Journal: PLoS Genetics

Article Title: Mechanistic insights into global suppressors of protein folding defects

doi: 10.1371/journal.pgen.1010334

Figure Lengend Snippet: (A) CcdB proteins exhibit biphasic refolding kinetics with a fast and slow phase whereas (B) unfolding of CcdB proteins follows single exponential kinetics. The experimental kinetic traces obtained at different GdnCl concentrations are shown in black, while the fits are shown in red. (C) Interaction between native (black), native protein in GdnCl (grey) and refolded (white) CcdB mutant proteins and labeled GyrA14 analyzed by MST. (D) The difference in apparent thermal melting temperatures ( Δ T m = T m M u t a n t − T m W T ) for native proteins in GdnCl (cyan), and refolded proteins (grey). The error bars wherever shown represent the standard deviation from two independent experiments, each performed in duplicates. (E) Binding of 500 nM WT and mutant CcdB to immobilised GyrA14 measured by passing the same concentrations of the analyte (CcdB proteins), after heat stress at two different temperature (40 and 80°C), followed by cooling back to 25°C. A room temperature control (25°C) was also used. The residual active fraction was calculated as described in the materials section. (F) The SEC profiles of a few of the CcdB mutants are shown. The PIM L36A shows aggregation as well as degradation as compared to the WT, and E11R and S12G suppressors. The L36A-S12G has a similar profile like the WT and S12G. Kinetic parameters from the fits are listed in S3 Table and values extrapolated to zero denaturant are listed in S4 Table .

Article Snippet: Briefly, the amount of CcdB protein expressed on the yeast cell surface was estimated by chicken anti-HA antibodies from Bethyl labs (1˸600 dilution) and the GyrA14 binding activity on the yeast cell surface was estimated by incubating the induced CcdB mutants with 100 nM FLAG tagged GyrA14, followed by washing with FACS buffer and subsequent incubation with mouse anti-FLAG antibodies, at a dilution ratio of 1˸300 as described previously [ ].

Techniques: Mutagenesis, Labeling, Standard Deviation, Binding Assay, Control

Journal: PLoS Genetics

Article Title: Mechanistic insights into global suppressors of protein folding defects

doi: 10.1371/journal.pgen.1010334

Figure Lengend Snippet: (A) Analysis of yeast cell surface expression and GyrA14 binding of different CcdB mutants and WT. CcdB WT and mutant plots (blue), are overlaid with plot of uninduced cells (black). In the last two panels, V20F-M32T, L36A-M32T plots (red) are overlaid with plots of V20F-L42E, L36A-L42E (purple) and V20F-S43T, L36A-S43T (green), only M32T is able to suppress the deleterious effects of the V20F and L36A mutations. (B-I) Kinetic and thermodynamic characterisation of CcdB mutants. (B) Thermal unfolding profiles of 5 μM of CcdB-WT, M32T, L42E and S43T mutants carried out by nanoDSF. (C) Equilibrium GdnCl denaturation profiles of 5 μM of CcdB-WT, M32T, L42E and S43T mutants carried out by nanoDSF. (D) Difference in thermal ΔT m ( Δ T m = T m M u t a n t − T m W T ) (in cyan), and thermodynamic stability assayed by chemical denaturation, ΔΔG° ( Δ Δ G ° = Δ G M u t a n t ° − Δ G W T ° ) (in grey) of the CcdB mutants. (E) Thermal unfolding profiles of 5 μM of native proteins in 1.5 M GdnCl (dotted lines) and refolded CcdB proteins in the same concentration of GdnCl (dashed lines). (F-G) The observed rate constants and amplitudes of the fast phase (black) and slow phase (grey) of refolding (1.5 M GdnCl) of WT and CcdB mutants (see also S9 Table ). (H-I) The observed rate constants and amplitudes of unfolding (3.5 M GdnCl) of WT and CcdB mutants. The error bars wherever shown represent the standard deviation from two independent experiments, each performed in duplicates.

Article Snippet: Briefly, the amount of CcdB protein expressed on the yeast cell surface was estimated by chicken anti-HA antibodies from Bethyl labs (1˸600 dilution) and the GyrA14 binding activity on the yeast cell surface was estimated by incubating the induced CcdB mutants with 100 nM FLAG tagged GyrA14, followed by washing with FACS buffer and subsequent incubation with mouse anti-FLAG antibodies, at a dilution ratio of 1˸300 as described previously [ ].

Techniques: Expressing, Binding Assay, Mutagenesis, Nano Differential Scanning Fluorimetry, Concentration Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) 1×10 6 SL-1 and SL-3 cells were cultured for 24 hr, whole-cell lysate (WCL) and supernatant (SUP) were prepared and subjected to cytokine array analyses. Each cytokine has one pair of duplicate spots. A1-2, A23-24, and F1-2 are experimental positive control, and F23-24 is an experimental negative control. (B) C57BL/6 mice were intranasally inoculated with 5×10 4 PFU of MHV68-H2bYFP or mock inoculated with PBS. At day 16 post-infection, splenocytes were isolated and subjected to flow cytometry, the flow plot represented the strategy gating YFP+ MHV68 infected cells (left panel); serum was prepared from 10 virus-infected mice or mock-infected mice, followed by IL16 ELISA assay (right panel). Histograms represented mean ±SD of 10 individual mice (two experiments, n = 5 for each experiment). p value was determined by two-tailed unpaired t-test. (C) WT MEFs were infected with MHV68 at an MOI of 1, total RNA was isolated from infected cells harvested at the indicated time points and subjected to qRT-PCR analyses with specific primers corresponding to IL16 and MHV68 ORF50 gene. The relative RNA amount was normalized to GAPDH in each sample. Histograms represented the mean of three independent biological replicates ±SD, p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Cell Culture, Positive Control, Negative Control, Infection, Isolation, Flow Cytometry, Virus, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) Intracellular staining of IL16 in splenocytes isolated from IL16+/+, IL16+/-, and IL16-/- mice. (B) Immunoblot detection of IL16 expression in splenocytes isolated from IL16+/+, IL16+/-, and IL16-/- mice. (C) Representative flow plots showed flow cytometric analyses of splenocytes from IL16+/+ (WT) and IL16-/- (KO) mice. (D) The statistic analyses of CD4+ T, CD8+ T, and B cells (Left panel); follicular (FC) B, marginal zone (MZ) B, and mature B cells (Right panel) in splenocytes from WT and IL16 KO mice. Histograms represented mean ±SD of 8 individual mice (two experiments, n = 4 for each experiment). ns = not significant.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Staining, Isolation, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO MEFs were infected with MHV68 at an MOI of 1 or 0.05. The infected cells were harvested at the indicated time points and immunoblot analyses were performed with specific antibodies as indicated. Actin was used as a loading control. (B) WT and IL16 KO MEFs were infected with MHV68 at an MOI of 5 or 0.05. The supernatant was harvested at the indicated times and viral titers were determined by TCID50 assay. Results are means from triplicate samples. Error bars represented standard deviations. ns = not significant. (C) WT and IL16 KO mice were intranasally infected with 5×10 4 PFU of MHV68. Lungs of infected mice were collected at day 4 and 7 post-infection. Virus titers were determined by TCID50 assay. Data represented one of two independent experiments with 5 or 7 mice per group. ns = not significant. Each symbol represented an individual mouse. The horizon line indicated geometric mean titer. (D) Vector or IL16-expressing plasmids with Flag tag were transfected into BHK21 cells for 24 hr, followed by MHV68 infection at an MOI of 5 or 0.05. The infected cells were harvested at the indicated time points and immunoblot analyses were performed with specific antibodies as indicated.–and + represents the cells transfected with vector and IL16-expressing plasmids with Flag tag, respectively. (E) Supernatant was harvested at the indicated times and viral titers were determined by TCID50 assay. Results were means from triplicate samples. Error bars represented standard deviations. ns = not significant.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Western Blot, Control, TCID50 Assay, Virus, Plasmid Preparation, Expressing, FLAG-tag, Transfection

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and three IL16 KO single clones were stimulated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr, respectively. Immunoblot analyses were performed with the indicated antibodies. GAPDH was used as a loading control. (B) WT and IL16 KO cells (clone E6) were stimulated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the indicated antibodies. GAPDH was used as a loading control. MHV68 viral genome was determined by qPCR with the primers specific to the MHV68 ORF50 coding region. The relative copy of the MHV68 viral genome was normalized to GAPDH in each sample. (C) The mRNA expression of MHV68 viral gene ORF73, ORF50, ORF59, and ORF25 was determined by qRT-PCR. The relative RNA amount was normalized to GAPDH in each sample. Histograms represented the mean of three independent biological replicates ±SD, p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Clone Assay, Western Blot, Control, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) The diagram showed the potential cleavage sites of IL16. (B) 293T cells were transfected with IL16 or mutants with Flag tag. At 48 hr post-transfection, the supernatant was collected and subjected to IL16 ELISA assay; whole-cell lysates (WCL) and supernatant (SUP) were prepared and subjected to immunoblot analyses with the indicated antibodies. (C) IL16 KO SL-1 cells were transfected with vector (Vec), IL16-, or IL16(D516A)-expressing plasmid with Flag tag, followed by anti-mouse Ig(G+M) treatment for 48 hr. The whole-cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Transfection, FLAG-tag, Enzyme-linked Immunosorbent Assay, Western Blot, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were inoculated intranasally with 5×10 4 PFU of MHV68-H2bYFP. Mice inoculated with 5×10 4 PFU of WT MHV68 were used as a control to gate YFP+ cells. Splenocytes were isolated at day 16 and day 18 post-infection. (A) Representative flow plots showing the identification of MHV68-infected YFP+ cells. (B) Frequency of YFP+ cells at day 16 post-infection. Results were compiled from two independent experiments with 8–9 mice per group. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency of infected cells. ns = not significant. (C) Frequency of splenocytes capable of reactivating virus by ex-vivo assay at day 16 post-infection. Serial dilutions of splenocytes were plated on MEFs and the presence of reactivating virus was determined by the presence of cytopathic effect (CPE). Representative results were from two independent experiments with 8–9 mice per group. (D) Frequency of YFP+ cells at day 18 post-infection. Results were compiled from two independent experiments with 10–12 mice per group. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency of infected cells. ns = not significant. (E) Frequency of splenocytes capable of reactivating virus by ex-vivo assay at day 18 post-infection. Data were generated from two independent experiments, 5 to 6 mice per experiment per group.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Control, Isolation, Infection, Virus, Ex Vivo, Generated

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were intranasally inoculated with 5×10 4 PFU of MHV68-H2bYFP and splenocytes were harvested at day 16 post-infection. (A) Representative flow plots showing the identification of MHV68-infected YFP+ cells. (B) Representative flow plots of YFP+ germinal center B cells (CD19 + CD95 + GL-7 + YFP + ). (C) Quantitation of the percentage of YFP+ germinal center B cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (D) Representative flow plots of YFP+ plasma cells (CD3 - YFP + B220 low CD138 + ). (E) Quantitation of the percentage of YFP+ plasma cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (F) Quantitation of the percentage of total germinal center B cells (CD19 + CD95 + GL-7 + ). (G) Quantitation of the percentage of total plasma cells (CD3 - B220 low CD138 + ). ns = not significant. p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Quantitation Assay, Clinical Proteomics, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were inoculated intranasally with 5×10 4 PFU of MHV68-H2bYFP. Splenocytes were isolated at day 16 post-infection and subjected to flow cytometry analyses. (A) Representative flow plots of CD4+ and CD8+ T cells from infected WT and IL16 KO mice. (B) Quantitation of the percentage of CD4+ and CD8+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (C) Representative flow plots of IFN-γ+CD4+ T cells from infected WT and IL16 KO mice. (D) Quantitation of the percentage of IFN-γ+CD4+ and CD44+CD4+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (E) Quantitation of the percentage of IL4+CD4+ and IL2+CD4+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (F) Representative flow plots of IFN-γ+ CD8+ T cells from infected WT and IL16 KO mice. (G) Quantitation of the percentage of IFN-γ+CD8+ and TNF-α+CD8+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. ns = not significant. p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Isolation, Infection, Flow Cytometry, Quantitation Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) The murine M12 B lymphoma cells were transfected with renilla reporter, a luciferase reporter (pGL2) driven by RTA proximal promoter (RTAp), together with IL16-expressing plasmid with Flag tag or vector alone (Vec). Luciferase activity was normalized to renilla activity and Luciferase value was reported as fold increase in luciferase activity over basal promoter activity. Each sample was done in triplicate (two independent experiments). IL16 expression was detected by immunoblot with a Flag antibody. (B) M12 cells were transfected with RTAp together with vector (Vec), IL16-, IL16(D506A)- or IL16(D516A)-expressing plasmid with Flag tag. At 24 hr post-transfection, transfected cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 12 hr. IL16, IL16 (D506A), and IL16 (D516A) expression was detected by immunoblot with Flag antibody. Luciferase value was reported as fold increase in luciferase activity over basal promoter activity. Each sample was done in triplicate (two independent experiments).

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, FLAG-tag, Activity Assay, Western Blot

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO SL-1 cells were treated with (+) or without (-) 20 μM JAKs inhibitor AG490 for 1 hr, followed by stimulation with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. GAPDH was used as a loading control. (B) WT and IL16 KO SL-1 cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. (C) Quantitation of phosphorylated STAT relative to total STAT based on immunoblot detection in C using the ImageJ image analysis software.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Western Blot, Control, Quantitation Assay, Software

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO SL-1 cells were pretreated with DMSO, 40 μM, or 80 μM STAT5 inhibitor (STAT5-I) for 1 hr, followed by stimulation with anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. (B) SL-1 cells were transfected with vector, STAT3-expressing plasmid, or STAT3C-expressing plasmid with Flag tag, followed by anti-mouse Ig(G+M) (5 μg/mL) treatment for 48 hr. Immunoblot analyses were performed with the indicated antibodies. (C) WT and IL16 KO SL-1 cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the specific antibodies as indicated. (D) Quantitation of phosphorylated STAT3(Y705) relative to total STAT3 and p21 relative to GAPDH based on immunoblot detection in C using the ImageJ image analysis software. (E) WT and IL16 KO SL-1 cells were treated with DMSO or Orthovanadate (50 μM) in the presence or absence of anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the specific antibodies as indicated.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, FLAG-tag, Quantitation Assay, Software

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: MHV68 infection induces IL16 production, which, in turn, increases STAT3(Y705) phosphorylation, subsequently reduces p21 expression, and inhibits MHV68 reactivation. Meanwhile , IL16 partially inhibits RTA promoter activity and STAT3(S727) phosphorylation, contributing to the inhibition of MHV68 reactivation. Ultimately, MHV68-induced IL16 helps to maintain MHV68 latency.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Phospho-proteomics, Expressing, Activity Assay, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is an epithelial cytokine expressed in alveolar epithelium and airway basal cells in human healthy and asthmatic lungs. (A) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in the LungMAP single-cell human lung atlas. Uniform manifold projection (UMAP) plots show the clustering of 347,970 lung cells (10 single-cell datasets, 148 normal human lung samples from 104 donors: adult, child, and adolescent). Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B and C) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in epithelial cells from human healthy (B) and asthmatic (C) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (B; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (C; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas and downloaded from https://asthma.cellgeni.sanger.ac.uk .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Generated, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Single-cell RNA-seq analysis of IL33 and TSLP expression in human lungs and gating strategy for analysis of mouse lung epithelial cells by flow cytometry. (A and B) Single-cell RNA-seq analysis of IL33 and TSLP expression in epithelial cells from human healthy (A) and asthmatic (B) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (A; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (B; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas , and downloaded from https://asthma.cellgeni.sanger.ac.uk . (C) Gating strategy of Epcam + epithelial cells and CD31 + endothelial cells in the lung of a naïve WT mouse. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with two distinct rat IgG1 isotype controls (rat IgG1 clone eBRG1, D, red; rat IgG1 clone RB40.34, E, red) for the anti-TL1A antibody (rat IgG1, MAB7441, clone 293327). Double staining was performed with antibodies against RAGE (D, green) or IL-33 (E, green). Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Control, Immunohistofluorescence, Staining, Double Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is expressed in mouse alveolar epithelium at steady state. (A) Visualization of Tnfsf15 (TL1A) expressing cells in the LungMAP single-cell mouse lung atlas. UMAP plots show the clustering of 95,658 lung cells (17 samples from late developmental stage to postnatal day 28). The different cell types in the lungs of naïve mice are indicated on the left. Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B) Single-cell RNA-seq analysis of Tnfsf15/TL1A and Il33 gene expression in mouse lung epithelium. UMAP plots show clustering and cell type annotation of 12,536 mouse lung epithelial cells (seven samples from the emergence of the alveolus to postnatal day 28) . The number and percentage of epithelial cells expressing Tnfsf15/TL1A , Il33 , or both are indicated on the right. Results are visualized using ShinyCell and are based upon data obtained by and downloaded from http://www.lungmap.net . (C) Flow cytometry analysis of cell surface TL1A expression on live CD31 + CD45 − endothelial cells and Epcam + CD31 − CD45 − epithelial cells in the lung of a naïve wild type C57BL/6J mouse at steady state. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with antibodies against TL1A (D and E) and RAGE (D) or IL-33 (E) proteins. A tyramide signal amplification (TSA)-based immunofluorescence method was used to detect TL1A-expressing cells in situ. Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Expressing, Generated, RNA Sequencing, Gene Expression, Flow Cytometry, Immunohistofluorescence, Staining, Amplification, Immunofluorescence, In Situ

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: High throughput proteomic analyses of lung ILC2s stimulated ex vivo with IL-33 and/or TL1A. (A) Flow cytometry of cultured lung ILC2s ex vivo. Representative histograms of ST2, CD90.2, Sca-1, CD25, ICOS, KLRG1, and DR3 expression at the surface of cultured ILC2s, 3 days after ILC2 cell isolation from the lung and ex vivo culture in the presence of IL-2. Phenotypic analysis was performed on live Lin – CD45 + cells. (B–D) Large-scale label-free proteomic analyses of mouse lung ILC2s after ex vivo overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plots of IL-33-stimulated ILC2s (B) or TL1A-stimulated ILC2s (C) compared with non-stimulated cells (NS; in culture with IL-2 alone). Volcano plot of IL-33/TL1A-stimulated ILC2s compared to IL-33-stimulated cells (D). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS and IL33 stimulation; n = 3 for TL1A and IL33/TL1A stimulations), using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Representative examples of proteins found modulated in each comparison are shown in color. (E) Flow cytometry of cultured lung ILC2s after 14 h of co-stimulation with IL-33 and TL1A in the presence of IL-2 (ILC2 culture used in ). Intracellular cytokine staining revealed that >99% of ILC2s co-expressed IL-9 and IL-13 intracellularly. Phenotypic analysis was performed on live Lin − CD45 + CD90.2 + cells.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: High Throughput Screening Assay, Ex Vivo, Flow Cytometry, Cell Culture, Expressing, Cell Isolation, Quantitative Proteomics, Comparison, Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A synergizes with IL-33 to induce an IL-9-producing ILC9 phenotype in lung ILC2s. (A and B) Large-scale label-free proteomic analyses of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice and cultured with IL-2 prior to overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plot of IL-33/TL1A-stimulated ILC2s (ILC9 cells) compared with nonstimulated cells (NS; in culture with IL-2 alone) (A). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS; n = 3 for IL33/TL1A stimulation) using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Examples of proteins modulated in both IL-33/TL1A-stimulated ILC2s and IL-33-stimulated ILC2s are shown in blue. Proteins shown in red are representative of molecules specifically modulated in IL-33/TL1A-stimulated ILC2s (A). Heat-map of fold changes of selected proteins in three independent biological replicates (B). (C–K) Analysis of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice , and cultured with IL-2 prior to 14 h stimulation with rIL-2 ± rIL-33 ± rTL1A. Flow cytometry analysis of live Lin − CD45 + cells (C, E, and J), frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) (D and K), and MFI fold change of IL-9 in ILC2s (E), after cytokines treatment and restimulation by PMA, ionomycin, and brefeldin A (4 h, C–E) or brefeldin A (4 h, J and K). Concentration of IL-9 secreted by ILC2s, measured by ELISA (F). Relative STAT5 mRNA expression levels measured by real-time qPCR (G). Samples were normalized to the expression of HPRT and are shown relative to IL-2-stimulated ILC2s. Immunoblot analysis of activated phosphorylated STAT5 (pSTAT5) and α-tubulin (H) or β-actin (I); Arrowheads indicate the migration of the protein of interest; cropped images. Cultured ILC2s were treated with rIL-2 + rIL-33 + rTL1A and increasing doses of a STAT5 inhibitor (STA5i, CAS 285986-31-4) or control vehicle (DMSO) (I–K). Numbers inside outlined areas (C) indicate percent of cells in the relevant gate. Each symbol represents an individual biological replicate (D–G and K). Data are pooled from six (D and E), six to eight (F) or three (G and K) independent experiments, or are representative of six (C and E) or three (H–J) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (D–G and K): ns not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Isolation, Cell Culture, Quantitative Proteomics, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Migration, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s ex vivo. (A) Analysis of cultured lung ILC2s 14 h after ex vivo stimulation by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Flow cytometry analysis of live Lin − CD45 + cells and frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) after cytokine treatment and incubation with brefeldin A (4 h), without restimulation by PMA and ionomycin. Numbers inside outlined area indicate percent of cells in the relevant gate and data are representative of eight independent experiments. (B) Concentration of IL-9 secreted by ILC2s treated with rIL-2 (20 ng/ml) and various concentrations of rIL-33 and rTL1A measured by ELISA. (C and D) MFI of nuclear factor IRF4 (C) and flow cytometry (D) of ILC2s 14 h after ex vivo stimulation of cultured ILC2s by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Numbers inside outlined areas (D) indicate percent of cells in the relevant gate and data are representative of three independent experiments. (E) Immunoblot analysis of JunB and α-tubulin14 h after cytokine stimulation of lung ILC2s; Arrowheads indicate the migration of the protein of interest; cropped image. Data are representative of three independent experiments. (F–H) Relative mRNA expression levels by real time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed relative to IL-2-stimulated ILC2s (F) or relative to HPRT mRNA quantity (G and H). (I and J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-33 + rTL1A ± rIL-2 ± rIL-7 ± rTSLP. Frequency of IL-9 high ILC2s (Lin − CD45 + CD90.2 + cells), after cytokines treatment and re-stimulation by PMA, ionomycin and brefeldin A (4 h, I). Concentration of IL-9 secreted by ILC2s, measured by ELISA (J). (K) Concentration of IL-9 (ELISA) secreted by ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rIL-4 ± rTGF-β. Each symbol represents an individual biological replicates with n = 2–5 independent experiments (A–C and F–K). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (A, C, and F–J) or Dunnett’s (B and K) multiple-comparisons tests: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. In H, all significant P values are annotated with stars, all other comparisons are not significant. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Ex Vivo, Cell Culture, Flow Cytometry, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Migration, Expressing

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A induce phenotypic changes in cultured lung ILC2s at the protein and mRNA levels. (A–J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rTL1A. MFI of the indicated cell surface markers determined by flow cytometry (A, B, D, and E). Relative mRNA expression levels of various genes (C and F–I), including genes characteristic of ILC1s or ILC3s (I), determined by real-time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed as relative to HPRT mRNA quantity. Concentration of IL-5 or IL-13 in cell supernatants, measured by ELISA assay (J). Each symbol represents an individual biological replicate from independent experiments (A–J). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test (B, E, and J) or one-way ANOVA followed by Tukey’s multiple-comparisons test (A, C, D, and F–I): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. In I, all significant P values are annotated with stars, all other comparisons are not significant.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Cell Culture, Ex Vivo, Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A cooperates with IL-33 for induction of IL-9 high ILC2s in vivo. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B) Gating strategy of IL-9 high IL-5 + IL-13 + ILC2s. (C–I) Flow cytometry of IL-5 + IL-13 + ILC2s gated on live ILCs (Lin − CD45 + CD90.2 + cells) (C) and IL-9 high ILC2s gated on live IL-5 + IL-13 + ILC2s (E), frequency of lung IL-5 + IL-13 + ILC2s among live ILCs (D), IL-9 high ILC2s among live IL-5 + IL-13 + ILC2s (F), and IL-9 high IL-13 + ILC2s among live ILCs (G) or IL-9 high ILCs (H), and concentration of IL-9 in BAL fluids (ELISA assay, I) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate and data are representative of two independent experiments (C and E). Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (D) or Dunnett’s (F, G, and I) multiple-comparisons tests: ns, not significant, ** P < 0.01, **** P < 0.0001. (J) Frequency of lung eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells, at day 7 after a single i.n. exposure to rIL-33 or rIL-33 plus rTL1A. Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test: * P < 0.05. (K and L) Multiphoton imaging (K) and intravital microscopy (L) of whole lungs of INFER IL-9 fluorescent reporter mice, with detection of IL-9-eGFP + ILC2s (green) and staining of blood vessels (red) and collagen fibers (blue), 16–18 h after a single i.n. administration of IL-33/TL1A combination (1 μg rIL-33 plus 5 μg rTL1A). To increase the numbers of lung IL-9 high ILC2s accessible to in vivo imaging, the single i.n. exposure to IL-33/TL1A combination was performed after prior expansion of lung ILC2s by repeated i.p. injections of IL-33 (K and L). Multiphoton image (K) is a 3D reconstitution of stitched images (7 × 7 tiles and 181 z-stack). Time-lapse images (L) illustrate the migratory behavior of IL-9-eGFP + ILC2s. Time in h/min/s. Scale bars: K, 300 μm; L, 20 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Imaging, Intravital Microscopy, Staining, In Vivo Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s in vivo. (A) Gating strategy and representative flow cytometry plots of live lung ILCs (live Lin − CD45 + CD90.2 + cells), live lung IL-5 + IL-13 + ILC2s (live IL-5 + IL-13 + ILCs) and live lung IL-9 high ILC2s (live IL-9 high IL-5 + IL-13 + ILC2s) in vivo in wild type (WT) C57BL/6J mouse, 14 h after a single i.n. administration of rIL-33 (1 μg) and rTL1A (5 μg). (B) Verification of the absence of contamination of the IL-5 + IL-13 + ILC2s and IL-9 high ILC2s populations by TCR + cells (T cells and NKT cells) using anti-TCRβ and anti-TCRγδ antibodies. (C) Confirmation of the expression of IL-5 and IL-13 in live Lin − CD3/TCR − NK1.1 − CD45 + CD90.2 + lung ILCs using antibodies against CD3/TCR and NK1.1 with a different fluorescence from the Lin cocktail (CD4, CD19, CD45R, CD11b, CD11c, Ter119, Ly6G, FcεRI). (D and E) Frequency of lung IL-9 high Lin − cells among live CD45 + cells (D), and flow cytometry of IL-9 high IL-13 + ILC2s (live IL-9 high IL-13 + Lin − CD45 + CD90.2 + cells) (E) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate. (F) Frequency of lung IL-9 high Lin − cells among live CD45 + cells of WT mice pretreated with six daily i.p. injections of rIL-33 (days 1–6) prior to one i.n. injection of PBS or rIL-33 and/or rTL1A (day 7). Flow cytometry analyses were performed on day 8. (G) Frequency of IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αIL-2 mAb) or without (Iso, isotype control mAb) IL-2 blockade. (H and I) Analysis of IL-9 and TL1A release in BAL fluids by ELISA at different time points after the third exposure to A. alternata in a chronic exposure model (repeated i.n. administration of 12.5 μg A. alternata at days 0, 3, and 6). Each symbol represents an individual mouse and data are pooled from two (D and G) or three (F, H, and I) independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (G) or one-way ANOVA followed by Dunnett’s multiple-comparison test (D, F, H, and I): * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Expressing, Fluorescence, Injection, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s accumulate around blood vessels after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by multiphoton imaging in the cleared lung of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. 360° rotation of a 3D static representation at a frame rate of 25 fps (500 frames per 20 sec).

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s migrate along collagen fibers after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by lung intravital multiphoton imaging of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A functions as an epithelial alarmin rapidly released after allergen exposure. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B–F) Analysis of TL1A and IL-33 release in BAL fluids after a single allergen exposure. TL1A (B and E), IL-33 (C and F), and LDH (D) levels in BAL fluids were determined by ELISA (B, C, E, and F) or LDH (D) assays, 15 min (B–D) or at different time points (E and F) after a single i.n. administration of A. alternata extract (12.5 μg). Each symbol represents an individual mouse and data are pooled from two independent experiments (B–F). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (B–D) or Dunnett’s (E and F) multiple-comparisons tests: ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G–K) Analysis of TL1A release in cell supernatants after exposure of TL1A-expressing cells to A. alternata or bee venom phospholipase A2 (PLA2). U2OS epithelial cells transfected with a mouse TL1A-Flag expression vector (mTL1A-Flag vector) or control vector were analyzed by indirect immunofluorescence microscopy with anti-mTL1A and anti-Flag antibodies (G). Scale bar, 20 μm. TL1A (H and J) and LDH (I and K) levels in cell supernatants were determined by ELISA (H and J) or LDH cytotoxicity assays (I and K) 15 min after treatment with A. alternata extract ( A. alternata , H and I) or 1 h after treatment with bee venom PLA2 (J and K). NT, not treated. Each symbol represents an individual biological replicate and data are pooled from three independent experiments (H–K). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (treatment versus NT): ** P < 0.01, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Control, Immunofluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A is important for early induction of IL-9 high ILC2s after allergen exposure. (A) Treatment schedule of naïve WT mice. (B) IL-9 mRNA levels in the lungs analyzed by qPCR at different time points after a single allergen exposure. Data are expressed as relative to IL-9 mRNA levels in mice treated with PBS. (C–H) Flow cytometry and frequency of IL-9 high Lin − cells among live CD45 + cells (C and D) and IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) (E and F), flow cytometry (G), and MFI of IRF4 expression in ILC2s (H), in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αTL1A mAb) or without (Iso, isotype control mAb) TL1A blockade. Numbers inside outlined areas indicate the percent of cells in the relevant gate (C, E, and G) and data are representative of two (G) or three (C and E) independent experiments. Each symbol represents an individual mouse and data are pooled from three (D and F) or two (B and H) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (B) or unpaired two-tailed Student’s t tests (D, F, and H): ns, not significant, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: ILC9 cells have an increased capacity to initiate IL-5-dependent allergic airway inflammation. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice by a single i.v. adoptive cell transfer of classical IL-33-activated ILC2s (ILC2) or IL-33/TL1A-activated ILC2s (ILC9). (B–H) Flow cytometry (B and D) and frequency of eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells from BALF (C and F) or lung (E and G), and number of Red5 + ILC2s or ILC9s in total lung of mice (H), at day 7 after a single i.v. adoptive transfer of 5 × 10 5 ILC2s or ILC9s in separate host mice. Adoptively transferred ILC2s and ILC9s were prepared from Rag2 −/− mice ( Il5 +/+ cells) (B–E) or Red5 mice ( Il5 −/− cells) (F–H). Control mice received an intravenous injection of PBS. Red5 + cells indicate the activity of the Il5 promoter. Each symbol represents an individual mouse and data are representative (B and D) or pooled (C and E–H) from two independent experiments. (I–K) Live imaging of ILC2s and ILC9 cells in the lung. Lung intravital microscopy was performed 1–4 h after adoptive transfer of 6 × 10 5 of each cell type in the same host (green, classical IL-33-activated ILC2s-CFSE + ; red, IL-33/TL1A-activated ILC9 cells-CTO + ) (I). Imaging of the migratory behavior of ILC2s and ILC9 cells in the lung (J) and cell quantification from lung intravital microscopy data (K). Time-lapse images, 2 h after adoptive cell transfer (J). A maximum intensity projection of stitched images (2 × 2 tiles and 18 z-stack) is shown (K). Time in h/min/s. Scale bars: J, 20 μm; K, 100 μm. Lung intravital microscopy data are representative (J and K) or analyzed (K) from three adoptive transfer experiments on four mice. Data are expressed as mean (±SEM) with P values determined by paired two-tailed Student’s t test (K) or one-way ANOVA followed by Tukey’s multiple-comparisons test (C and E–H): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Adoptive Transfer Assay, Control, Injection, Activity Assay, Imaging, Intravital Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Adoptively transferred ILC2s and ILC9s are equally recruited to the lung and exhibit an ameboid-like mode of migration. IL-33-activated ILC2s (CFSE/green), IL33/TL1A-activated ILC9s (CTO/red), blood vessels (Evans Blue/dark blue), and collagen fibers (second harmonic generation/light blue) were observed by lung intravital multiphoton imaging 2 h after intravenous adoptive transfer (6 × 10 5 cells). Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Migration, Imaging, Adoptive Transfer Assay

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 maintains BTIC self-renewal. ( a ) Morphology of self-renewing spheres and immunofluorescence of neuronal and glial lineage markers in differentiated cells. Scale bar=200 μm. ( b ) Immunofluorescence of ING5 in undifferentiated (upper panels) and cells differentiated for 5 days (lower panels). Scale bar=20 μm. ( c , d ) The mRNA levels ( c ) and protein levels ( d ) of ING5 decrease during differentiation in BT 189 cells. n =4. ( e ) Immunoblotting of ING5 protein in the BT 12, and BT 134 lines during differentiation. ( f ) ING5 overexpression increases sphere formation rates and average volumes in BTIC sphere formation assays ( n =3, ** P <0.01, * P <0.05). Scale bar=400 μm. ( g ) ING5 knockdown by siRNA decreases sphere formation rates and sphere sizes of BTICs ( n =4, ** P <0.01, * P <0.05). Scale bars=400 μm. ( h ) (Left) Sphere formation rates for cell lines stably expressing shRNAs against ING5 (shR1 and shR2-ING5) or control non-targeting shRNA (shR-ctr). ( n =4, ** P <0.01, * P <0.05). (Right) Fluorescence of the RFP reporter in stable cell lines superimposed with differential interference contrast (DIC) images. Scale bar=100 μm. ( i ) RT-qPCR analysis of stem cell core transcription factors and stem cell markers after ING5 overexpression. ( j , k ) Western blot analysis of the neural stem cell marker Nestin and neuronal lineage marker Tubb3 in response to ING5 overexpression ( j ) and knockdown ( k ).

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Western Blot, Over Expression, Knockdown, Stable Transfection, Expressing, Control, shRNA, Fluorescence, Quantitative RT-PCR, Marker

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 increases the stem cell pool and inhibits differentiation. ( a ) Flow cytometry analysis of CD133/CD44 positive cells in iPB cell lines (top panels) and CD44 positive cells in shRNA cell lines (bottom panels), gated by isotype control. ( b ) (Left) Mitotic pair analysis of the three division modes: symmetric proliferating (sym-pro), symmetric differentiating (sym-diff) and asymmetric (asym) cell division, in iPB cell lines. Over 150 pairs were counted for each group in one experiment. n =3, * P <0.05. (Right) An example of cell division symmetry based on the distribution of stem cell factor Nestin with the Red arrow indicating symmetric differentiating division and the white arrow asymmetric division. ( c ) Morphological changes of iPB cells before (Day 0) and after (Days 1–3) differentiation induced by 1% FBS. Scale bar=100 μm. ( d ) After differentiation for 5 days, immunofluorescence of Nestin and Tubb3 in shRNA cell lines with an RFP reporter. Scale bar=200 μm. ( e ) Western blot of Nestin and Tubb3 in differentiated shRNA cell lines corresponding to cells shown in d .

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Flow Cytometry, shRNA, Control, Immunofluorescence, Western Blot

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 activates mitogenic pathways to promote self-renewal. ( a ) The sphere formation rates of iPB-ctr and iPB-ING5 overexpressing cells at three successive passages in the absence of EGF and FGF treatment. ( n =3, ** P <0.01, *** P <0.001). ( b ) DIC images of spheres from the tertiary sphere passage in iPB cells. Scale bar=400 μm. ( c ) Sphere formation rate under treatment with protein kinase inhibitors ( n =3, * P <0.05). ( d ) Protein and phosphorylated protein levels of effectors in the PI3K and MEK pathways. Cells were treated with PX-866 at 1 μ M and PD184352 at 2 μ M for 48 h. ( e ) Immunofluorescence of Nestin and Tubb3 in differentiated iPB control (Left panels) or iPB-ING5 cells (Right panels) treated with 1 μ M PX-866 or 2 μ M PD184352. Scale bar=200 μm. ( f ) Flow cytometry analysis of the CD133 positive population in PX-866 (1 μ M ) and PD184352 (2 μ M ) treated BTIC 189 cells, gated by isotype control.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Control, Flow Cytometry

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: The FSH pathway transduces effects of ING5 on stem cell properties. ( a ) Sphere formation assay for cells treated with calcium modulators and FSHR blocking antibody (Anti-FSHR) at the indicated concentrations ( n =3, * P <0.05 and ** P <0.01 compared to iPB-ctr/DMSO; # P <0.05 and ## P <0.01 compared to iPB-ING5/DMSO). ( b ) IPA downstream function analysis indicates the FSH pathway is elevated by ING5. Genes positively correlated with this function were listed with fold changes. ( c ) RT-qPCR of genes related to hormone and steroidogenesis functions. ( n =3, * P <0.05, ** P <0.01) ( d ) The expression levels of FSHB and FSHR genes in BT 189 cells before and after differentiation for 1–5 days. ( e ) Immunostaining for ING5 and FSHR in iPB cells. Scale bar=100 μm. ( f ) Flow cytometry analysis of CD133 positive cells in BT 189 cells treated with FSHR neutralizing antibody or IgG control, gated by isotype control. ( g ) Immunofluorescence of Nestin and Tubb3 shows inhibition of the FSH pathway induces neuronal differentiation. Scale bar=200 μm. ( h ) FSH recombinant protein treatment at indicated concentrations increases sphere formation rates in shRNA cell lines ( n =3, * P <0.05 and ** P <0.01 compared to untreated shR-ctr; # P <0.05 compared to untreated shR-ING cells). ( i ) FSH recombinant protein treatment induces sphere-forming abilities in iPB-ctr cells but not in ING5 overexpressing cells ( n =3, ** P <0.01). ( j ) Sphere formation rates for cells treated with Anti-FSHR or BAPTA alone, and the combination of both ( n =3, * P <0.05). ( k ) FSH treatment at 5 ng/ml for 3 days induces the expression of OCT4 and Nestin in BT 189 cells.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Tube Formation Assay, Blocking Assay, Quantitative RT-PCR, Expressing, Immunostaining, Flow Cytometry, Control, Immunofluorescence, Inhibition, Recombinant, shRNA

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: PHD motif is required for the function of ING5 in BTICs and ING5 levels negatively correlate with survival of GBM. ( a ) Sphere formation assays in iPB cell lines overexpressing wild-type (ING5-FLAG) and PHD-deleted ING5 (ΔPHD) ( n =3, ** P <0.01). ( b ) Western blot shows the protein levels of endogeneous ING5, wildtype ING5 with a FLAG tag and PHD-deleted ING5 (black arrows) in three iPB cell lines. ( c ) ChIP analysis of ING5 binding to promoters of target genes presented as fold enrichment relative to IgG controls. The endogenous ING5 in BT 189 cells, overexpressed ING5 with a Flag tag in iPB-ING5 cells and overexpressed PHD-deleted ING5 protein with a Flag tag were immunoprecipitated by the ING5 antibody and Flag antibody respectively. The upper panels are the schematic representation of the location of the primer sets and promoter regions enriched for ING5 binding were shown in red. ( d ) Kaplan–Meier survival analysis of TCGA GBM patients with high and low levels of ING5 expression (stratified by mean value, n =114). ( e , f ) ING5 expression levels negatively correlate with survival of the Proneural subtype ( n =24) and the Classical subtype ( n =30) of GBM patients. ( g ) The relationship of ING5 levels to survival in the SOX2-low group of patients (ING5, SOX2 stratified by median values, n =61). ( h ) Model for how ING5 functions in the maintenance of BTIC self-renewal. In the absence of growth factors, ING5 induces FSH and calcium signaling by promoting transcription of the FSH receptor and ligand genes, and various plasma membrane calcium channel genes. The FSH and calcium signaling pathways further activate PI3K/AKT and MEK/ERK signaling to induce stem cell features and the expression of stemness factors OCT4, OLIG2 and Nestin. Gene activation by ING5 is dependent on its PHD motif to target ING5-associated histone acetyltransferase complexes to the promoters.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Western Blot, FLAG-tag, Binding Assay, Immunoprecipitation, Expressing, Clinical Proteomics, Membrane, Protein-Protein interactions, Activation Assay

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Cartoon of AC9-POPDC complex. ( B , C ) Proximity ligation assay was performed with HEK293 cells expressing pCDNA3 (background control), POPDC1-Myc alone, and YFP-AC9 in the presence of either POPDC1-Myc or POPDC2-Myc. The interaction between AC9 and Gβγ served as a positive control . ( B ) Images of PLA signal (red) and DAPI (blue). ( C ) Mean cellular fluorescence intensity was quantified by high content microscopy and shown as Box and Whisker plots. Kruskal-Wallis One Way ANOVA analysis was performed (n=7 experiments, P=0.003 between groups) with multiple comparisons by Bonferroni t-test (*P<0.05) ( D ) Quantification of COS7 cells expressing BiFC constructs for AC9, POPDC 1-3. The expressed proteins tagged with VN (top line) and VC (bottom line) are shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments, ***P<0.001 between groups) with multiple comparisons to control by Dunn’s method (**P<0.01, *P<0.05) ( E ) Representative live-cell images of indicated BiFC combinations in HEK293 cells (n>20 cells; scale bar is 10 um). Quantification of POPDC1-VN:AC9-VC is shown in and .

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Proximity Ligation Assay, Expressing, Positive Control, Fluorescence, Microscopy, Whisker Assay, Construct

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Cell lysates from HEK293 cells expressing Flag-AC9 in the presence or absence of Myc-tagged POPDC1 or −2 were subjected to co-immunoprecipitation (Co-IP) with anti-MYC and assayed for AC activity with 300 nM Gαs-GTPγS. Kruskal-Wallis One Way ANOVA analysis on Ranks was performed (n=6 experiments, P=0.003 between all groups) with multiple comparisons to AC9 control by Dunn’s method (**P<0.01). ( B ) A portion of the lysates and Co-IP from ( A ) were subjected to western blot (WB) analysis with anti-MYC (POPDC) and anti-FLAG (AC9). Membranes isolated from Sf9 cells expressing Flag-AC9 served as a positive WB control. Note, POPDC protein runs as multiple bands (48-65 kDa) with altered sizes/patterns in different tissues due to changes in glycosylation patterns . Molecular weight markers are denoted as M. Quantitation of Flag-AC9 WB by One Way ANOVA, n=3-4 experiments, with comparisons by Tukey test, **P<0.01). ( C ) HEK293 cells expressing Flag-AC9 +/- POPDC1-Myc or POPDC2-Myc were subjected to Co-IP with anti-FLAG and subjected to WB analysis with anti-MYC and anti-FLAG. Quantitation of anti-MYC WB by One Way ANOVA, n=3 experiments, with comparisons by Tukey test, **P<0.01). ( D ) POPDC1 does not interact with control TM proteins. HEK293 cells expressing POPDC1-Myc +/- GFP-tagged AC9, EGFR, or LAMP1 were subjected to Co-IP with anti-MYC. Western blotting of lysates and Co-IPs for GFP (top) and Myc (bottom) are shown (n=3 experiments). ( E ) Schematic of POPDC1 truncations. ( F ) BiFC of AC9 and indicated POPDC1 truncations in COS-7 cells. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments, ***P<0.001 between groups) with multiple comparisons by Tukey test (**P=0.008). ( G ) Co-IP with anti-MYC in COS-7 cells expressing Flag-AC9 and indicated Myc-tagged POPDC1 truncations. Western blotting with anti-AC9 and anti-MYC (POPDC1) of Co-IP and lysates is shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=3-5 experiments, P=0.003), with multiple comparisons by Dunn’s method (*P<0.05).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Activity Assay, Western Blot, Isolation, Molecular Weight, Quantitation Assay

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Co-localization of CFP-TREK-1 with the BiFC signal from AC9-VN:AC9-VC homodimer (top) and POPDC1-VN:AC9-VC complex (bottom) in HEK293 cells. Scale bar is 10 um. ( B ) The lifetime distribution of Cerulean-tagged proteins +/- the indicated YFP-tagged proteins expressed in HEK293 cells are displayed as box and whisker plots with all outliers shown. Mann-Whitney Rank Sum Test was performed (***P<0.001; n=cell number indicated on each bar). ( C ) Co-IP of Myc-tagged POPDC1 (anti-MYC) pulls down both Flag-AC9 and GFP-tagged TREK-1 (n=3 experiments). ( D ) Co-IP of TREK-1 (anti-GFP) pulls down Flag-AC9 and endogenous POPDC1. AKAP79 is not pulled down in complex (n=3 experiments). Quantification of Flag-AC9 WB (IP/total expression in lysate) is shown to the right of ( C ) and ( D ).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Whisker Assay, MANN-WHITNEY, Co-Immunoprecipitation Assay, Expressing

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) BiFC signal between the indicated VN- and VC-tagged proteins expressed in HEK293 cells (POPDC1, P1). Kruskal-Wallis One-Way ANOVA on Ranks (n=5 experiments, P<0.001 between groups) with multiple comparison to VN and VC controls by Student-Newman-Keuls tests (*P<0.05). ( B ) Percent decrease of BiFC signal in ( A ) with ISO treatment (10 μM, 10 min at 37°C). Box and whisker plots are shown. Paired t-test was performed for each condition (vehicle versus ISO) using raw data prior to calculation of percent of vehicle control (n=6; *P<0.05; **P<0.01). ( C ) ISO dose-response curves for BiFC interactions between POPDC1:AC9, TREK-1:AC9, and TREK-1 with a catalytically inactive AC9 (TREK-1:AC9d). Data were analyzed by Two-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4; *P<0.05). ( D ) TREK-1:AC9 BiFC signal is absent in COS-7 cells lacking detectable POPDC1 (TREK-1:TREK-1 BiFC is shown as a positive control). One-way ANOVA with multiple comparisons by Holm Sidak method (n=3 experiments; ***P<0.001); ( E ) Overexpression of POPDC1 truncation of Popeye domain (POPDC1-Δ172), but not WT POPDC1, abolishes ISO reduction of TREK-1:AC9 BiFC signal. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4 experiments; ***P<0.001 compared to control). ( F ) Model of cAMP effects on AC9-POPDC1-TREK-1 complex formation.

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Whisker Assay, Positive Control, Over Expression

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) IP-AC assay with IgG versus anti-TREK-1 in heart homogenates from WT and Adcy9 -/- mice; AC activity stimulated with 300 nM Gαs-GTPγS. IP-AC with anti-AKAP150 is shown as a positive control. Two-way ANOVA (overall P<0.001, n=3 mice per genotype) with multiple comparisons by Holm-Sidak test; # P<0.001 for indicated comparison or to respective IgG controls. IgG versus anti-TREK-1 in Adcy9 -/- was analyzed by Student’s t-test (**P=0.009, n=3). ( B ) IP-AC assay with IgG versus anti-TREK in WT and Popdc1 -/- mouse heart homogenates; AC activity stimulated as in (A). Statistics as in A, n=4 mice per genotype, #P<0.001. Paired t-test within Popdc1 -/ - , P=0.123. ( C ) Total AC activity in heart homogenates from WT or Popdc1 -/- ( P1 -/- ) mice stimulated with 300 nM Gαs-GTPγS or 100 μM calcium and 300 nM calmodulin. Analyzed by Student’s t-test (n=4, n.s.) ( D ) Cell lysates from HEK293 cells expressing AC1 or AC8 in the presence or absence of Myc-tagged POPDC1 were subjected to co-IP with anti-MYC and assayed for AC activity with 100 μM calcium and 300 nM calmodulin. Student’s t-test (n=3 experiments, **P=0.0011 and *P=0.012 for AC1 and AC8, respectively). (E) IP-AC assay with IgG versus anti-TREK-1 in WT and Popdc1 -/- mouse heart homogenates; AC activity stimulated 100 μM calcium and 300 nM calmodulin. Student’s t-test (*P=0.03; **P=0.005, n=4 mice per genotype).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Activity Assay, Positive Control, Expressing, Co-Immunoprecipitation Assay