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: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Representative images of eGFP-gephyrin expressed in HEK cells that were fixed and probed using DARPin-FLAG clones or commercial antibody clone 3B11. Shown is eGFP and FLAG signal provided by the control (E3_5) and gephyrin-binding DARPin-FLAG clones (e.g., 27B3). The relative signal between eGFP and FLAG for a given cell is plotted, and the slope compared between clones to assess relative binding. ( B ) Quantification of binder labeling of eGFP-tagged gephyrin WT versus S268A/S270A and S268E/S270E phospho-mutants overexpressed in HEK293T cells. ( C ) Quantification of binding to overexpressed full-length (P1 variant) gephyrin or GC or E domains only. ( D ) Quantification of binding to eGFP-tagged gephyrin P1 isoform or isoforms including the C3 or C4a cassettes. Statistics : one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data points represent the slope calculated from at least 25 cells in three independent experiments. All panels: mean and SD are presented. Figure 6—figure supplement 2—source data 1. Values and statistical analysis performed to generate graphs in .

Article Snippet: Cell line (human) , HEK293T , ATCC , CRL 11268 , Used for in-cell DARPin binding screen.

Techniques: Clone Assay, Control, Binding Assay, Labeling, Variant Assay

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet:

Article Snippet: Cell line (human) , HEK293T , ATCC , CRL 11268 , Used for in-cell DARPin binding screen.

Techniques: Recombinant, High Throughput Screening Assay, Selection, Subcloning, Construct, Expressing, FLAG-tag, Plasmid Preparation, Sequencing, Fluorescence, Binding Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Diagram of gephyrin function at the inhibitory postsynapse via its scaffolding role. ( B ) Gephyrin domain structure and location of key phosphoserine residues S268 and S270, the commonly used antibody clone for detection of gephyrin (Ab7a) is phospho-S270-specific. ( C ) The antibody Ab7a does not detect gephyrin clusters colocalized with the γ2 GABA A receptor subunit (GABRG2) in a phospho-null mouse model where S268 and S270 are mutated to alanines. ( D ) DARPins are an order of magnitude smaller than conventional antibodies and achieve target binding specificity by varying the sequence of ankyrin repeats (A.R.) with variable residues (magenta). ( E ) DARPin library design, with residues in magenta randomized in the original design and additional residues randomized in the caps (green). An N3C structure is shown with the N-terminal cap as a green ribbon and the C-terminal cap as a cyan ribbon with green side chains. ( F ) Schematic of anti-gephyrin DARPin selection and screening. ( G ) Structure of DARPin-FLAG clones used for initial validation experiments contain an N-terminal His 8 tag and C-terminal FLAG tag for purification and detection, respectively. ( H ) Coomassie-stained gel of the nonbinding control (E3_5) and eight anti-gephyrin DARPin binders. Figure 1—source data 1. Raw image and annotated uncropped Coomassie gel from .

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques: Scaffolding, Binding Assay, Sequencing, Selection, Clone Assay, Biomarker Discovery, FLAG-tag, Purification, Staining, Control

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: Native gephyrin in fixed hippocampal neuron cultures (DIV15) probed using DARPin-FLAG clones, subsequently detected with anti-FLAG antibodies, and compared to staining with commercial anti-gephyrin antibody clone Ab7a. ( A ) Representative images of DARPin-FLAG clones 27B3, 27D3, 27F3, and 27G2 gephyrin puncta colocalized to Ab7a signal compared to the control DARPin E3_5. ( B ) Higher-magnification images of dendrite segments showing detected DARPin-FLAG signal colocalized with presynaptic VGAT. ( C ) Colocalization analysis indicating the fraction of gephyrin puncta that colocalize with VGAT along a proximal dendrite segment (>30 neurons/group pooled across three experiments). ( D ) Average puncta size identified by antibody Ab7a or DARPin-FLAG clones averaged by cell (pooled across neurons, >1100 synapses/group pooled across three experiment). Statistics : ( C, D ) one-way ANOVA, Tukey’s post-hoc test comparing all groups ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05. Figure 2—source data 1. Data and statistical analysis to generate the violin plot in .

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques: Clone Assay, Staining, Control

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: Fixed embryonic E17 rat hippocampal neuron cultures (DIV15) were stained using DARPin-FLAG clones and subsequently detected with anti-FLAG antibodies and compared to staining with commercial anti-gephyrin antibody clone Ab7a or 3B11. ( A ) DARPin-FLAG control (E3_5) and clone 27D5 with no synaptic labeling. ( B ) DARPin-FLAG clones and antibody 3B11 that demonstrate high background labeling. ( C ) DARPin-FLAG clones with highly specific inhibitory synapse labeling.

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques: Staining, Clone Assay, Control, Labeling

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Representative images of DARPin-hFc 27G2 (but not antibody Ab7a) labeling gephyrin puncta in both wildype (WT) and phospho-mutant gephyrin S268A/S270A mutant mouse brain tissue (somatosensory cortex layer 2/3). ( B ) Representative images from hippocampal neuron culture showing the relative Ab7a signal (indicating S270 phosphorylation) varies by synapse and between neurons. ( C ) Representative image showing DARPin-hFc 27G2 binding at synaptic puncta in primary hippocampal neuron culture is preserved after inhibition of CDKs following 24 hr treatment with 5 µM aminopurvalanol (PurvA) while Ab7a staining is severely reduced. ( D ) The relative fluorescence intensity at individual synapses (pooled from 30 neurons per group) showing a pronounced decrease in the average Ab7a/DARPin-hFc 27G2 intensity ratio. Quantification of Ab7a/DARPin-hFc 27G2 fluorescence signal averaged across cells pooled from three independent experiments, n = 30 cells/group. ( E ) Representative images of hippocampal neuron culture used for quantification of relative Ab7a/DARPin-hFc labeling of clusters on the soma, proximal dendrites, or the axon-initial segment (A.I.S.) (AnkG). ( F ) Ab7a/DARPin intensity ratio of individual synapses pooled from 45 cells over three independent experiments showing a decrease in A.I.S. cluster Ab7a staining. Lower: quantification indicates significantly reduced A.I.S. Ab7a labeling of clusters compared to dendritic or somatic compartments. Statistics : ( D ) one-way ANOVA; ( F ) repeated-measures one-way ANOVA. All panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Mean and SD are presented. Figure 3—source data 1. Values and statistical results used to generate .

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques: Labeling, Mutagenesis, Phospho-proteomics, Binding Assay, Inhibition, Staining, Fluorescence

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: The ratio of fluorescent intensity signal between pS270-specific antibody Ab7a and the phosphorylation nonspecific DARPin-hFc 27G2 indicates that Ab7a labeling is variable between clusters within and between individual synapses and neurons. Each data represents one cluster analyzed from six individual example neurons with different patterns of relative Ab7a reactivity. Median and SD are indicated in red. Figure 3—figure supplement 4—source data 1. Values used to plot .

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques: Phospho-proteomics, Labeling

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: Embryonic E17 rat hippocampal neuron cultures were transfected with plasmid expressing EGFP-gephyrin at days in vitro (DIV) 8 and were fixed and stained using DARPin-hFc clones, antibody Ab7a, or no binder at DIV 15 and analyzed for EGFP-gephyrin cluster size along the principle dendrite. Median puncta size averaged by principal dendrite are presented from 6 to 10 neurons pooled across three independent experiments per group. Statistics: One-way ANOVA, n.s., no significant difference. Figure 3—figure supplement 5—source data 1. Values and statistical analysis used to plot .

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques: Transfection, Plasmid Preparation, Expressing, In Vitro, Staining, Clone Assay

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Left: the relative Ab7a to DARPin-hFc 27G2 fluorescence intensity in the mouse hippocampus area CA1 shows layer-specific variability. Right: colorized gephyrin puncta indicating relative S270 phosphorylation as seen from hotter (more red/yellow) coloration. ( B ) Distribution of relative gephyrin phosphorylated at S270 (p270) at puncta between hippocampal lamina. Data pooled between six adult mice, three sections analyzed per mouse encompassing 14,000–47,000 gephyrin puncta per layer. ( C ) Analysis of the median relative gephyrin pS270 ratio between hippocampal layers (data pooled between sections per mouse, n = 6 mice quantified). ( D ) Example distribution of gephyrin pS270 signal by puncta size in the CA1 stratum pyramidale, with a population of large, hypophosphorylated clusters outlined. ( E ) Representative image of s. pyramidale with hot colors indicating gephyrin clusters with elevated phosphorylation; arrows indicate trains of large hypophosphorylated clusters. ( F ) Representative image showing large DARPin-identified gephyrin clusters apposed to presynaptic VGAT-containing terminals with corresponding low Ab7a antibody signal. ( G ) Representative image indicating gephyrin clusters on the A.I.S. (AnkG) colocalize with the α 2 GABA A receptor subunit. ( H ) Representative images of gephyrin puncta identified using cluster analysis software in WT and S268A/S270A phospho-null mutant mice in the hippocampus using identical imaging parameters. ( I ) Violin plots indicating the distribution of gephyrin puncta sizes (14,000–47,000 puncta per group, pooled across 5–6 mice per group). ( J ) Analysis of the median puncta size between hippocampal layers and genotypes indicating larger gephyrin clusters in mutant mice. Statistics : ( C ) one-way ANOVA, ( J ) mixed-effects analysis comparing hippocampal lamina (horizontal bars) and genotypes (angled bars). All panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Median and SD are presented. Figure 4—source data 1. Data and statistical analysis presented in .

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques: Fluorescence, Phospho-proteomics, Software, Mutagenesis, Imaging

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: Extended example distribution of signal from adult brain tissue from . including the s. oriens, pyramidale, radiatum, and stratum lacunosum moleculare (S.L.M.). ( A ) Ab7a versus DARPin-hFc 27G2 puncta intensity. ( B ) Ab7a/DARPin-hFc 27G2 intensity ratio plotted by puncta size. Figure 4—figure supplement 1—source data 1. Data and statistical analysis presented in .

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques:

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-gephyrin Ab7a (rabbit monoclonal) , Synaptic Systems , 147 008; RRID :AB_2619834 , IF/ICC used at 1:2000.

Techniques: Recombinant, High Throughput Screening Assay, Selection, Subcloning, Construct, Expressing, FLAG-tag, Plasmid Preparation, Sequencing, Fluorescence, Binding Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Diagram of gephyrin function at the inhibitory postsynapse via its scaffolding role. ( B ) Gephyrin domain structure and location of key phosphoserine residues S268 and S270, the commonly used antibody clone for detection of gephyrin (Ab7a) is phospho-S270-specific. ( C ) The antibody Ab7a does not detect gephyrin clusters colocalized with the γ2 GABA A receptor subunit (GABRG2) in a phospho-null mouse model where S268 and S270 are mutated to alanines. ( D ) DARPins are an order of magnitude smaller than conventional antibodies and achieve target binding specificity by varying the sequence of ankyrin repeats (A.R.) with variable residues (magenta). ( E ) DARPin library design, with residues in magenta randomized in the original design and additional residues randomized in the caps (green). An N3C structure is shown with the N-terminal cap as a green ribbon and the C-terminal cap as a cyan ribbon with green side chains. ( F ) Schematic of anti-gephyrin DARPin selection and screening. ( G ) Structure of DARPin-FLAG clones used for initial validation experiments contain an N-terminal His 8 tag and C-terminal FLAG tag for purification and detection, respectively. ( H ) Coomassie-stained gel of the nonbinding control (E3_5) and eight anti-gephyrin DARPin binders. Figure 1—source data 1. Raw image and annotated uncropped Coomassie gel from .

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Scaffolding, Binding Assay, Sequencing, Selection, Clone Assay, Biomarker Discovery, FLAG-tag, Purification, Staining, Control

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: Native gephyrin in fixed hippocampal neuron cultures (DIV15) probed using DARPin-FLAG clones, subsequently detected with anti-FLAG antibodies, and compared to staining with commercial anti-gephyrin antibody clone Ab7a. ( A ) Representative images of DARPin-FLAG clones 27B3, 27D3, 27F3, and 27G2 gephyrin puncta colocalized to Ab7a signal compared to the control DARPin E3_5. ( B ) Higher-magnification images of dendrite segments showing detected DARPin-FLAG signal colocalized with presynaptic VGAT. ( C ) Colocalization analysis indicating the fraction of gephyrin puncta that colocalize with VGAT along a proximal dendrite segment (>30 neurons/group pooled across three experiments). ( D ) Average puncta size identified by antibody Ab7a or DARPin-FLAG clones averaged by cell (pooled across neurons, >1100 synapses/group pooled across three experiment). Statistics : ( C, D ) one-way ANOVA, Tukey’s post-hoc test comparing all groups ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05. Figure 2—source data 1. Data and statistical analysis to generate the violin plot in .

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Clone Assay, Staining, Control

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: Fixed embryonic E17 rat hippocampal neuron cultures (DIV15) were stained using DARPin-FLAG clones and subsequently detected with anti-FLAG antibodies and compared to staining with commercial anti-gephyrin antibody clone Ab7a or 3B11. ( A ) DARPin-FLAG control (E3_5) and clone 27D5 with no synaptic labeling. ( B ) DARPin-FLAG clones and antibody 3B11 that demonstrate high background labeling. ( C ) DARPin-FLAG clones with highly specific inhibitory synapse labeling.

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Staining, Clone Assay, Control, Labeling

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Representative images of DARPin-hFc 27G2 (but not antibody Ab7a) labeling gephyrin puncta in both wildype (WT) and phospho-mutant gephyrin S268A/S270A mutant mouse brain tissue (somatosensory cortex layer 2/3). ( B ) Representative images from hippocampal neuron culture showing the relative Ab7a signal (indicating S270 phosphorylation) varies by synapse and between neurons. ( C ) Representative image showing DARPin-hFc 27G2 binding at synaptic puncta in primary hippocampal neuron culture is preserved after inhibition of CDKs following 24 hr treatment with 5 µM aminopurvalanol (PurvA) while Ab7a staining is severely reduced. ( D ) The relative fluorescence intensity at individual synapses (pooled from 30 neurons per group) showing a pronounced decrease in the average Ab7a/DARPin-hFc 27G2 intensity ratio. Quantification of Ab7a/DARPin-hFc 27G2 fluorescence signal averaged across cells pooled from three independent experiments, n = 30 cells/group. ( E ) Representative images of hippocampal neuron culture used for quantification of relative Ab7a/DARPin-hFc labeling of clusters on the soma, proximal dendrites, or the axon-initial segment (A.I.S.) (AnkG). ( F ) Ab7a/DARPin intensity ratio of individual synapses pooled from 45 cells over three independent experiments showing a decrease in A.I.S. cluster Ab7a staining. Lower: quantification indicates significantly reduced A.I.S. Ab7a labeling of clusters compared to dendritic or somatic compartments. Statistics : ( D ) one-way ANOVA; ( F ) repeated-measures one-way ANOVA. All panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Mean and SD are presented. Figure 3—source data 1. Values and statistical results used to generate .

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Labeling, Mutagenesis, Phospho-proteomics, Binding Assay, Inhibition, Staining, Fluorescence

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: Embryonic E17 rat hippocampal neuron cultures were transfected with plasmid expressing EGFP-gephyrin at days in vitro (DIV) 8 and were fixed and stained using DARPin-hFc clones, antibody Ab7a, or no binder at DIV 15 and analyzed for EGFP-gephyrin cluster size along the principle dendrite. Median puncta size averaged by principal dendrite are presented from 6 to 10 neurons pooled across three independent experiments per group. Statistics: One-way ANOVA, n.s., no significant difference. Figure 3—figure supplement 5—source data 1. Values and statistical analysis used to plot .

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Transfection, Plasmid Preparation, Expressing, In Vitro, Staining, Clone Assay

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Left: the relative Ab7a to DARPin-hFc 27G2 fluorescence intensity in the mouse hippocampus area CA1 shows layer-specific variability. Right: colorized gephyrin puncta indicating relative S270 phosphorylation as seen from hotter (more red/yellow) coloration. ( B ) Distribution of relative gephyrin phosphorylated at S270 (p270) at puncta between hippocampal lamina. Data pooled between six adult mice, three sections analyzed per mouse encompassing 14,000–47,000 gephyrin puncta per layer. ( C ) Analysis of the median relative gephyrin pS270 ratio between hippocampal layers (data pooled between sections per mouse, n = 6 mice quantified). ( D ) Example distribution of gephyrin pS270 signal by puncta size in the CA1 stratum pyramidale, with a population of large, hypophosphorylated clusters outlined. ( E ) Representative image of s. pyramidale with hot colors indicating gephyrin clusters with elevated phosphorylation; arrows indicate trains of large hypophosphorylated clusters. ( F ) Representative image showing large DARPin-identified gephyrin clusters apposed to presynaptic VGAT-containing terminals with corresponding low Ab7a antibody signal. ( G ) Representative image indicating gephyrin clusters on the A.I.S. (AnkG) colocalize with the α 2 GABA A receptor subunit. ( H ) Representative images of gephyrin puncta identified using cluster analysis software in WT and S268A/S270A phospho-null mutant mice in the hippocampus using identical imaging parameters. ( I ) Violin plots indicating the distribution of gephyrin puncta sizes (14,000–47,000 puncta per group, pooled across 5–6 mice per group). ( J ) Analysis of the median puncta size between hippocampal layers and genotypes indicating larger gephyrin clusters in mutant mice. Statistics : ( C ) one-way ANOVA, ( J ) mixed-effects analysis comparing hippocampal lamina (horizontal bars) and genotypes (angled bars). All panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Median and SD are presented. Figure 4—source data 1. Data and statistical analysis presented in .

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Fluorescence, Phospho-proteomics, Software, Mutagenesis, Imaging

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Coomassie-stained acrylamide gel indicating abundant gephyrin precipitated both by the antibody 3B11 and DARPin-hFc 27B3, 27F3, and 27G2 without signal in antibody (IgG) or DARPin (E3_5) controls. Lower bands correspond to IgG or DARPin-hFc protein. ( B ) Immunoblot of gephyrin precipitated with different binders probed with the antibody 3B11. Figure 5—figure supplement 1—source data 1. Raw Coomassie gel images and immunoblots from .

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Staining, Acrylamide Gel Assay, Western Blot

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: A ) Mouse brain tissue lysate preparation diagram. ( B ) Liquid chromatography tandem mass spectrometry (LC-MS/MS) and interactome determination methodology workflow indicating thresholds for consideration of interacting proteins. ( C ) Scale-free interaction networks (STRING) of gephyrin interactors identified from pulldowns using the commercial antibody 3B11, or DARPin-hFc 27B3, 27F3, and 27G2 compared to control conditions (containing antibody control IgG or the control DARPin-hFc E3_5). Nodes represent unique gephyrin interactors – red nodes indicate known (canonical) gephyrin interactors. ( D ) Venn diagram of the overlap in identified interactors from gephyrin complexes isolated using different DARPin-hFc clones; bottom indicates coverage compared to an extensive gephyrin interactome determined using BioID labeling and 22 canonical gephyrin interactors identified from the literature. ( E ) Consensus interactome of proteins identified by all DARPin-hFc clones and colored by protein ontology. Canonical gephyrin interacting proteins are indicated by blue font, and bold font indicates interactors also identified by the antibody clone 3B11. Asterisks indicate proteins previously identified by BioID . Italic font indicates interactors exclusively identified by DARPins. Edges connecting protein nodes indicate putative interactions (STRING analysis), and node circle size indicates relative protein abundance averaged across all experiments. Figure 5—source data 1. List of interactors and relative abundance of detected proteins used to construct interaction networks and Venn diagrams in .

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Isolation, Clone Assay, Labeling, Quantitative Proteomics, Construct

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Representative images of eGFP-gephyrin expressed in HEK cells that were fixed and probed using DARPin-FLAG clones or commercial antibody clone 3B11. Shown is eGFP and FLAG signal provided by the control (E3_5) and gephyrin-binding DARPin-FLAG clones (e.g., 27B3). The relative signal between eGFP and FLAG for a given cell is plotted, and the slope compared between clones to assess relative binding. ( B ) Quantification of binder labeling of eGFP-tagged gephyrin WT versus S268A/S270A and S268E/S270E phospho-mutants overexpressed in HEK293T cells. ( C ) Quantification of binding to overexpressed full-length (P1 variant) gephyrin or GC or E domains only. ( D ) Quantification of binding to eGFP-tagged gephyrin P1 isoform or isoforms including the C3 or C4a cassettes. Statistics : one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data points represent the slope calculated from at least 25 cells in three independent experiments. All panels: mean and SD are presented. Figure 6—figure supplement 2—source data 1. Values and statistical analysis performed to generate graphs in .

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Clone Assay, Control, Binding Assay, Labeling, Variant Assay

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet:

Article Snippet: Neurons were prepared for DARPin-FLAG or DARPin-hFc staining and immunostaining as with HEK293T cultures, with the exception that endogenous gephyrin was analyzed using the anti-gephyrin antibody clone Ab7a (Sysy 147 011) or clone 3B11 (Sysy 147 111).

Techniques: Recombinant, High Throughput Screening Assay, Selection, Subcloning, Construct, Expressing, FLAG-tag, Plasmid Preparation, Sequencing, Fluorescence, Binding Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: Fixed embryonic E17 rat hippocampal neuron cultures (DIV15) were stained using DARPin-FLAG clones and subsequently detected with anti-FLAG antibodies and compared to staining with commercial anti-gephyrin antibody clone Ab7a or 3B11. ( A ) DARPin-FLAG control (E3_5) and clone 27D5 with no synaptic labeling. ( B ) DARPin-FLAG clones and antibody 3B11 that demonstrate high background labeling. ( C ) DARPin-FLAG clones with highly specific inhibitory synapse labeling.

Article Snippet: Antibody , Anti-gephyrin 3B11 (mouse monoclonal) , Synaptic Systems , Cat# 147111; RRID :AB_887719 , IF/ICC used at 1:1000.

Techniques: Staining, Clone Assay, Control, Labeling

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Coomassie-stained acrylamide gel indicating abundant gephyrin precipitated both by the antibody 3B11 and DARPin-hFc 27B3, 27F3, and 27G2 without signal in antibody (IgG) or DARPin (E3_5) controls. Lower bands correspond to IgG or DARPin-hFc protein. ( B ) Immunoblot of gephyrin precipitated with different binders probed with the antibody 3B11. Figure 5—figure supplement 1—source data 1. Raw Coomassie gel images and immunoblots from .

Article Snippet: Antibody , Anti-gephyrin 3B11 (mouse monoclonal) , Synaptic Systems , Cat# 147111; RRID :AB_887719 , IF/ICC used at 1:1000.

Techniques: Staining, Acrylamide Gel Assay, Western Blot

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: A ) Mouse brain tissue lysate preparation diagram. ( B ) Liquid chromatography tandem mass spectrometry (LC-MS/MS) and interactome determination methodology workflow indicating thresholds for consideration of interacting proteins. ( C ) Scale-free interaction networks (STRING) of gephyrin interactors identified from pulldowns using the commercial antibody 3B11, or DARPin-hFc 27B3, 27F3, and 27G2 compared to control conditions (containing antibody control IgG or the control DARPin-hFc E3_5). Nodes represent unique gephyrin interactors – red nodes indicate known (canonical) gephyrin interactors. ( D ) Venn diagram of the overlap in identified interactors from gephyrin complexes isolated using different DARPin-hFc clones; bottom indicates coverage compared to an extensive gephyrin interactome determined using BioID labeling and 22 canonical gephyrin interactors identified from the literature. ( E ) Consensus interactome of proteins identified by all DARPin-hFc clones and colored by protein ontology. Canonical gephyrin interacting proteins are indicated by blue font, and bold font indicates interactors also identified by the antibody clone 3B11. Asterisks indicate proteins previously identified by BioID . Italic font indicates interactors exclusively identified by DARPins. Edges connecting protein nodes indicate putative interactions (STRING analysis), and node circle size indicates relative protein abundance averaged across all experiments. Figure 5—source data 1. List of interactors and relative abundance of detected proteins used to construct interaction networks and Venn diagrams in .

Article Snippet: Antibody , Anti-gephyrin 3B11 (mouse monoclonal) , Synaptic Systems , Cat# 147111; RRID :AB_887719 , IF/ICC used at 1:1000.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Isolation, Clone Assay, Labeling, Quantitative Proteomics, Construct

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet: ( A ) Representative images of eGFP-gephyrin expressed in HEK cells that were fixed and probed using DARPin-FLAG clones or commercial antibody clone 3B11. Shown is eGFP and FLAG signal provided by the control (E3_5) and gephyrin-binding DARPin-FLAG clones (e.g., 27B3). The relative signal between eGFP and FLAG for a given cell is plotted, and the slope compared between clones to assess relative binding. ( B ) Quantification of binder labeling of eGFP-tagged gephyrin WT versus S268A/S270A and S268E/S270E phospho-mutants overexpressed in HEK293T cells. ( C ) Quantification of binding to overexpressed full-length (P1 variant) gephyrin or GC or E domains only. ( D ) Quantification of binding to eGFP-tagged gephyrin P1 isoform or isoforms including the C3 or C4a cassettes. Statistics : one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data points represent the slope calculated from at least 25 cells in three independent experiments. All panels: mean and SD are presented. Figure 6—figure supplement 2—source data 1. Values and statistical analysis performed to generate graphs in .

Article Snippet: Antibody , Anti-gephyrin 3B11 (mouse monoclonal) , Synaptic Systems , Cat# 147111; RRID :AB_887719 , IF/ICC used at 1:1000.

Techniques: Clone Assay, Control, Binding Assay, Labeling, Variant Assay

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-gephyrin 3B11 (mouse monoclonal) , Synaptic Systems , Cat# 147111; RRID :AB_887719 , IF/ICC used at 1:1000.

Techniques: Recombinant, High Throughput Screening Assay, Selection, Subcloning, Construct, Expressing, FLAG-tag, Plasmid Preparation, Sequencing, Fluorescence, Binding Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Downregulation of TAP2 reduces the surface levels of peptide-HLA complexes in lung cancer cells. A Outline of the experimental strategy used to measure changes in the levels of selected HLA-peptide complexes in lung cancer cells with or without TAP1 and/or TAP2 downregulation using flow cytometry. B-G A549 lung cancer cells were transfected with scrambled/control siRNA or with TAP1 and/or TAP2 targeting siRNAs and left untreated (black histograms) or stimulated with IFNγ (blue histograms) or IFNγ + TNFα (red histograms). Panels B-D show the surface levels of HLA-A2-HER2 369-377 , and panels E–G show the levels of HLA-A2-MAGE3 271-279 . H–K A549 TAP2 knockout (KO) cells were transfected with an empty vector (KO + EV) or with a vector containing FLAG-TAP2 (KO + TAP2) and left untreated (black) or stimulated with cytokines IFNγ (blue) or IFNγ + TNFα (red). I, Graphs showing the levels of TAP2 protein analyzed by flow cytometry with or without TAP2 gene elimination, J-K, Surface levels of HLA-A2-HER2 369-377 or HLA-A2-MAGE3 271-279 in A549 cells with or without TAP2 gene elimination. For panels B-G and I-K, an isotype control antibody (IgG) was used as a background signal reference. Data presented as the mean ± s.d.; *, p < 0.05; **, p < 0.01; ***, p < 0.001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. For panels B-G, Scr transfected cells were used as a control for statistical comparison, and for I-K parental wild type (WT) cells were compared with TAP2 deleted cells (KO) and TAP2 deleted plus EV (KO + EV) or with TAP2 deleted with posterior TAP2 transfection (KO + TAP2). MFI, mean fluorescent intensity; si, siRNA; scr, scrambled; Tx, treatment; ns, not significant

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Transfection, Control, Knock-Out, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: TAP2 downregulation protects cancer cells from tumor antigen-specific CD8 T-cell killing. A , B Schema and outline of the experimental strategy used to measure tumor antigen-specific killing of lung cancer cells by cognate CD8 + T-cells using flow cytometry, LDH release and MTT assay. C-L A549 lung cancer cells were transfected with scrambled siRNA or with TAP1 and/or TAP2 targeting siRNAs; and left untreated (black) or stimulated with IFNγ (blue) or IFNγ + TNFα (red). After treatment, target lung tumor cells were co-cultured with effector (CD8 + T-cells) cells in the ratios of 1:0, 1:2 and 1:5, respectively. Panel C shows the flow cytometry gating strategy to assess cell apoptosis in cancer cell/T-cell co-cultures using the markers CD3 (for CD8 + T-cells), EpCAM (for tumor cells) and Annexin V. Panels D-F show the percentage of EpCAM + and Annexin V + apoptotic cancer cells. Panels G-I show the percent of LDH release, and panels J-L represent the cellular viability using MTT assay. M-P A549 TAP2 knockout (KO) cells transfected with empty vector (KO + EV) or FLAG-TAP2 (KO + TAP2) and left untreated or stimulated with IFNγ or IFNγ + TNFα were co-cultured with tumor antigen specific CD8 + T-cells at different target cell (tumor): effector (CD8 + T-cell) cell ratios. An isotype control antibody (IgG) was used as a background signal reference. Data are presented as the mean ± s.d.; * , p < 0.05; ** , p < 0.01; *** , p < 0.001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. For panels D-L, Scr transfected cells were used as a control for statistical comparison, and for N-P wild type (WT) compared with KO and KO + EV or with KO + TAP2 cells. si, siRNA; scr, scrambled; E, effector CD8 T cells; T, target tumor cells; Tx, treatment; ns, not significant; WT, wild type. See also supplementary Fig. S5-S6

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, MTT Assay, Transfection, Cell Culture, Knock-Out, Plasmid Preparation, Control, Two Tailed Test, Comparison

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Downregulation of TAP2 alters intracellular immunomodulatory pathways via SOCS1 upregulation and TAP2 downregulation in human NSCLC is due to epigenetic changes. A A549 cells were transfected with scrambled siRNA or with TAP2 targeting siRNAs followed by targeted transcriptomic analysis. Graph shows differentially expressed genes in TAP2 silenced versus control cells ranked based on low (blue) to high (red) transcript expression. B , C SOCS1 protein expression by flow cytometry in A549 cells. B, Cells were transfected with scrambled siRNA or with TAP2 targeting siRNAs C, Cells were transfected with empty vector or with the full-length TAP2 including a FLAG octapeptide. D-G, A549 cells were transfected with TAP1/2 siRNAs as indicated in 3A and stimulated with IFNγ ( D , E ) or with IFNγ plus TNFα ( F , G ). Volcano plots representing differential expression of IFNγ pathway signature genes. H A549 cells were transfected with TAP1/2 siRNAs followed by stimulation with IFNγ plus TNFα. Phosphoprotein levels were measured and ranked based on low (blue) to high (red) expression in TAP2 silenced vs control cells. I-M Normal adjacent to tumor (NAT) and lung tumor tissues surgically resected from NSCLC patients with low TAP2 protein levels. I Schematic of the strategy for obtaining and analyzing single cell preparations. J Volcano plot showing the differential gene expression of paired lung cancer relative to NAT using RNA sequencing. K Fold change of the mRNA expression of TAP2 and SOCS1 in tumor samples relative to NAT, L Heatmap of ATAC-seq analysis representing chromatin accessibility in the TAP2 promoter region. M Predicted transcription factor (TF) binding sites with the highest affinity scores (log2 count) for the TAP2 promoter region. Data are presented as the mean ± s.d. ** , p < 0.01 determined by two-tailed unpaired Student’s t-test. FC, fold change; MFI, mean fluorescent intensity; si, siRNA; scr, scrambled; TFs, transcription factors. See also supplementary Fig. S7

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Control, Expressing, Flow Cytometry, Plasmid Preparation, FLAG-tag, Quantitative Proteomics, Gene Expression, RNA Sequencing, Binding Assay, Two Tailed Test

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Myeloid cell-derived IL-4 reduces TAP2 expression via epigenetic remodeling in lung cancer. A TCGA NSCLC cohort analysis of TAP2 expression stratified by the median IL-4 mRNA levels. B A549 cells were treated with IL-4 for 0-24 h and TAP2 protein levels were measured using flow cytometry. C-I A549 cells were treated with IL-4, IL-4 + IFNγ or IL-4 with IFNγ + TNFα. C, TAP2 protein levels measured by flow cytometry, D, SOCS1 protein levels by flow cytometry, E, Surface levels of HLA-A2-HER2 369-377 complexes and F, HLA-A2-MAGE3 271-279 complexes measured by flow cytometry. G-I A549 cells (target) were treated with IL-4 ± IFNγ + TNFα and incubated with effector tumor antigen-specific CD8 + T-cells to measure, G, cancer cell killing by Annexin V positivity, H, LDH release and I, cellular viability using MTT assay. J-M Fluorescence images and signal measurement from multiplexed spatial analysis of protein and mRNA transcripts in NSCLCs from Cohorts #1 and #2. J-K, Representative captions of cytokeratin (CK, green), IL-4 mRNA (red), CD11b mRNA (white), TAP2 protein (yellow) and nuclei (blue). L, expression levels of IL-4 mRNA measured selectively in CK + tumor cells or in CD11b + myeloid cells. M, expression levels of TAP2 protein in CK + tumor cells stratified by the median IL-4 mRNA expressed in CD11b + myeloid cells. N-R A549 cells were treated with IL-4 ± IFNγ + TNFα and analyzed using ATAC-seq. N, ATAC-seq promoter peak enrichment values of the TAP2 gene. O, comparative analysis of the TAP2 ATAC-seq promoter peak enrichment after different cytokine treatments in A549 cells. P, heatmap of ATAC-seq analysis representing chromatin accessibility in the TAP2 promoter region after cytokine treatments. Q, promoter peak enrichment values of the SOCS1 gene. R, comparative analysis of the SOCS1 ATAC-seq promoter peak enrichment after cytokine treatments. Promoter regions were considered as the DNA sequences between the gene Transcription Start Site (TSS, + 3 kb) and Transcription End Site (TES, -3 kb). S-U Autologous single cell suspension cultures including cancer and immune cells from primary NSCLC tissues stimulated with IFNγ + TNFα followed by incubation with IL-4Rα (αIL-4R) or PD-1 blocking antibodies (αPD-1). S, levels of CD8 + /CD25 + T-cells measured by flow cytometry. T, percentage of EpCAM + /Annexin V + apoptotic cancer cells in autologous cell suspensions. U, percentage of EpCAM + /Annexin V + apoptotic cancer cells in autologous cell suspensions with selective elimination of CD3 + T-cells (TECS). Isotype (IgG) was used as a background signal control. Data are presented as the mean ± s.d. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. B-I and S-U, untreated cells were used as a control. E, effector CD8 + T-cells; FPKM, fragments per kilobase million; ns, not significant; MFI, mean fluorescent intensity; T, tumor; S, stroma. See also supplementary Figs. S8-S12

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Incubation, MTT Assay, Fluorescence, Suspension, Blocking Assay, Control, Two Tailed Test

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Restoration of TAP2 protein expression induces tumor cell surface antigenicity in lung cancer cells. A Schematic showing the strategy for high throughput screening of pharmacologic agents using TAP2 and PD-L1 immunostaining in A549 cells. B-D Representative plots showing the upregulation of TAP2 protein (red) or PD-L1 protein (blue) or both (green) in A549 cells treated with individual compounds from the Pharmakon 1600 library (B), Enzo 640 FDA library (C) and Enzo epigenetic library (D). The scores for each compound were calculated as TAP2 protein level fold change and normalized to the vehicle control (DMSO) treated group. E Summary of TAP2 protein inducer compounds selected based on representation of all 3 libraries. F-G Dose–response curves to determine EC 50 values for TAP2 protein upregulation in A549 cells using selected compounds shown in E. H Experimental outline for SAHA/Vorinostat treatment and analysis of A549 cells. I Levels of TAP2 protein measured by flow cytometry. J surface HLA-A2-HER2 369-377 complexes and K surface HLA-A2-MAGE3 271-279 levels measured by flow cytometry. L Levels of apoptotic cancer cell death (Annexin V staining) and cell viability (LDH release and MTT assay) of parental A549 cells co-incubated with tumor antigen-specific effector CD8 + T-cells using different effector to target cell ratios (1:0, 1:2 and 1:5) with or without treatment with 3.5 µM SAHA. An isotype control antibody (IgG) was used as a background signal reference. Data are presented as the mean ± s.d.; *p < 0.05; **p < 0.01; ****, p < 0.0001 determined by two-tailed unpaired Student’s t-test. FC, fold change; ns, non-significant; No-Tx, no treatment; MFI, mean fluorescent intensity; TKI, tyrosine kinase inhibitor. See also supplementary Fig. S13

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, High Throughput Screening Assay, Immunostaining, Control, Flow Cytometry, Staining, MTT Assay, Incubation, Two Tailed Test

Journal: eLife

Article Title: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

doi: 10.7554/eLife.80895

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-mouse Alexa Cy3 (goat polyclonal) , Jackson ImmunoResearch Labs , JAC 115-165-166; RRID :AB_2338692 , IF/ICC used at 1:500.

Techniques: Recombinant, High Throughput Screening Assay, Selection, Subcloning, Construct, Expressing, FLAG-tag, Plasmid Preparation, Sequencing, Fluorescence, Binding Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: (A) Schematic representation of the domain structures of mitophagy receptor proteins, highlighting the LIR motifs (pink color) and the C-terminal transmembrane (TM) domain (blue color). (B) Clustal Omega alignment of BCL2L13 orthologs across species showing conservation of reported phosphorylation sites. Note that S261 lies at the start of the second of two repeat elements (purple hue) which precede S275 and the LIR motif (pink hue). Reported phosphorylation sites in mass spectrometry datasets are highlighted in yellow. (C) Immunoblots of WT (NT) or CRISPR/Cas9 RNP-BCL2L13 knockout (KO) MEF cell lines bearing stable lentiviral expression of indicated BCL2L13 cDNAs, treated with 20 µM CCCP for 1 hour. These blots demonstrate that the phospho-specific antibodies detect BCL2L13 phosphorylation specifically at serine 261 (S261) and serine 275 (S275) in MEF cells, with phosphorylation levels increasing following CCCP treatment. Arrows indicate endogenous mouse Bcl2l13 (predicted to be 7 kDA smaller versus human). (D) Phosphorylation of endogenous Bcl2L13 on Ser261 and Ser275 compared to the regulation of other mitophagy receptor protein levels. Immunoblots with indicated antibodies on WT or BCL2L13 KO MEF cells, treated with DMSO vehicle (veh) or 10 µM 991 or 20 µM CCCP or 1 µM rotenone or 1mM DFP. (E) Quantification of BCL2L13 Ser 261 (left) and BCL2L13 Ser 275 (right) from the immunoblot shown in panel D. Densitometric analysis was performed to assess protein expression levels. Phospho-protein levels were quantified and normalized to total protein levels.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Phospho-proteomics, Mass Spectrometry, Western Blot, CRISPR, Knock-Out, Expressing

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: (A) Immunoblot of U2OS cells stably expressing flag-tagged BCL2L13, treated with various autophagy and mitophagy-inducing compounds: 20 µM CCCP, 1 mM DFP, 10 µM 991, 100 ng/ml rotenone, and 1 µM INK128 for 1 hour. After treatment, cells were lysed in 2% CHAPS buffer, and the lysates were subjected to flag immunoprecipitation (IP). (B) Protein sequence coverage map of the BCL2L13 samples submitted for mass spectrometry analysis. GluC digestion resulted in 75% coverage of the BCL2L13 protein sequence. (C) The table summarizes the spectral counts for each detected phosphorylation site. Notably, three phosphorylation sites — S275, S259, and S261 — show a significant increase in spec count after CCCP treatment compared to DMSO vehicle. These sites match the consensus kinase motifs for ULK1- or TBK1-like kinases, as highlighted in the red box. (D) Immunoblots of MEF cells from wild-type (WT) or CRISPR-Cas9 RNP-mediated BCL2L13 knockout (KO) lines, treated with 20 µM CCCP at the indicated time-point to validate the phospho-specific antibody developed by CST. These blots demonstrate that the antibody specifically detects BCL2L13 at serine 275 (S275) in MEF cells, with phosphorylation levels increasing following treatment with CCCP.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Western Blot, Stable Transfection, Expressing, Immunoprecipitation, Sequencing, Mass Spectrometry, Phospho-proteomics, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: ( A ) Diagram of the Mito-QC construct: mCherry-GFP tandem mitophagy assay. ( B) Representative histogram of ratiometric flow cytometry analysis before and after 4 hours of CCCP in WT MEF cells stably expressing the mitophagy reporter Mito-QC (“MEF-QC”). (C) MEF-QC cells were treated with CCCP or DFP at the indicated time points. Following treatment, cells were harvested in flow buffer, and mitophagy was quantified using flow cytometry based on the mCherry/GFP ratio. ( D ) HEK293A-QC cells treated and analyzed as in panel C. (E) Immunoblot of protein levels of the mitophagy receptors in MEF and HEK293A following CCCP or DFP at the indicated timepoints. ( F) Human and mouse BCL2L13 CRISPR-Cas9 deletion was assessed in MEF-QC and HEK293A-QC cells. (G) WT or CRISPR-Cas9 mediated BCL2L13 knockout (KO) HEK293-QC cells were treated with CCCP or DFP at the indicated time points. Following treatment, cells were harvested in flow buffer, and mitophagy was quantified using flow cytometry based on the mCherry/GFP ratio. ( H) WT or BCL2L13 KO MEF-QC cells were treated with CCCP or DFP at the indicated time points and mitophagy was quantified using flow cytometry based on the mCherry/GFP ratio as in panel G. (I) Immunoblot analysis of mitophagy receptors in WT and BCL2L13 KO MEFs reveals differential regulation of ubiquitin-independent mitophagy receptors MEF cells exhibit differential regulation of the ubiquitin-independent mitophagy receptors. (J) Immunoblot analysis of mitophagy receptors in WT and BCL2L13 KO HEK293A cells. Ratiometric flow cytometry data (mCherry/GFP ratio) are presented as the mean ± SEM for biological replicates (N = 3). Statistical significance was assessed using Two-Way ANOVA. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001).

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Construct, Mitophagy Assay, Flow Cytometry, Stable Transfection, Expressing, Western Blot, CRISPR, Knock-Out, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: (A) Immunoblots of MEF cells from wild-type (WT) or CRISPR-Cas9 RNP-mediated TBK1 knockout (KO) lines, treated with vehicle or TBK1 activator LPS for 1 hour. (B) Immunoblot analysis of wild-type (WT) MEFs, TBK1 knockout (KO) MEFs, and AMPK knockout (AMPKDKO) MEFs treated with vehicle (Veh), LPS, CCCP, or MK8722. (C) Quantification of BCL2L13 Ser 261 (left) and BCL2L13 Ser 275 (right) from the immunoblot shown in B. Densitometric analysis was performed to assess protein expression levels. Phospho-protein levels were quantified and normalized to total protein levels and loading control, Actin.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Western Blot, CRISPR, Knock-Out, Expressing, Control

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: (A) Immunoblot analysis of CRISPR/Cas9 knockouts of individual mitophagy receptors, and triple and quadruple deletions in MEFs. (B) MitoQC functional mitophagy assay after CCCP in MEFs from panel A, as analyzed by flow cytometry. Quantitative analysis was performed on the ratiometric flow cytometry data (mCherry/GFP). Data are presented as mean ± SEM (n = 3). Statistical significance was determined using one-way ANOVA. Asterisks indicate significant differences compared to the control group (* p < 0.05). (C) Representative confocal micrographs of Control (NT), BCL2L13 KO, Triple KO, and Quad KO MEFs treated with 20 µM CCCP for the indicated timepoints and immunostained for HSP60 and Rab7. Zoomed-in regions highlight co-localization of HSP60 and Rab7 (indicated by blue arrows). (D) Mitolysosome formation was quantified as RAB7+ mitochondrial fragments using CellProfiler. Data points represent experimental averages of more than 200 cells per condition (n = 3). Statistical significance was determined using one-way ANOVA. Asterisks indicate significant differences compared to the control group (* p < 0.05). (E) Model for the regulation of BCL2L13. After mitochondrial damage, AMPK activates Parkin and BCL2L13, while simultaneously suppressing NIX and BNIP3 activation by ULK1. In contrast, under hypoxic conditions, ULK1-dependent phosphorylation and activation of BNIP3, NIX, and FUNDC1 are critical events, and neither Parkin nor BCL2L13 appears to be involved. Thus, Bcl2L13 is currently the only ubiquitin-independent mitophagy receptor known to be acutely activated by mitochondrial damage.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Western Blot, CRISPR, Functional Assay, Mitophagy Assay, Flow Cytometry, Control, Activation Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: Using MitoQC flow cytometry analysis and quantitative co-locatization of HSP60 and RAB7 by high-throughput indirect immunofluorescence imaging, as in . (A) Endogenous mouse BCL2L13 levels (NT cells) alongside stable expression of WT human Flag-tagged BCL2L13 and Serine-to-Alanine mutants in the Quad KO MEFs. Endogenous BCL2L13 protein levels are comparable to the flag-tagged addbacks. Note that human BCL2L13 runs ∼20kD larger than mouse BCL2L13. (B) Mitophagy in Quad KO MEFs stably expressing the mitophagy reporter (mito-QC) with addback of BCL2L13 WT point mutants was analyzed using flow cytometry. Quantitative analysis of ratiometric flow cytometry (mCherry/GFP). Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. Asterisks indicate statistically significant differences compared to the control group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001). (C) Control (NT), Triple KO, and Quad KO MEF stably expressing with indicated BCL2L13 cDNAs were treated with 20 µM CCCP for 1H or 4H timepoints and immunostained for HSP60 and Rab7, as detailed in . Mitolysosome formation was quantified as RAB7+ mitochondrial fragments using CellProfiler. Bar graphs represent the mean and standard error of >200 cells/condition.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Flow Cytometry, High Throughput Screening Assay, Immunofluorescence, Imaging, Expressing, Stable Transfection, Control