Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: GSKJ4 and A-485 treatments modulate Notch receptors expression and activity. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (A) TALL-1 or (C) MOLT3 cells treated for 48 h with 2 μM GSKJ4 or with DMSO. (B) Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panel) and HA and β-actin protein levels (lower panel) in TALL-1 cells transfected with HA-tagged EZH2 expression vector (HA-EZH2) or with the empty control vector. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (D) TALL-1 or (E) MOLT3 cells treated for 48 h with 5 μM A-485 or DMSO. Data represent mean values of three biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 3) * P < 0.05, ** P < 0.01, *** P < 0.001. Uncropped western blots related to this figure are displayed in .

Article Snippet: The expression vector PIRVNeoSV containing the human c-Myc cDNA coding sequence (c-Myc) was kindly provided by Dr. Giuseppe Giannini (Sapienza University, Rome, Italy). pCMV3-HA vector containing the human EZH2 coding sequence (HA-EZH2) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot

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: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Photograph of the cut section of an ovarian sclerosing stromal tumor (SST; left) displaying classic SST appearance with yellow tissue at periphery and white, central fibrotic depression, and micrographs of hematoxylin & eosin stained representative section at low (top right) and high (bottom right) magnification. Scale bars, 1 cm (left), 200 μm (top right), 50 μm (bottom right). b Schematic representation of the FHL2-GLI2 fusion transcript including the exons and domains involved. The breakpoint of the 5′ and 3′ partner genes are represented as black vertical lines. Spanning reads are depicted and aligned to the predicted junction sequence. c Schematic representation showing the Reads Per Kilobase per Million (RPKM) mapped read counts of each GLI2 exon. The GLI2 fusion breakpoint is represented as a red dashed line. d Fluorescence in situ hybridization (FISH) of two representative SSTs using a three-color FHL2-GLI2 probe, with 5′ GLI2 (orange), 3′ GLI2 (red), and 5′ FHL2 (green), showing the presence of the FHL2-GLI2 fusion (white arrows). e Representative Sanger sequencing electropherograms of the genomic FHL2-GLI2 breakpoint. f RNA in situ hybridization (RNA-ISH) using custom FHL2-GLI2 probes (red) showing the chimeric FHL2-GLI2 mRNA expression in two representative SSTs harboring the FHL2-GLI2 fusion. g Frequency of the FHL2-GLI2 fusion gene and GLI2 rearrangements in 26 SSTs from this study. h Frequency of the FHL2-GLI2 fusion gene and GLI2 rearrangements in 26 SSTs and frequency of the FHL2-GLI2 fusion gene in 48 other ovarian sex cord-stromal tumors from this study. aGCT, adult-type granulosa cell tumor. i Frequency of FHL2-GLI2 fusion gene in 26 SSTs from this study and in 9950 tumors from 33 cancer types from The Cancer Genome Atlas (TCGA). AML acute myeloid leukemia, PCPG pheochromocytoma and paraganglioma.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Staining, Sequencing, Fluorescence, In Situ Hybridization, RNA In Situ Hybridization, Expressing

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY), and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. b Representative images of colony formation assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 5 mm; top). Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2 or FHL2-GLI2. The migratory effects/wound area was assessed at 0 and 24 h (Scale bar, 500 μm; top) and quantified (bottom). In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Colony Assay, Wound Healing Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Quantitative assessment of CALB2 transcripts in immortalized mesenchymal stem cells (MSCs) and HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. b Representative western blot analysis of calretinin protein levels in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (bottom) of protein levels as compared to control. c Representative confocal micrographs of immunofluorescence analysis of calretinin (green) and 4–6-diamidino-2-phenylindole (DAPI, blue) in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 50 μm). Quantification (bottom) of calretinin intensity/cell relative to control. d Quantitative assessment of FOXL2 transcripts in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. In a – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Differential gene expression analysis of human sclerosing stromal tumors (SSTs) subjected to RNA-sequencing ( n = 8, this study) and high-grade serous ovarian carcinomas ( n = 16; The Cancer Genome Atlas) and other sex cord-stromal tumors (SCSTs, n = 11; this study). Gene expression fold-change is color-coded according to the legend. Only genes significantly differentially expressed ( P < 0.05; two-tailed unpaired t -test) are shown. CPM, count per million. b Expression levels of Sonic Hedgehog (SHH) pathway genes in human SSTs ( n = 11) and other sex-cord stromal tumors ( n = 9) as defined using NanoString. Expression levels and SHH enrichment scores are color-coded according to the legends. *** P < 0.001, Wilcoxon rank test. Hierarchical clustering was performed using complete linkage and Euclidian distance. c Quantitative assessment of the Sonic Hedgehog pathway PTCH1 and GLI1 transcripts in immortalized mesenchymal stem cells (MSCs), HEK-293 and medulloblastoma (DAOY) cells and of PTCH1 and CCND1 transcripts in human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. d Representative western blot analysis of PTCH1 and GLI1 protein expression in MSC, HEK-293 and DAOY cells and of PTCH1 and CCND1 in BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (below) of protein levels as compared to control. In c – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Gene Expression, RNA Sequencing, Two Tailed Test, Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Representative confocal micrographs of immunofluorescence analysis of FLAG (red), 4–6-diamidino-2-phenylindole (DAPI, blue), and GFP (green) in HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Scale bars, 10 μm. b GLI response element (GLI-RE) luciferase reporter assay of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (top), and GLI-RE promoter activity in HEK-293 transiently transfected with control, FHL2-GLI2 and FHL2-GLI2 with R338A-K339A mutations in the Zinc Finger 5 of GLI2 required for DNA binding (FHL2-GLI2 Mutated; bottom). SV40-Renilla was used to normalize transfection efficiency. c Immunoprecipitation assay with SUFU antibody of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Western blot analysis using anti-FLAG and anti-SUFU antibodies, and tubulin as loading control (left). GLI-RE promoter activity in HEK-293 stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 transfected with SUFU or control (right). d Cell titer blue proliferation assay of HEK-293, DAOY and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 20 µM GANT61 or vehicle control (DMSO). GANT, GANT61. e FLAG Chromatin Immunoprecipitation (ChIP) assay of GLI1 and PTCH1 promoters (promoter 1 and 2) in MSC and HEK-293 cells stably expressing either control or FHL2-GLI2. GLI1 and PTCH1 gene body and MYOD1, gene promoters not under GLI regulation, were used as negative controls. In b – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; *P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Reporter Assay, Activity Assay, Transfection, Binding Assay, Immunoprecipitation, Western Blot, Proliferation Assay, Chromatin Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY) and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2) or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). b Representative images of colony formation assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with Vismodegib 500 nM or vehicle control (DMSO). Scale bars, 5 mm. Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). The migratory effect/wound area was assessed at 0 and 24 h and quantified compared to DMSO (bottom). Vismo, Vismodegib. Scale bars, 500 μm. In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Colony Assay, Wound Healing Assay, Two Tailed Test

Journal: eLife

Article Title: The nanoscale organization of the Nipah virus fusion protein informs new membrane fusion mechanisms

doi: 10.7554/eLife.97017

Figure Lengend Snippet: ( A ) Representative images of 293T cell–cell fusion induced by NiV-G and NiV-F-WT, L53D, V108D, or Q393L. 293T cells were co-transfected with plasmids coding for NiV-G and empty vector (NC) or NiV-F constructs. Cells were fixed at 18 hr post-transfection. Arrows point to syncytia. Scale bar: 10 μm. ( B ) Relative levels of 293T cell–cell fusion in ( A ). Five fields per experiment were counted from three independent experiments. Data are presented as mean ± SEM. p value was obtained by Welch’s test. *p < 0.05; ****p < 0.0001. ( C ) The cell surface expression levels of NiV-F-WT, L53D, V108D, and Q393L on 293T cells were measured by flow cytometry. Mean fluorescence intensity (MFI) values were calculated by FlowJo and were normalized to that of F-WT. Data are presented as mean ± SEM of three independent experiments. Statistical significance was determined by the unpaired t -test with Welch’s correction (ns: p > 0.05). Values were compared to that of the NiV-F-WT. ( D ) NiV-F processing of F-WT, L53D, V108D, Q393L in 293T cells. 293T cells were transfected by F-WT and the mutants. The cell lysates were analyzed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blotting after 28 hr post-transfection. F0 and F2 were probed by M2 monoclonal mouse anti-FLAG antibody. GAPDH was probed by monoclonal mouse anti-GAPDH. ( E ) Relative entry levels of VSV/NiV pseudovirions expressing NiV-G-HA and NiV-F-FLAG (WT; solid black line) or FLAG-tagged NiV-F-L53D (L53D), V108D (V108D), or Q393L (Q393L; dotted red line). The negative control (NC), the recombinant VSV pseudoviruses without glycoproteins, is shown as a dotted gray line. The relative light units (RLUs) of the lysates of infected Vero cells were quantified 18–24 hr post-infection and plotted against the number of viral genomes/ml over 3 logs of viral input. Data shown are mean ± SEM from one representative experiment (of three). ( F ) The result of a representative western blot analysis of VSV/NiV pseudovirions. 4 × 10 8 copies VSV/NiV pseudovirions were separated by a denaturing 10% SDS–PAGE and probed against NiV-G-HA (rabbit anti-HA) and NiV-F-FLAG (mouse anti-Flag). ( G ) The VLPs expressing NiV-M-Bla, G-HA, and the FLAG-tagged WT or mutant NiV-F were allowed to bind to the target HEK293T cells loaded with CCF2-AM dye at 4°C. The Blue/Green (B/G) ratio was measured at 37°C for 4 hr at a 3-min-interval. The average background was subtracted, and the results were normalized to the maximal B/G ratio of WT VLPs. Results from one representative experiment (of three) are shown. ( H ) The result of a representative western blot analysis of NiV VLPs. Equal volumes of VLPs were separated by a denaturing 10% SDS–PAGE and probed against NiV-G (rabbit anti-HA), NiV-F (mouse anti-Flag), and NiV-M (mouse anti-β-lactamase). Figure 3—figure supplement 1—source data 1. Related to . Figure 3—figure supplement 1—source data 2. Related to . Figure 3—figure supplement 1—source data 3. Related to . Figure 3—figure supplement 1—source data 4. Related to . Original files for western blot analysis. Figure 3—figure supplement 1—source data 5. Related to . PPTX files indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 6. Related to . Figure 3—figure supplement 1—source data 7. Related to . Original files for western blot analysis. Figure 3—figure supplement 1—source data 8. Related to . PPTX files indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 9. Related to . Figure 3—figure supplement 1—source data 10. Related to . Original files for western blot analysis. Figure 3—figure supplement 1—source data 11. Related to . PPTX files indicating the relevant bands and treatments.

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , ATCC , CRL-3216 , .

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Flow Cytometry, Fluorescence, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Negative Control, Recombinant, Infection, Mutagenesis

Journal: eLife

Article Title: The nanoscale organization of the Nipah virus fusion protein informs new membrane fusion mechanisms

doi: 10.7554/eLife.97017

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , ATCC , CRL-3216 , .

Techniques: Recombinant, FLAG-tag, Residue, Plasmid Preparation, Sequencing, Concentration Assay, Imaging, Luciferase, RNA Extraction, Software, Modification, Generated, Microscopy

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of ADAMTS17 shows location and targeting of exon 3 by CRISPR/Cas9 gRNA to induce nonhomologous end joining. The nucleotide and amino acid sequence of the ADAMTS17 WT allele (green) and after AT insertion (red) are indicated. The dinucleotide insertion induced a frameshift, which resulted in a premature stop codon after 12 amino acids. (B) Sanger sequencing traces of a PCR product generated with primers flanking exon 3 showing the AT insertion (underlined) in the Adamts17 KO. (C) Quantitative real-time PCR using cDNA prepared from WT and Adamts17 KO lung tissue as a template shows significant reduction of ADAMTS17 mRNA in the KO (n = 3). (D) Micrographs of ADAMTS17 immunostaining of sections through WT and Adamts17 KO skin (left), DKO growth plates (middle), and of primary DKO mouse skin fibroblasts (right). The signal in the dermis around hair follicles, in growth plate chondrocytes, and in fibroblasts and their ECM originating from the monoclonal ADAMTS17 antibody was strongly reduced in KO and DKO tissues and cells, indicating lack of ADAMTS17 protein in Adamts17 KO mice. (E) Pie chart showing Mendelian distribution of genotypes recovered from Adamts17 Het intercrosses at the time of genotyping (P7–P10) (n = 94 mice). (F) Breeding scheme to generate WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. (G) Pie chart showing distribution of genotypes recovered from Adamts10 Het; Adamts17 Het intercrosses at P7–P10 (n = 180 mice). Statistical analysis was performed using Chi square calculation. (H) Kaplan–Meier survival analysis of DKO mice. The numbers of observed dead/total mice for the individual genotypes are indicated in brackets. Statistical significance was determined using a log-rank test. (I) Whole mount images of WT, 10KO, 10KO;17Het mice at 4 wk of age show progressive reduction in body size. (J) Bar graphs showing body weights of 4-wk-old mice of the indicated genotypes. The number of mice is indicated below the genotypes. (I, K) Bar graphs showing body weight normalized to average femur length for the genotypes that were significantly different in (I). Bars in (C) indicate mean values and whiskers the SD. In (J, K) floating bars indicate the 25th–75th percentile range, lines the mean value, and whiskers the SD. (C, J, K) Statistical differences in (C) were determined using a two-sided t test and (J, K) were using a one-way ANOVA with post hoc Tukey test. a, P < 0.05 compared with WT.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: CRISPR, Sequencing, Generated, Real-time Polymerase Chain Reaction, Immunostaining

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Micrographs of Masson’s trichrome–stained cross sections through dorsal skin from 4-wk-old WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. ED, epidermis; D, dermis; HD, hypodermis; PC, panniculus carnosus. (B, C, D) Bar graphs showing quantification of overall skin thickness (B) and the thicknesses of the epidermis, dermis, hypodermis (C), and panniculus carnosus (p. carnosus, (D)). Individual data points represent multiple measurements along the different skin layers from n = 3 mice/genotype. (E) Stacked bar graphs showing the relative proportions of individual skin layers. The percentage values are indicated. (F) Bar graphs show the quantification of hair follicle numbers in the skin for each genotype. (G, H) Bar graphs showing normalized gene expression in fragments per kilobase of transcript per million mapped reads (FPKM) for Adamts10 and Adamts17 in individual skin cell types at E14.5 (G) and P5 (H). Data were extracted from the Hair-GEL database ( ; ). (I, J, K) Micrographs showing the localization of ADAMTS17 mRNA (red/dark purple) in WT skin cross sections at E13.5 (I), E16.5 (J), and P0 (K) detected by RNAscope in situ hybridization with a probe specific for ADAMTS17 mRNA. Sections were counterstained with hematoxylin. (L) Micrograph of ADAMTS17 immunostaining (green) of cross sections through WT skin. Nuclei were stained with DAPI (blue). (M) Micrographs of primary mouse skin fibroblasts after immunostaining for fibrillin-1 (red) and fibronectin (green). Nuclei were counterstained with DAPI (blue). (M, N) Quantification of mean fluorescence intensity from (M) (n = 4 biological replicates). In (B, C, D, F), floating bars indicate 25th–75th percentile range, lines the mean value and whiskers the SD. In (N), the bars represent the mean value and the whiskers the SD. Statistical differences in (B, C, D, F, N) were determined using a one-way ANOVA with post hoc Tukey test. a, P < 0.05 compared with WT; b, P < 0.05 compared with Adamts10 KO; P < 0.05 compared with Adamts17 KO.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Staining, Gene Expression, RNAscope, In Situ Hybridization, Immunostaining, Fluorescence

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of ADAMTS10 and ADAMTS17, which is identical. The degenerate (ADAMTS10) and canonical (ADAMTS17) furin-processing sites and the localization of the catalytic residue Glu-390 in ADAMTS17 (17) that was mutated into Ala to generate proteolytically inactive ADAMTS17-EA (17-EA) are indicated. The domain organization of the catalytic (17-PCD) and ancillary (17-AD) domain constructs is indicated. (B) Schematic representation of experimental design for coculture of human dermal fibroblasts (HDF) with HEK293 cells stably expressing 17- or 17-EA (left) or co-transfection of 17- or 17-EA–encoding plasmids with FN1 or COL6A2-encoding plasmids in HEK293 cells (right). (C) Volcano plot showing N-terminally labeled peptides identified by N-terminomics method TAILS in conditioned medium from ADAMTS17-expressing HEK293 cells cocultured with HDFs. Peptides present only in samples from WT ADAMTS17 (red) or enriched in conditioned medium from WT ADAMTS17 cocultures compared with the cocultures with proteolytically inactive ADAMTS17-EA suggest ADAMTS17 substrates. (D) Venn diagram showing overlap of ADAMTS17-cleaved proteins (TAILS) from coculture systems (left) and binding partners for the ADAMTS17 ancillary domain (17-AD) identified by yeast-2-hybrid screening with a human placenta–derived cDNA library (right). Note that fibronectin (FN1) and COL6 were independently identified in both screens. (E) Domain organization of fibronectin (FN1, NP_997647 ) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence) and the localization of the peptide identified by TAILS (red bar, red amino acid sequence). (F) MS2 spectrum of the N-terminally labeled FN1 peptide (GNSVNEGLNQPTDDSCFDPYTVSHYAVGDEWER) showing b- and y ions. (G) Western blot detection of endogenous fibronectin in conditioned medium (Med) and cell lysates (Lys) collected after coculture of 17- or 17-EA–expressing HEKs with HDFs. A monoclonal (green) and four different polyclonal (red) anti-fibronectin antibodies were used. (H) Western blot detection of recombinant fibronectin (rFN) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA with rFN in HEK293 cells. A polyclonal anti-fibronectin antibody (red) and a monoclonal anti V5-tag antibody (green) were used to detect rFN.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Residue, Construct, Stable Transfection, Expressing, Cotransfection, Labeling, Binding Assay, Derivative Assay, cDNA Library Assay, Sequencing, Western Blot, Recombinant

Journal: Life Science Alliance

Article Title: Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

doi: 10.26508/lsa.202503232

Figure Lengend Snippet: (A) Domain organization of COL6A2 (NP_0018403) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence). (B) Domain organization of COL6A3 ( NP_004360 ) showing the localization of the peptide identified by MS (red bar, red amino acid sequence). (C) MS2 spectrum of the N-terminally labeled ADAMTS17-digested COL6A3 peptide (SDDEVDDPAVELkQFGVAPF) showing b- and y ions. (D) Western blot of endogenous (end.) COL6 (red) in conditioned medium (Med) and cell lysate (Lys) collected from cocultures of 17- or 17-EA–expressing HEK293 cells with HDFs. (E) Micrographs of endogenous COL6A1 deposition (red) in the ECM of HDFs cocultured with 17- or 17-EA–expressing HEK293 cells. Nuclei were stained with DAPI (blue). (F) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates). (G) Micrographs of endogenous COL6A1 deposition (red) in the ECM of HDF after culture in the presence of conditioned medium from 17- or 17-EA–expressing HEK293 cells. Nuclei were stained with DAPI (blue). (H) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates). (I) Western blot of recombinant COL66A2 (rCOL6) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA and rCOL6A2 in HEK293 cells using a monoclonal anti FLAG-tag antibody (green). (J) Micrographs of HDFs cultured in the presence of 50 μg/ml of purified recombinant 17-PCD and 17-AD protein (see for domain organization) costained for endogenous COL6A1 (red) and the Myc-tag of the recombinant ADAMTS17 protein fragments (green). Nuclei were stained with DAPI (blue). (K) Micrographs of adult HDFs and Weill–Marchesani syndrome (WMS) patient–derived dermal fibroblasts (WMS-DF) for endogenous COL6A1 (red). Nuclei were counterstained with DAPI (blue). (L) Quantification of the mean fluorescence intensity of the COL6A1 signal (n = 3 replicates, 2–3 fields of view). (M) Western blot of endogenous COL6A1 (red) and GAPDH (green) in conditioned medium (Med) and cell lysate (Lys) collected from HDF and WMS-DF cultures. (N) Quantification of COL6A1 band mean fluorescence intensities normalized to GAPDH. In (E, G, M), bars represent the mean value and whiskers the SD. In (K), the floating bars indicate the 25th–75th percentile range, the lines the mean value, and whiskers the SD. Statistical differences in (E, G, K, M) were determined using a two-sided t test.

Article Snippet: Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS-201-012) were purchased from ATCC.

Techniques: Sequencing, Labeling, Western Blot, Expressing, Staining, Fluorescence, Recombinant, FLAG-tag, Cell Culture, Purification, Derivative Assay

Journal: Molecular Metabolism

Article Title: The novel long noncoding RNA Lnc19959.2 modulates triglyceride metabolism-associated genes through the interaction with Purb and hnRNPA2B1

doi: 10.1016/j.molmet.2020.100996

Figure Lengend Snippet: Purb acts as a transcriptional repressor to inhibit ApoA4 expression. (A) Western blot detected the ApoA4 and Purb expression levels after knockdown or overexpression of Purb in BRL-3A cells. (B) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of four truncated ApoA4 promoters with pcDNA3.1 vector or pc3.1- Purb in HEK293T cells. (C) The schematic diagram shows that Purb binding site (PNR) contained at 2000 bp of ApoA4 promoter was well conserved. (D) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of ApoA4 promoters of 2000 bp with pcDNA3.1 vector or pc3.1- Purb in different doses (range: 0–1.5 μg/mL) in HEK293T cells. (E) Dual-luciferase reporter assay detected relative luciferase activities after cotransfected with wt/mutApoA4 promoters of 2000 bp with pc3.1- Purb in HEK293T cells. (F) ChIP analysis of Purb interaction with the ApoA4 promoter. BRL-3A cells lysates were immunoprecipitated with anti- Purb or control mouse IgG antibody. (G–H) Overexpression of lnc19959.2 and control in BRL-3A cells, respectively, were incubated with the proteasome inhibitor MG-132 (10 μM) or the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) for 6 or 12 h. The protein level of Purb extracted from the whole cell was detected by western blot. (I–J) BRL-3A cells lysates were immunoprecipitated with an HA or Flag-specific antibody in BRL-3A cell lysates which were stably expressing ubiquitin with C-terminal HA tag or Purb with C-terminal Flag tag, respectively. And then they were analyzed by western blot with anti- Purb or anti-Ubiquitin. Bottom, the input of the cell lysates. (K) Western blot detects the ubiquitination levels in BRL-3A cells. Unpaired t -test was used to measure the statistical significance; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The BRL-3A rat liver cell line and HEK293T human renal epithelial cell lines (American Type Culture Collection) were cultured in glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA) at 37 °C with humidified air and 5% CO 2 .

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Luciferase, Reporter Assay, Cotransfection, Plasmid Preparation, Binding Assay, Immunoprecipitation, Control, Incubation, Stable Transfection, Ubiquitin Proteomics, FLAG-tag

Journal: Molecular Metabolism

Article Title: The novel long noncoding RNA Lnc19959.2 modulates triglyceride metabolism-associated genes through the interaction with Purb and hnRNPA2B1

doi: 10.1016/j.molmet.2020.100996

Figure Lengend Snippet: Cebpb activates the lnc19959.2 expression. (A) Heatmaps present gene expressions selected from sequence prediction software. (B–C) RT-qPCR analysis of lnc19959.2 expression levels in BRL-3A cells in the condition of knockdown or overexpression of Cebpb. (D) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of four truncated lnc19959.2 promoters with pcDNA3.1 vector or pc3.1-Cebpb in HEK293T cells. (E) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of lnc19959.2 promoters of 1500 bp with pcDNA3.1vector or pc3.1-Cebpb in different doses (range: 0–1.5 μg/mL) in HEK293T cells. (F) Schematic diagram shows that the part of lnc19959.2 promoter sequence of wt and mut of Cebpb binding site is located in −1500～-1000 bp. (G) Dual-luciferase reporter assay detected relative luciferase activities after being cotransfected with wt/mutlnc19959.2 promoters of 1500 bp with pc3.1-Cebpb in HEK293T cells. (H) ChIP analysis of Cebpb interacted with the lnc19959.2 promoter. BRL-3A cells lysates were immunoprecipitated with anti-Cebpb or control mouse IgG antibody. Unpaired t -test was used to measure the statistical significance; ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: The BRL-3A rat liver cell line and HEK293T human renal epithelial cell lines (American Type Culture Collection) were cultured in glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA) at 37 °C with humidified air and 5% CO 2 .

Techniques: Expressing, Sequencing, Software, Quantitative RT-PCR, Knockdown, Over Expression, Luciferase, Reporter Assay, Cotransfection, Plasmid Preparation, Binding Assay, Immunoprecipitation, Control

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: (A) Schematic of cell-free protein expression into synthetic lipid vesicles and subsequent electroformation with Vesicle Prep Pro (Nanion). ( B ) Full-length PKD2L1-GFP protein detected by western blot after cell-free expression into vesicles. ( C ) Monitored fluorescence over time of cell-free expressed PKD2L1-GFP and a non-fluorescent control plasmid produced in the presence or absence of lipid vesicles. Figure 1—source data 1. Original file for western blot analysis displayed in . Figure 1—source data 2. PDF file containing original western blot for , indicating the relevant bands and treatments.

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Expressing, Western Blot, Fluorescence, Control, Plasmid Preparation, Produced

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: (A) Top , fluorescence (488γ) standard curve determined with a recombinant GFP-tagged protein fit with a linear regression. PURExpress synthesized PKD2L1-GFP and PKD2-GFP were measured after 3 hr of expression at 37°C. (GFP standard curve N = 5, PKD2L1 and PKD2 N = 3 replicates). Bottom , average protomer and tetramer protein production from the PURExpress reaction. Error bars represent SEM. ( B ) Representative sequence coverage of PKD2L1-GFP (top) and PKD2-GFP (bottom) from tandem mass spectrometry spectra. ( C ) Mass spectrometry outputs identifying polycystin proteins. ( D, E ) Fluorescence-detection size-exclusion chromatography (FSEC) of polycystin proteins derived from recombinant and cell-free protein expression (CFE) sources. Recombinant human PKD2-GFP and PKD2L1-GFP protein was obtained from lysates of 0.5 × 10 6 HEK cells stably expressing the channels. Purified Aequorea Victoria GFP His-tag protein was obtained from Thermo Fisher Scientific. SUVs containing CFE-derived polycystins were lysed using dodecyl β- D -maltoside (DDM) prior to FSEC analysis (see methods).

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Fluorescence, Recombinant, Synthesized, Expressing, Sequencing, Mass Spectrometry, Size-exclusion Chromatography, Derivative Assay, Stable Transfection, Purification

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: Schematic of PKD2L1-SNAP incorporated into GUVs, followed by SNAP staining with cell permeable (Cell488), and cell impermeable (Surface647) SNAP-Tag marker.

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Staining, Marker

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: (A) Schematic of possible ion channel orientation outcomes from PKD2L1 cell-free expression (top) and hypothesized fluorescence results when Cell488 and Surface647 added (bottom). ( B ) Fluorescent confocal images from the SNAP-tagged vesicles. The scale bar represents 10 μm for all images. ( C ) Vesicle percentage depicts the percent of vesicles with each fluorescent output ( N = 65 vesicles).

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Expressing, Fluorescence

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: (A) Images of voltage-clamped giant unilamellar vesicles (GUVs) with incorporated PKD2L1-GFP channels. Left , establishing high-resistance seals in the on-cell patch configuration. Right , transitioning to the inside-our patch configuration. Scale bar = 20 μm. ( B ) Example unitary single-channel current records from GUVs reconstituted with or without PKD2L1 protein. Vesicles were patched using the symmetrical 150 mM K + conditions (see methods) and PKD2L1 single-channel current producing full and sub-conductances are colored black and blue, respectively. ( C ) Average single-channel current amplitudes. Conductance ( γ ) estimated by fitting the average single-channel currents to a linear equation. Error (gray) indicates SEM from N = 3–8 GUVs. Several replicates lacked single-channel openings at all test potentials. ( D ) PKD2L1 single-channel current recorded using asymmetric cationic solutions, with 150 mM K + in the bath and 150 mM NMDG + in the pipette. ( E ) Resulting average single-channel current amplitudes where no inward single-channel current was detected ( N = 3–4 GUVs).

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Transferring

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: Conductance properties of polycystins measured from giant unilamellar vesicle (GUV) and cilia membranes.

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Membrane, Expressing

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: ( A, B ) Top , images of voltage-clamped primary cilia from mouse hippocampal neurons and inner medullary kidney collecting duct cells (IMCD) harvested from transgenic mice expressing a fluorescent cilia reporter (ARL13B-EGFP tg ) . Scale bar = 20 μm. Previous work has genetically identified PKD2L1 and PKD2 as essential ion channel subunit in the primary cilia of the renal collecting duct cells and hippocampal neurons ( ; ). Bottom , average single-channel current amplitudes recorded from primary cilia using K + in the recording electrode solution. Conductance ( γ ) estimated by fitting the average single-channel currents to a linear equation. Error indicates SEM ( N = 6 cilia).

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Transgenic Assay, Expressing

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: Use of a reporter cell system to evaluate NKG2A/CD94 inhibition and its interaction with peptide‐HLA‐E complexes. (a) Schematic showing the interaction between NKG2A and the TCR/CD3 complex on the Jurkat reporter cells with HLA‐E‐peptide complexes and membrane‐bound anti‐CD3‐scFv on the T cell stimulator cells (K562S). (b) Surface expression of NKG2A on control and NKG2A/CD94 expressing reporter cells. (c) Schematic representation of a construct harbouring the sequences of the H1N1 leader, a 9‐mer peptide, GS‐linker (#1), β 2 ‐microglobulin, GS‐linker (#2) and HLA‐E*0103. In the table, the five different 9‐mer peptides used in this study are depicted. Flow cytometric analysis of T cell stimulator cells (K562S RFP). Left panel; HLA‐E expression on control K562S cells (open histogram) and K562S transduced to express peptide‐HLA‐E complexes (filled histograms). Right panel; expression of membrane‐bound anti‐CD3 Ab fragment (CD14‐stem) on K562 cells (open histogram) and K562S (with and without HLA‐E) cells (filled histograms). (d) Gating strategy of one representative experiment of control reporter cells and NKG2A/CD94 reporter cells stimulated with control K562S and K562S‐HLA‐E HLA‐G , K562S‐HLA‐E HLA‐B8 , K562S‐HLA‐E HLA‐A2 , K562S‐HLA‐E HLA‐C4 and K562S‐HLA‐E hsp60 . Open histogram: unstimulated cells. NFκB‐eCFP expression was measured via flow cytometry. (e) Control and NKG2A/CD94 expressing reporter cells were stimulated with the indicated K562S cells. Reporter activation is shown as the geometric mean of fluorescence intensity (gMFI) of NFκB activation. Lower panel: reporter activation is shown as fold induction (gMFI of K562S‐HLA‐E peptide ‐stimulated cells/gMFI control K562S‐stimulated cells). Results of seven independent experiments performed in duplicates are depicted. For statistical evaluation, a two‐way ANOVA followed by Bonferroni post hoc test was performed (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns p > 0.05).

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Inhibition, Membrane, Expressing, Control, Construct, Flow Cytometry, Activation Assay, Fluorescence

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: NKG2A expression on CD8 + T cells and natural killer (NK) cells. (a) Left panel; representative gating of NKG2A and NKG2C on NK cells and CD8 + T cells from freshly isolated PBMCs is shown. Right panel; expression of NKG2A and NKG2C on CD8 + T cells and NK cells; each dot represents one donor. Median is shown ( n = 10). (b) Freshly isolated PBMCs were stimulated with K562S cells expressing CD86 for 5 days. NKG2A and NKG2C expression was assessed on NK and CD8 + T cells. Each dot represents one donor. Median is shown ( n = 6). (c) PBMCs were co‐cultured for 5 days with the indicated K562S cell lines and proliferation (CSFE low ) was analysed for NK cells. Left panel; one representative experiment is shown. Right panel; data from several donors are depicted (HLA‐G and HLA‐C4 [ n = 8]; HLA‐B8 and HLA‐A2 [ n = 4]). Each data point represents the mean of triplicates of one donor. NK cell proliferation induced upon stimulation with K562S expressing the indicated HLA‐E‐complexes is normalized to the NK cell proliferation induced by control K562S cells (% of CFSE low of K562S‐HLA‐E peptide stimulated NK cells/% of CFSE low control K562S‐stimulated NK cells). For statistical evaluation, a one‐way ANOVA followed by Dunn's multiple comparison test was performed (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns p > 0.05). (d) Lysis of the indicated target cells upon 4 h co‐culture with PBMCs pre‐stimulated for 7–10 days in the presence of IL‐2 and IL‐15 to induce NK cell expansion. Effector: target cell ratio was 10:1 and monalizumab was used at a final concentration of 5 μg/ml. Data were normalized to control K562 cells (control K562 = 100% cell lysis). Median is shown ( n = 10). For statistical evaluation, a two‐way ANOVA followed by Bonferroni post hoc test was performed (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns p > 0.05).

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Expressing, Isolation, Cell Culture, Control, Comparison, Lysis, Co-Culture Assay, Concentration Assay

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: Evaluation of NKG2A antibodies in their binding and blocking capacities. (a) The EC 50 values were determined for the therapeutic NKG2A antibody monalizumab, as well as for the monoclonal antibodies clone Z199, clone REA110 and clone 131411. NKG2A expressing reporter cells were incubated with increasing concentrations ranging from 100 pg/ml to 10 μg/ml of the depicted antibodies. Antibody binding was detected with an APC‐conjugated anti‐human and anti‐mouse IgG antibody, respectively. EC 50 was calculated from six experiments performed in duplicate. (b) NKG2A expressing reporter cells were stimulated with control K562S and K562S‐HLA‐E HLA‐A2 in the presence or absence of monalizumab, Z199, REA110 and 131411 (all 1 μg/ml). Reporter activation is shown as fold induction (gMFI of K562S‐HLA‐E HLA‐A2 stimulated cells/gMFI control K562S‐stimulated cells). Results are shown of four (monalizumab) and two (Z199, REA110 and 131411) independent experiments performed in duplicates.

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Binding Assay, Blocking Assay, Bioprocessing, Expressing, Incubation, Control, Activation Assay

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: Identification of the epitopes of NKG2A antibodies. (a) Sequence alignment of NKG2A (A113 to L233) and NKG2C (A111 to L231). (b) Schematic representations of wild‐type NKG2A and NKG2C, as well as chimeric constructs of NKG2A‐C (#1, #2, #3), are depicted. Cell surface expression of the molecules was determined by using a Strep‐tag antibody. (c) Jurkat cells expressing the indicated molecules were incubated with the following NKG2A antibodies monalizumab, REA110, Z199, 131411, and a NKG2C antibody (134522), respectively (open histogram: the indicated antibodies on control cells). (d) Schematic representation of a mutated NKG2C construct (NKG2C mut ) and its cell surface expression detected with a Strep‐tag antibody. (e) Flow cytometric analysis of the depicted antibodies on cells expressing the NKG2C mut molecule (open histogram: the indicated antibodies on control cells). (f) Crystal structure of NKG2A (orange) and CD94 (green) (PDB ID code 3BDW). Positions A113, M189 (light green), E197 (light green) and K232 are highlighted with arrows . One representative experiment is shown (b–e).

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Sequencing, Construct, Expressing, Strep-tag, Incubation, Control

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: Assessment of functional EC 50 values of NKG2A blocking antibodies monalizumab and Z199. NKG2A/CD94 expressing reporter cells were stimulated with control K562S, K562S‐HLA‐E HLA‐G , K562S‐HLA‐E HLA‐B8 , K562S‐HLA‐E HLA‐A2 and K562S‐HLA‐E HLA‐C4 for 24 h in the presence of monalizumab (a) and Z199 (b) at concentrations ranging from 31.6 μg/ml to 10 ng/ml. White bars represent NKG2A inhibition in the absence of an antibody. Results are depicted of three (monalizumab) and two (Z199) independent experiments performed in duplicate. Left panels; data are normalized to control K562S cells in the absence or presence of the indicated antibody concentrations. Right panels; inhibition curves and half‐maximum effective concentrations (EC 50 ) were calculated for the NKG2A antibodies from normalized data.

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Functional Assay, Blocking Assay, Expressing, Control, Inhibition

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: EC 50 values and the 95% confidence intervals (CI) were determined for monalizumab and Z199 for their ability to block the HLA‐E peptide /NKG2A interaction in a functional assay.

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Blocking Assay, Functional Assay