dpp4 activity assay kit  (Danaher Inc)

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