-HAstV capsid antibody. Uninfected cells were sorted to determine the sgRNA counts by next-generation sequencing. ( B ) FcRn protein levels in Cas9-Caco2 cells disrupted for FCGRT or DPP4 using independent sgRNAs per gene or targeted with a control anti-GFP sgRNA. Two independent replicates are shown. ( C ) Representative histogram for surface expression of DPP4 in naïve Caco2 cells stained with anti-DPP4 antibody or isotype control antibody. ( D ) Representative histogram showing DPP4 expression in fixed, permeabilized Caco2 cells disrupted for FCGRT or DPP4 or B2M using gene specific sgRNAs or targeted with a control anti-GFP sgRNA. ( E ) Percentage of DPP4 expressing Caco2 cells disrupted for DPP4 using sgRNAs or targeted with a control anti-GFP sgRNA. (n=3) ( F ) Percentage of anti-HAstV capsid antibody stained Caco2 cells treated with PBS (n=5) or isotype antibody (n=5) or various concentrations of anti-DPP4 antibody (n=6) prior to HAstV1 infection. ( G ) Percentage of anti-HAstV capsid antibody-stained control (n=6), FCGRT (n=6) or DPP4 (n=6) knockout Caco2 cells transfected with HAstV1 RNA at 48h post-transfection. Results were analyzed using Kruskal-Wallis test with Dunn’s post-test (F and G) from two to three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: Caco2 cells were plated in 6-well plates at 2 million cells per well and treated for 24h with 500μM sitagliptin, 25μM vildagliptin, or 20μM, then lysed and processed using DPP4 Activity Assay Kit (Sigma MAK088).

Techniques: CRISPR, Expressing, Selection, Infection, Staining, Next-Generation Sequencing, Control, Knock-Out, Transfection

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Enrichment [-log10 Robust Rank Aggregation (RRA)] scores of positively-selected sgRNAs in HAstV1-negative cells sorted 24hpi of a genome-wide CRISPR-Cas9 Caco2 cell library compared to HAstV1-positive cells, calculated by MAGeCK. ( B,C ) Cas9-Caco2 cells disrupted for FCGRT (n=6-9) or B2M (n=9) using two independent sgRNAs per gene or targeted with a control anti-GFP sgRNA (n=9) were infected with HAstV1or HAstV8, then stained with anti-HAstV capsid antibody at 24hpi. ( D ) Mean fluorescent intensity (MFI) of DPP4 in negative and positive cell populations of Caco2 cells infected with HAstV1 (n=6) or HAstV8 (n=6) infected at 24hpi. ( E ) Cas9-Caco2 cells were disrupted for DPP4 (n=9) using two independent sgRNAs per gene or targeted with a control anti-GFP sgRNA (n=9), then assessed for infection using anti-HAstV capsid antibody 24hpi with HAstV1 or HAstV8. (F ) Percentage of anti-HAstV capsid antibody-stained Caco2 cells treated with PBS (n=4-5) or isotype control (n=4-6) or anti-DPP4 polyclonal antibody (n=6-7) for 12h hours prior to infection with HAstV1 or HAstV8. Results from three independent experiments were analyzed using the Kruskal-Wallis test with Dunn’s post-test (B to F). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: Caco2 cells were plated in 6-well plates at 2 million cells per well and treated for 24h with 500μM sitagliptin, 25μM vildagliptin, or 20μM, then lysed and processed using DPP4 Activity Assay Kit (Sigma MAK088).

Techniques: Genome Wide, CRISPR, Control, Infection, Staining

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A,B ) Enrichment scores of positively-selected sgRNAs in HAstV1-or HAstV8-positive cells sorted 24hpi from a surfaceome CRISPRa Caco2 cell library compared to unsorted cells, calculated by MAGeCK. ( C ) dCas9-Caco2 cells were targeted for DPP4 (n=8) using two independent sgRNAs per gene or a control anti-GFP sgRNA (n=8), then assessed for infection using anti-HAstV capsid antibody 24hpi with HAstV1 or HAstV8. ( D, E ) HAstV1 levels in HEK293T (293T) (n=6) or HEK293T stably expressing FCGRT (293T-FCGRT) (n=8) or DPP4 (293T-DPP4) (n=6) at 24hpi. ( F ) HAstV1 levels in H293T-DPP4 cells treated with isotype control (n=7) or anti-DPP4 antibody (n=7) prior to HAstV infection at 24hpi. Results from three independent experiments were analyzed using Kruskal-Wallis test with Dunn’s post-test (C) or Mann-Whitney test (D to F). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Bars indicate mean of all data points.

Article Snippet: Caco2 cells were plated in 6-well plates at 2 million cells per well and treated for 24h with 500μM sitagliptin, 25μM vildagliptin, or 20μM, then lysed and processed using DPP4 Activity Assay Kit (Sigma MAK088).

Techniques: Control, Infection, Stable Transfection, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Schematic of the CRISPR activation surfaceome screen. The human surfaceome library was introduced into dCas9 expressing Caco2 cells, followed by selection of transduced cells. After 10 days, pooled Caco2 cells were infected with HAstV1 or HAstV8 for 24h, followed by staining using anti-HAstV capsid antibody. We sorted top 3% of the HAstV capsid positive cells to determine the sgRNA counts by next-generation sequencing. ( B ) Representative histogram showing expression of DPP4 in dCas9-Caco2 cells transduced with sgRNAs for DPP4 overexpression. ( C ) FcRn protein levels in dCas9-Caco2 transduced with sgRNA for overexpressing FcRn. Two independent replicates are shown. ( D ) FcRn protein levels in 293T and 293T-FCGRT cells. Two independent replicates are shown. ( E ) Representative histogram showing surface expression of DPP4 in 293T cells stained with anti-DPP4 antibody or isotype control antibody. ( F ) Abundance of DPP4 and HAstV capsid positive 293T and 293T-DPP4 cells infected with HAstV1. ( G, H ) HEK293T or HEK293T-DPP4 cells were transfected with FCGRT (n=5) , DPP4 (n=5) and/or B2M (n=5) plasmids then 48h later were infected and stained with anti-HAstV capsid antibody at 24 hpi. Results were analyzed using Kruskal-Wallis test with Dunn’s post-test (G and H) from three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: Caco2 cells were plated in 6-well plates at 2 million cells per well and treated for 24h with 500μM sitagliptin, 25μM vildagliptin, or 20μM, then lysed and processed using DPP4 Activity Assay Kit (Sigma MAK088).

Techniques: CRISPR, Activation Assay, Expressing, Selection, Infection, Staining, Next-Generation Sequencing, Transduction, Over Expression, Control, Transfection

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) HAstV1 was tested against immobilized FcRn and mAb 8E7 via surface plasmon resonance. ( B ) Maturation process of HAstV VP90 structural protein. ( C ) VP25 protein from HAstV1 (left) and HAstV8 (right) was immobilized and tested against 2-fold dilutions of FcRn ranging from 8μM to 62.5nM via biolayer interferometry. ( D, E ) HAstV1 levels at 24hpi in Caco2 cells treated with PBS (Mock) (n=3-6), non-specific control protein (n=6-7), or soluble FcRN (s-FCRN) (n=7) or soluble DPP4 (s-DPP4) (n=6) prior to infection. Results from 2-3 independent experiments were analyzed using Kruskal-Wallis test with Dunn’s post-test (F and G). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: Caco2 cells were plated in 6-well plates at 2 million cells per well and treated for 24h with 500μM sitagliptin, 25μM vildagliptin, or 20μM, then lysed and processed using DPP4 Activity Assay Kit (Sigma MAK088).

Techniques: SPR Assay, Control, Infection

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A-C ) Gel filtration and SDS-PAGE profiles of HAstV1 VP25 ( A ) HAstV8 VP25 ( B ) and HAstV1 VP34 ( C ). HAstV1 and HAstV8 VP25 were eluted from a HiLoad 16/600 Superdex 200 column and HAstV1 VP34 was eluted from a Superdex 75 Increase 10/300 column. ( D ) Soluble hDPP4 was expressed via transfection in Expi293 cells and eluted via gel filtration on Superdex 200 Increase 10/300 column (left) and further analyzed using MALS (right). The MALS curve (black) is plotted with the derived molecular weight (red) of 191,3000 Dalton ± 0.848%. ( E-G ) Binding experiments testing full-length spike protein from MERS-CoV, ( E ) VP25 from HAstV1 and HAstV8 ( F , left and right, respectively), and VP34 from HAstV1 ( G ) against DPP4. In all cases, each protein was immobilized via biosensor and tested against 2-fold dilutions of DPP4 from either 1μM to 62.5nM ( E, G ) or 1μM to 15.625nM ( F ).

Article Snippet: Caco2 cells were plated in 6-well plates at 2 million cells per well and treated for 24h with 500μM sitagliptin, 25μM vildagliptin, or 20μM, then lysed and processed using DPP4 Activity Assay Kit (Sigma MAK088).

Techniques: Filtration, SDS Page, Transfection, Derivative Assay, Molecular Weight, Binding Assay

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A,B ) HAstV1 and HAstV8 infection at 24hpi in Caco2 and 293T-FCGRT cells treated with PBS (n=6) or nipocalimab (n=6) prior to infection. ( C, D ) HAstV1 and HAstV8 infection at 24hpi in Caco2 or 293T-DPP4 cells treated with PBS (n=6-8) or sitagliptin (n=6-8) prior to infection. ( E ) HAstV1 levels at 24hpi in HIEs differentiated in monolayers on transwell inserts treated with PBS (n=7), nipocalimab (n=9), anti-DPP4 antibody (n=6) and sitagliptin (n=6) prior to infection. Results from three independent experiments were analyzed using Mann-Whitney test (A to D) or Kruskal-Wallis test with Dunn’s post-test (E). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Bars indicate mean of all data points.

Article Snippet: Caco2 cells were plated in 6-well plates at 2 million cells per well and treated for 24h with 500μM sitagliptin, 25μM vildagliptin, or 20μM, then lysed and processed using DPP4 Activity Assay Kit (Sigma MAK088).

Techniques: Infection, MANN-WHITNEY

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Percentage of anti-HAstV capsid antibody-positive Caco2 cells at 24hpi treated with PBS (n=6) or various concentrations of nipocalimab (n=6) prior to HAstV1 infection. ( B ) Percentage of anti-HAstV capsid antibody-positive Caco2 cells treated with PBS (n=6) or vildagliptin (n=6) or teneligliptin (n=6) prior to HAstV1 or HAstV8 infection. ( C ) Fluorescent 7-Amino-4-Methyl Coumarin (AMC) released by DPP4 enzymatic activity in Caco2 cells treated with PBS (Control; n=4) or DMSO or DPP4 inhibitors (n=4). Results were analyzed using Kruskal-Wallis test with Dunn’s post-test ( A ) from three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: Caco2 cells were plated in 6-well plates at 2 million cells per well and treated for 24h with 500μM sitagliptin, 25μM vildagliptin, or 20μM, then lysed and processed using DPP4 Activity Assay Kit (Sigma MAK088).

Techniques: Infection, Activity Assay, Control