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: 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: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Overview of work in the present study. The study began with an analysis of whole-exome sequencing (WES) data comparing rare genetic variant frequencies between late-onset Alzheimer’s disease (LOAD) cases and non-AD controls. Subsequent analyses focused on the MUC6 variable number of tandem repeat (VNTR) region. The particular variants identified initially to be associated with LOAD risk were later removed from the consensus variant calls, presumably because this VNTR region is extremely challenging for high-throughput sequence characterization methods. Polymorphism in the MUC6 VNTR region was associated with phospho-tau (pTau) pathology and with altered AP2A2 expression. Immunohistochemical analyses showed that AP2A2 protein was often colocalized with pTau tangles in LOAD brains. Green boxes indicate new data and analytic results.

Article Snippet: The following plasmids were used for transfections: pCMV6-XL5 empty vector; pCMV6-XL5-MYC/DDK [Flag-tag]-AP2A2, and pCMV6-XL5 -Tau (no tag)—all from Origene (Rockville, MD).

Techniques: Sequencing, Variant Assay, High Throughput Screening Assay, Expressing, Immunohistochemical staining

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Plot of rare variant analysis in Alzheimer’s Disease Sequencing Project (ADSP) whole-exome sequencing (WES) data, indicating genetic variants associated with late-onset Alzheimer disease (LOAD) risk in the MUC6 variable number of tandem repeats (VNTR) region. (A) p Values are shown from single rare variant analyses in terms of association with LOAD phenotype. Several genetic variants were initially reported to be associated with LOAD risk at p < 1 × 10 −10 (red circles). These specific genetic variant calls later failed quality control when different data quality filters were applied. (B) Panel depicts this genomic region in schematic form. The initial genetic variants were present in a unique, exonic, and polymorphic VNTR region that is annotated inside the MUC6 gene and within 4000 bases of the 3′-untranslated region (UTR) of the AP2A2 gene. Whereas these specific SNP calls in the MUC6 VNTR region were the initial reason for studying this genetic locus, the subsequent experiments focused on the polymorphic size of the VNTR region and the association with pTau proteinopathy.

Article Snippet: The following plasmids were used for transfections: pCMV6-XL5 empty vector; pCMV6-XL5-MYC/DDK [Flag-tag]-AP2A2, and pCMV6-XL5 -Tau (no tag)—all from Origene (Rockville, MD).

Techniques: Variant Assay, Sequencing

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Comparison of AP2A2 transcript levels (a proxy for gene expression) from cerebellum of 2 groups (n = 15 in each group) stratifying on the size of largest MUC6 VNTR region. The 2 groups were matched for age at death, sex, severity of Alzheimer disease neuropathologic changes, and postmortem interval ( Table 3 ). Fold-change of detected AP2A2 transcript relative to a different transcript was calculated based on quantitative PCR results. Regardless of the normalization method, there was a trend for the subjects with larger VNTR regions (red squares) to have lower average levels of detected AP2A2 transcripts in comparison to the individuals with smaller VNTR regions (blue circles). This difference was statistically significant when AP2B1 levels were used for normalization (A) , but was only a nonsignificant trend when GAPDH (B) was used for normalization. Comparisons were performed using unpaired 2-tailed Student t -tests.

Article Snippet: The following plasmids were used for transfections: pCMV6-XL5 empty vector; pCMV6-XL5-MYC/DDK [Flag-tag]-AP2A2, and pCMV6-XL5 -Tau (no tag)—all from Origene (Rockville, MD).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Brightfield immunohistochemical staining of AP2A2 in human brain sections. Shown are representative results from staining of temporal neocortex. Panels (A) and (B) show results from a nondemented aged subject with minimal Alzheimer-type pathology. Panel (A) and ( B ; higher magnification) show the relatively even, neuronal staining for AP2A2. Panels (C) and (D) show representative results from a demented subject with severe Alzheimer’s disease pathology. Note that the staining pattern of AP2A2 is here reminiscent of neurofibrillary tangles (green arrowhead), as well as some scattered, darkly AP2A2-immunoreactive structures in the neuropil (black arrows). Scale bars: A , C = 200 microns; B , D = 25 microns. Panel (E) shows the results of Western blots using the same AP2A2 antiserum—immunoblots of HEK-293 cell extracts are shown. This antiserum recognizes a prominent band at the predicted (∼104 kDa) size that is augmented by transfection with a pCMV-AP2A2 plasmid. The human brain extracts (shown here are Western blots from the cerebellum of 1 control and 1 LOAD brain) stained a similar banding pattern.

Article Snippet: The following plasmids were used for transfections: pCMV6-XL5 empty vector; pCMV6-XL5-MYC/DDK [Flag-tag]-AP2A2, and pCMV6-XL5 -Tau (no tag)—all from Origene (Rockville, MD).

Techniques: Immunohistochemical staining, Staining, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: AP2A2 and pTau colocalization, detected using immunofluorescence and digitally quantified in a convenience sample of 5 LOAD cases. (A) A representative example of AP2A2 and pTau (immunostained with PHF1 antibody) staining using epifluorescence microscopy, as well as the HALO software generated digital markup showing the area of staining that was AP2A2 + , pTau + , and AP2A2 + pTau + double positive. In the colocalized markup, the red indicates pTau + , the green indicates AP2A2 + , and the yellow indicates AP2A2 + pTau + double positive. (B) and (C) Confocal z-stack images show cells double positive for pTau and AP2A2 at higher magnification. The arrow and arrowhead indicate the same cells (B) , shown in the orthogonal projection images in (C) . (D) Panel shows the percentage of the tissue in each of the regions of interest that was positive staining. The number of AP2A2 + pTau + double positive cells in relation to the number of AP2A2 + is shown in (E) , and in relation to the pTau + cells in (F) , as quantified by confocal microscopy in the hippocampus and neocortex. Data are plotted as mean ± SD for the 5–10 z-stacks included for each region and for each slide.

Article Snippet: The following plasmids were used for transfections: pCMV6-XL5 empty vector; pCMV6-XL5-MYC/DDK [Flag-tag]-AP2A2, and pCMV6-XL5 -Tau (no tag)—all from Origene (Rockville, MD).

Techniques: Immunofluorescence, Staining, Epifluorescence Microscopy, Software, Generated, Confocal Microscopy

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Unlike in cases with LOAD pathology, AP2A2 does not colocalize extensively with phospho-TDP-43 in brains with limbic-predominant age-related TDP-43 proteinopathy neuropathologic changes (LATE-NC), nor with pTau in progressive supranuclear palsy (PSP) brains. (A) A representative example of AP2A2 and phospho-TDP-43 staining using epifluorescence microscopy shows the lack of colocalization of AP2A2 with phospho-TDP-43 in a brain with both AD neuropathologic changes (ADNC) and LATE-NC. (B) AP2A2 was also not colocalized with pTau (immunostained with antibody PHF1) in the caudate nucleus of subjects with autopsy-proven PSP. Quantitative analyses of these results are shown in and .

Article Snippet: The following plasmids were used for transfections: pCMV6-XL5 empty vector; pCMV6-XL5-MYC/DDK [Flag-tag]-AP2A2, and pCMV6-XL5 -Tau (no tag)—all from Origene (Rockville, MD).

Techniques: Staining, Epifluorescence Microscopy

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene

doi: 10.1093/jnen/nlz116

Figure Lengend Snippet: Western blots show results of experiments in cultured cells testing whether AP2A2 and Tau proteins can be coimmunoprecipitated. Flag-AP2A2 and Tau expressing plasmids were transfected into cultured HeLa cells, separately and together. The lysates, Anti-Flag M2 coimmunoprecipitation (Co-IP), and nonimmunized mouse serum (NMS) controls were immunoblotted for AP2A2 (A) , Tau (B) , AP2B1 (C) , and tubulin as a control (D) . Molecular weights (kDa) are indicated on the left of the blots. A band labeled by the AP2A2 antibody (∼104 kDa) was present without transfection, and that signal was augmented in the lysate and in IPs where Flag-AP2A2 plasmids were transfected, indicating that Flag-AP2A2 transfection was successful. Anti-Flag M2 beads pulled down the Flag-tagged AP2A2. There were no augmented signals of Tau protein in the M2-IP product of the AP2A2 and Tau cotransfection when probed with Tau antibody DA9, indicating that AP2A2 and Tau proteins were not coimmunoprecipitated. Bands at ∼55 kDa (near to Tau) in the NMS and M2-IP lanes were likely immunoglobulin protein and were present in all the immunoblots. As a positive control, endogenous AP2B1, a known binding partner of AP2A2 with the same molecular weight of ∼104 kDa, was Co-IP’d with AP2A2. Gel portions where the proteins were predicted to be present, according to their known molecular weights, are shown with a red asterisk for each protein. Complete Western blots are shown in .

Article Snippet: The following plasmids were used for transfections: pCMV6-XL5 empty vector; pCMV6-XL5-MYC/DDK [Flag-tag]-AP2A2, and pCMV6-XL5 -Tau (no tag)—all from Origene (Rockville, MD).

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Co-Immunoprecipitation Assay, Labeling, Cotransfection, Positive Control, Binding Assay, Molecular Weight

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Heatmap of expression data of several ZFPs identified using quantitative real-time PCR (qPCR). ( B ) Quantification of mRNA coding for ZNFX1 in different macrophages following H37Rv infection (MOI = 2), using qPCR. hpi, hours postinfection; RQ, relative quantification. ( C ) Western blot analysis of ZNFX1 expression in H37Rv-infected BMDMs. ( D ) Immunohistochemistry detection and statistical analysis of ZNFX1 expression in the lung tissues and LNs of patients with CI or TB ( n = 4). A 2-way ANOVA with Holm-Šídák post hoc test ( B ) or an unpaired t test ( D ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Infection, Quantitative Proteomics, Western Blot, Immunohistochemistry

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Schematic diagram of the time points of assays during the in vivo evaluation of Znfx1 –/– -induced immune responses against M . tuberculosis infection. CK, cytokine. ( B ) In vivo M . tuberculosis load in the lung and spleen tissues of Znfx1 –/– mice at 1 and 4 weeks after H37Rv infection ( n = 5). ( C ) H&E staining of the lung and spleen tissues of Znfx1 –/– mice. The splenic MGCs were quantified ( n = 5, with 30 randomly selected fields of view for statistics). Yellow arrows indicate MGCs. ( D ) Measurement of NO production indicated as the concentration of NO 2 – in the lung and spleen tissues 1 week postinfection ( n = 5). ( E ) Luminex multiplex assays of cytokine expression in the peripheral blood of mice 1 week after H37Rv infection ( n = 5). ( F ) ELISA of cytokine expression in the lung and spleen of mice 1 week after H37Rv infection ( n = 5). A 2-way ANOVA with Holm-Šídák post hoc test ( B ) or an unpaired 2-tailed t test was used ( C – F ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: In Vivo, Infection, Staining, Concentration Assay, Luminex, Multiplex Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Flow cytometry analysis of red fluorescence–positive WT and Znfx1 –/– BMDMs infected with H37Rv carrying the red fluorescence protein (RFP) gene (i.e., H37Rv-RFP) at MOI = 10 at 2 hpi ( n = 3). ( B ) Flow cytometry analysis of green fluorescence–positive WT and Znfx1 –/– BMDMs incubated with FITC-conjugated latex beads ( n = 4). ( C ) CFU assays of intracellular M . tuberculosis levels in H37Rv-infected WT and Znfx1 –/– BMDMs at MOI = 5 ( n = 4). ( D ) Flow cytometry analysis of CD80, CD86, MHC-II, and CD206 expression on the surface of H37Rv-infected WT and Znfx1 –/– BMDMs at MOI = 2 at 24 hpi ( n = 3). ( E ) Western blot assay of the regulatory effects of ZNFX1 on activation of signaling pathways following M . tuberculosis infection at MOI = 5. p-, phosphorylated. ( F ) Western blot assay of the regulatory effects of ZNFX1 on activation of the autophagy-associated mTOR signaling pathways. An unpaired 2-tailed t test ( A and B ) or a 2-way ANOVA with Holm-Šídák post hoc test ( C – F ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Flow Cytometry, Fluorescence, Infection, Incubation, Expressing, Western Blot, Activation Assay, Protein-Protein interactions

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Western blot assay of the regulatory effects of ZNFX1 on AMPK activation. mpi, minutes postinfection. ( B ) Western blot assay of the regulatory effects of ZNFX1 on the levels of p62 and LC3-I/II conversion. ( C ) Immunofluorescence assays of LC3 puncta in Znfx1 –/– BMDMs treated with BafA1, followed by infection with H37Rv-RFP ( n = 3, with 10 randomly selected fields of view for statistics). ( D ) Double-staining immunofluorescence assays of LC3 in F4/80 + macrophages in the lung and spleen tissues of WT and Znfx1 –/– mice following H37Rv infection ( n = 5, with 5 randomly selected fields of view for statistics). “Pearson’s R value” refers to Pearson’s correlation coefficient. ( E ) CFU assays of M . tuberculosis load in WT and Znfx1 –/– BMDMs treated with rapamycin ( n = 5). A 2-way ANOVA with Holm-Šídák post hoc test ( A – D ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Western Blot, Activation Assay, Immunofluorescence, Infection, Double Immunofluorescence Staining

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) High-throughput RNA sequencing analysis of H37Rv-infected (MOI = 2) WT and Znfx1 –/– BMDMs at 6 and 24 hpi identified Prkaa2 as the downstream target of ZNFX1. ( B and C ) Expression levels of Prkaa2 and its coded protein AMPKα2 following H37Rv (MOI = 2) in WT and Znfx1 –/– BMDMs, using qPCR ( B ) and Western blotting ( C ), respectively. ( D ) Double-staining immunofluorescence assays of p-AMPK in F4/80 + macrophages in the lung and spleen tissues of WT and Znfx1 –/– mice following H37Rv infection ( n = 5, with 5 randomly selected fields of view for statistics). “Pearson’s R value” refers to Pearson’s correlation coefficient. ( E ) Flow cytometry analysis of red fluorescence–positive WT and Znfx1 –/– BMDMs treated with EX229, followed by infection with H37Rv-RFP (MOI = 10, n = 4). ( F and G ) CFU assays of intracellular M . tuberculosis levels in H37Rv-infected (MOI = 5) WT and Znfx1 –/– BMDMs ( F ) and ZNFX1 -silenced hMDMs ( G ) following EX229 treatment ( n = 4). si, siRNA. ( H ) Flow cytometry analysis of CD80, CD86, and MHC-II expression on the surface of WT and Znfx1 –/– BMDMs treated with EX229 and infected with H37Rv (MOI = 2, n = 3). A 2-way ANOVA with Holm-Šídák post hoc test ( B and E – H ) or an unpaired 2-tailed t test ( D ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: High Throughput Screening Assay, RNA Sequencing, Infection, Expressing, Western Blot, Double Immunofluorescence Staining, Flow Cytometry, Fluorescence

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: WT and Znfx1 –/– mice were treated with EX229 and infected with H37Rv. ( A ) CFU assays of M . tuberculosis load in the lung and spleen ( n = 5). ( B ) ELISA of cytokine expression in the lung and spleen of mice 1 week after H37Rv infection ( n = 5). ( C ) Assays of NO production in the lung and spleen 1 week postinfection ( n = 5). ( D and E ) H&E staining of the lung and spleen. The MGCs in the spleen were quantified. Yellow arrows indicate MGCs ( n = 5, with 30 randomly selected fields of view for statistics). A 2-way ANOVA with Holm-Šídák post hoc test ( A – C and E ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Western blotting of ZNFX1 in the cytosol, membrane, and nucleus fraction of BMDMs. ( B ) qPCR analysis of Prkaa1 and Prkaa2 in Znfx1 –/– BMDMs treated with DRB ( n = 3). ( C ) qPCR analysis of Prkaa2 in WT and Znfx1 –/– BMDMs infected with LV- Prkaa2 ( n = 3). LV, lentiviral. ( D ) RIP assay using anti-ZNFX1 antibody and qPCR analysis of the association between ZNFX1 protein and Prkaa1 and Prkaa2 mRNA in BMDMs ( n = 3). ( E ) RNA pulldown assay using biotinylated Prkaa2 and GFP transcripts and Western blotting of the association between Prkaa2 mRNA and ZNFX1 protein in BMDMs. ( F ) Schematic diagram of recombinant plasmids carrying full-length or various truncated forms of the Znfx1 gene accompanied by a FLAG tag. ( G ) RIP assay using anti-FLAG antibody and qPCR analysis of the association between full-length or truncated forms of ZNFX1 protein and Prkaa2 mRNA in HEK293T cells transfected with various Znfx1 expression plasmids ( n = 5). ( H ) qPCR analysis of Prkaa2 in HEK293T cells transfected with various Znfx1 expression plasmids and treated with DRB ( n = 3). ( I ) CFU assay of M . tuberculosis load in WT and Znfx1 –/– BMDMs transfected with the F2 truncated form of Znfx1 ( n = 4). ( J ) Schematic diagram of the molecular mechanism of ZNFX1 in the regulation of autophagy against M . tuberculosis infection. OE, overexpression. A 2-way ANOVA with Holm-Šídák post hoc test ( D and I ) or a 1-way ANOVA followed by multiple-comparison test ( G ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Western Blot, Membrane, Infection, Recombinant, FLAG-tag, Transfection, Expressing, Colony-forming Unit Assay, Over Expression, 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 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: Nature reviews. Neuroscience

Article Title: The Molecular Biology of FMRP: New Insights into Fragile X Syndrome

doi: 10.1038/s41583-021-00432-0

Figure Lengend Snippet: High throughput identification of FMRP targeted mRNAs

Article Snippet: Van Driesche 2019 ( 35 ) mouse cerebellar Purkinje cells (Pcp2-Cre) and granule cells (NeuroD1-Cre), Anti-GFP mAbs (HtzGFP19C8 and HtzGFP19F7, Heiman 2008 ( 91 ); from FMRP-cTag mice 135 high confidence FMRP CLIP targets in Purkinje cells; 259 in granule cells, they are distinct Long, coding sequences none has ranked targets based on TRAP-seq of same cell types; Synaptic functions; microtubule, Cell signaling, GTPase signaling in Purkinje cells, GABAergic transmission in cerebellar granule cells Sawicka 2019 ( 36 ) mouse CA1 neurons (Camk2a-Cre) Anti-GFP mAbs (HtzGFP19C8 and HtzGFP19F7, Heiman 2008 ( 91 ); from FMRP-cTag mice 327; different from cb granule neurons Long, coding sequences none has ranked targets based on TRAP-seq of same cell types; Signaling, transcription, synaptic development and function, microtubule organization, axon transport, autism, circadian rhythm Ascano 2012 ( 42 ) Human HEK293 cell lines Transfected HA-tagged FMRP, anti-HA ab (MMS-101P, Convance), PAR-CLIP >6000 coding sequence and 3’UTR ACUK, WGGA many Tran 2019 ( 43 ) Human postmortem adult human frontal cortex Anti-FMRP pAb (MBL, RN016P), CLIP-seq 4895 binds to A-to-I edited site; predominantly 3’UTR ACUG Synaptic functions, (focus was editing in autism, not FMRP targets) Li 2020 ( 44 ) Human human iPSC differentiated dorsal and ventral forebrain NPCs and neurons (four cell types) FLAG tag knocked into human FMR1 locus, FMRP-FLAG, anti-FLAG ab (Sigma Aldrich F1804), CLIP-seq 1653 total (1232 in dNPCs, 1234 in vNPCs, 629 in dNeurons, 721 in vNeurons coding sequences, long genes CUAC; UGGA; GAUG, AGGU, CAGC; unclear about G4 Neurogenesis, axon and dendrite morphogenesis, cytoskeleton, microtubule, RNA transport, histone modification (especially in NPCs), autism.

Techniques: High Throughput Screening Assay, Binding Assay, Sequencing, Transmission Assay, Activity Assay, Transfection, FLAG-tag, Modification

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: Nature reviews. Neuroscience

Article Title: The Molecular Biology of FMRP: New Insights into Fragile X Syndrome

doi: 10.1038/s41583-021-00432-0

Figure Lengend Snippet: High throughput identification of FMRP targeted mRNAs

Article Snippet: Van Driesche 2019 ( 35 ) , mouse , cerebellar Purkinje cells (Pcp2-Cre) and granule cells (NeuroD1-Cre), , Anti-GFP mAbs (HtzGFP19C8 and HtzGFP19F7, Heiman 2008 ( 91 ); from FMRP-cTag mice , 135 high confidence FMRP CLIP targets in Purkinje cells; 259 in granule cells, they are distinct , Long, coding sequences , none , has ranked targets based on TRAP-seq of same cell types; , Synaptic functions; microtubule, Cell signaling, GTPase signaling in Purkinje cells, GABAergic transmission in cerebellar granule cells.

Techniques: High Throughput Screening Assay, Binding Assay, Sequencing, Transmission Assay, Activity Assay, Membrane, Transfection, FLAG-tag, Modification

Journal: Nature reviews. Neuroscience

Article Title: The Molecular Biology of FMRP: New Insights into Fragile X Syndrome

doi: 10.1038/s41583-021-00432-0

Figure Lengend Snippet: TRAP (translating ribosome affinity purification)-seq. Cells or tissues are transduced with an epitope-tagged ribosomal protein (green), which can be expressed in specific cells or tissues (step 1). The cells/tissues are then subjected to immunoprecipitation with antibody against the epitope (usually HA, FLAG, or GFP) (step 2); the co-precipitating RNA is then de-proteinized and sequenced (blue line) (step 3). Note that mRNAs associated with few or no ribosomes are not immunoprecipitted (red line). TRAP-seq has been performed in WT and Fmr1-deficient mouse brain35, 55 and has identified mRNAs under translational control by FMRP in specific neuron subtypes.

Article Snippet: Van Driesche 2019 ( 35 ) , mouse , cerebellar Purkinje cells (Pcp2-Cre) and granule cells (NeuroD1-Cre), , Anti-GFP mAbs (HtzGFP19C8 and HtzGFP19F7, Heiman 2008 ( 91 ); from FMRP-cTag mice , 135 high confidence FMRP CLIP targets in Purkinje cells; 259 in granule cells, they are distinct , Long, coding sequences , none , has ranked targets based on TRAP-seq of same cell types; , Synaptic functions; microtubule, Cell signaling, GTPase signaling in Purkinje cells, GABAergic transmission in cerebellar granule cells.

Techniques: Affinity Purification, Transduction, Immunoprecipitation, Control