Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of Tensin-3 as a MALT1 substrate that controls B cell adhesion and lymphoma dissemination.

doi: 10.1073/pnas.2301155120

Figure Lengend Snippet: Fig. 2. MALT1 cleaves human and mouse Tensin-3 after R614 and R645. (A) Immunoblot analysis of 293T cells transfected with the indicated combinations of VSV-tagged MALT1, untagged Tensin-3 or FLAG-tagged BCL10, and given either no treatment (−) or treated for 16 h (+) with z-LVSR-fmk. Tensin-3 cleavage was assessed using antibodies directed against the NT or CT part of Tensin-3. (B) Immunoblot analysis of 293T cells transfected with the indicated combinations of VSV-tagged MALT1 or MALT1 C464 (a catalytically inactive mutant) and untagged Tensin-3. (C) Overview of the domain structure of Tensin family members. ABD: actin-binding domain; SH2: src homology domain 2; PTB: phosphotyrosine binding domain. (D) Immunoblot analysis of 293T cells transfected with the indicated combinations of GFP-tagged Tensin-1, -2, -3, and -4 (CTEN), VSV-tagged MALT1 and FLAG-tagged BCL10. (E) Immunoblot analysis of 293T cells transfected with the indicated combinations of STREP-tagged MALT1, FLAG-tagged human Tensin-3 (hTensin-3) or FLAG-tagged BCL10 constructs. (F) Overview of the domain structure of Tensin-3 and the position of the cleavage sites R614 and R645. ABD: actin-binding domain; SH2: src homology domain 2; PTB: phosphotyrosine binding domain. (G) Tensin-3-deficient BJAB cells were transduced with the indicated Tensin-3 constructs and incubated for 1 h with (+) or without (−) PMA and ionomycin (P+I). Tensin-3 cleavage was analysed by western blotting using an antibody directed against the NT part of Tensin-3. (H) Immunoblot analysis of 293T cells transfected with the indicated combinations of STREP-tagged MALT1, FLAG-tagged mouse Tensin-3 (mTensin-3), hTensin-3, or VSV-tagged BCL10 constructs. Black and open arrowheads indicate uncleaved and cleaved forms, respectively, of Tensin-3. Tubulin served as loading control, * indicates nonspecific bands. Data in all panels are representative of two independent experiments.

Article Snippet: Antibodies used in this study included polyclonal rabbit anti- hTensin- 3 (C2, Santa Cruz, directed against the C terminus), affinity purified rabbit antihTensin- 3 (generated against a GST- Tensin- 3 fusion protein comprising amino acids 418 to 577 of hTensin- 3), rabbit anti- BCL10 (H197, Santa Cruz Biotechnology), D ow nl oa de d fr om h ttp s: //w w w .p na s. or g by 4 9.

Techniques: Western Blot, Transfection, Mutagenesis, Binding Assay, Construct, Transduction, Incubation, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of Tensin-3 as a MALT1 substrate that controls B cell adhesion and lymphoma dissemination.

doi: 10.1073/pnas.2301155120

Figure Lengend Snippet: Fig. 5. Constitutive cleavage leads to decreased Tensin-3 levels, promoting dissemination of xenografted ABC DLBCL. (A and B) Immunoblot analysis of the status of Tensin-3 cleavage in lysates of the indicated GCB or ABC DLBCL cell lines (A), incubated for 24 h with or without z-LVSR-fmk (B). For the HBL-1 cell line, ten times more lysate was loaded for the Tensin-3 blot compared to the other cell lines. (C) Immunoblot analysis for Tensin-3 and cleaved BCL10 in lysates of MCL cell lines characterized by constitutive activation of the NF-κB2 or NF-κB1 pathway. (D and E) Tumor growth of xenografted HBL-1 cells stably transduced with control or Tensin-3-specific sgRNA (D) and weight of tumors excised from mice at day 24 (E). (F and G) Flow cytometric analysis of the presence of hCD20+ HBL-1 cells in the BM (F) or in the spleen (G) of NSG mice 24 d after xenografting of tumor cells into the flanks of the animals (n = 11 and 15 for control and Tensin-3 silenced cells, respectively). Data are representative of two (A–D) independent experiments. (E–G) show pooled data of 2 independent experiments. In (F and G), data were normalized to control samples. Bars represent means ± SEM; differences were statistically significant with P < 0.05 (unpaired t test) unless indicated otherwise (*P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant P > 0.05).

Article Snippet: Antibodies used in this study included polyclonal rabbit anti- hTensin- 3 (C2, Santa Cruz, directed against the C terminus), affinity purified rabbit antihTensin- 3 (generated against a GST- Tensin- 3 fusion protein comprising amino acids 418 to 577 of hTensin- 3), rabbit anti- BCL10 (H197, Santa Cruz Biotechnology), D ow nl oa de d fr om h ttp s: //w w w .p na s. or g by 4 9.

Techniques: Western Blot, Incubation, Activation Assay, Stable Transfection, Transduction, Control

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/eLife.79826

Figure Lengend Snippet: This video shows HEK293T BCL10-KO cells reconstituted with BCL10-mScarlet expressing EYFP-p65. Upon addition of PMA, cells acquire BCL10 puncta (left panel) and p65 nuclear translocation (middle panel). Right panel, Hoechst staining. Scale bar: 10 µm.

Article Snippet: Antibody , anti-BCL10 (Rabbit polyclonal) , Cell Signaling Technology , C78F1 , Simple Western (1:10).

Techniques:

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/eLife.79826

Figure Lengend Snippet: This video shows optogenetic induction of CARD9 CARD -miRFP670-Cry2 clusters (top panel). After a few frames, CARD9 clusters induce BCL10-mScarlet (bottom panel) nucleation, which goes on to polymerize independently of CARD9 clusters. Thus, revealing a pre-existing state of supersaturation. Scale bar: 10 µm.

Article Snippet: Antibody , anti-BCL10 (Rabbit polyclonal) , Cell Signaling Technology , C78F1 , Simple Western (1:10).

Techniques:

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/eLife.79826

Figure Lengend Snippet:

Article Snippet: Antibody , anti-BCL10 (Rabbit polyclonal) , Cell Signaling Technology , C78F1 , Simple Western (1:10).

Techniques: Transfection, Construct, Plasmid Preparation, Expressing, Simple Western, Recombinant, Software

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/elife.79826

Figure Lengend Snippet: Figure 1. Assembly of the CARD-BCL10-MALT1 (CBM) signalosome drives all-or-none activation of nuclear transcription factor-κB (NF-κB). (A) Schematic diagram of binary activation in a population of cells. Higher doses of stimulation increase the probability, but not the magnitude, of activation by each cell. (B) β-Glucan stimulation of THP-1 WT and BCL10-KO monocytes transduced with an NF-κB transcriptional reporter. Monocytes were stimulated for 24 hr then measured for T-Sapphire expression via flow cytometry. The graph shows the mean ± standard deviation (SD) fraction of cells positive for NF-κB expression of three biological replicates. The inset shows the histogram of T-Sapphire expression indicating the distinct negative and positive populations of THP-1 WT cells upon stimulation with 10 μg/ml β-glucan. (C) HEK293 WT and BCL10-KO cells were transduced with an NF-κB transcriptional reporter and stimulated with PMA for 24 hr then measured for T-Sapphire expression via flow cytometry. The inset shows the histogram of T-Sapphire expression indicating the distinct negative and positive populations of HEK293T WT cells upon stimulation with 5 ng/ml PMA. Shown are means ± SD of three biological replicates. (D) THP-1 BCL10-KO monocytes were transduced with a doxycycline-inducible BCL10-mEos3.2 construct. Cells were imaged for BCL10 expression 24 hr after Dox induction. Images show BCL10 puncta only in cells treated with β-glucan for 16 hr (see also Figure 1—figure supplement 3). In contrast, BCL10 E53R does not form puncta regardless of stimulation. Scale bar: 10 µm. (E) Quantification of the number of THP-1 cells with BCL10 puncta after β-glucan stimulation. Each dot represents the average of 4 independent experiments with more than 50 cells each. Shown are means ± SD. (F) Endogenous untagged CARD9 and BCL10 form high molecular weight species in THP-1 cells stimulated with β-glucan for 24 hr. After treatment, cells were lysed, and the protein extracts were resolved by size exclusion chromatography followed by capillary immunodetection.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) HEK293T American Type Culture Collection # CRL- 3216 Cell line (Homo sapiens) THP- 1 American Type Culture Collection # TIB- 202 Strain, strain background (Saccharomyces cerevisiae) rhy1713 Khan et al., 2018 Parent strain for DAmFRET experiments Strain, strain background (Saccharomyces cerevisiae) rhy1734 Khan et al., 2018 Parent strain for DAmFRET experiments Transfected construct (Homo sapiens) m1.0151 This paper Plasmid for expressing Hs CARD9- (G4S)mCardinal from CMV promoter Transfected construct (Homo sapiens) m1.0012 This paper Plasmid for expressing Hs BCL10- (EA3R)4- mEos3.2 from CMV promoter Antibody anti- BCL10 (Rabbit polyclonal) Cell Signaling Technology C78F1 Simple Western (1:10) Antibody anti- CARD9 (Mouse monoclonal) Santa Cruz Biotechnology B- 12: sc- 46677 Simple Western (1:150) Recombinant DNA reagent pCW57.1 Addgene #41393 Recombinant DNA reagent pLV- EF1a- IRES- Hygro Addgene #85134 Chemical compound, drug PMA BioVision 1544–5 Software, algorithm GraphPad Prism Version 9 for Windows

Techniques: Activation Assay, Transduction, Expressing, Flow Cytometry, Standard Deviation, Construct, High Molecular Weight, Size-exclusion Chromatography, Immunodetection

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/elife.79826

Figure Lengend Snippet: Figure 2. The adaptor protein BCL10 creates a nucleation-mediated switch. (A) Domain architecture of CARD-BCL10-MALT1 (CBM) proteins. Death fold domains are colored green. Mutations discussed in the text are indicated. (B) DAmFRET plot of CARD9 expressed in yeast cells, showing moderate AmFRET indicative of oligomer formation at all concentrations. (C) Left, DAmFRET plot of BCL10 expressed in yeast cells, showing a bimodal distribution between cells containing only monomers and cells containing polymers. The discontinuity and overlapping range of protein concentration for the two populations indicates that polymerization is rate limited by a nucleation barrier. Right, schema of cells in the two populations, showing how self-assembly of partially photoconverted mEos3.1-fused proteins causes a FRET signal. (D) DAmFRET plot of MALT1 expressed in yeast cells, showing no AmFRET, indicating a monomeric state of the protein at all concentrations. (E) Structural alignment of CARD9CARD monomers in its soluble (6E26, green) or polymerized (6N2P, blue) forms, showing no major conformational differences between them. (F) Structural alignment of BCL10CARD monomers in its soluble (2MB9, green) or polymerized (6BZE, blue) forms, showing multiple conformational differences that span a-helices 1, 2, 5, and 6. (G) Structural alignment of MALT1DD monomers in its soluble (2G7R, green) and polymerized (together with BCL10, 6GK2, blue) forms, showing no major conformational differences. (H) Weibull fits to DAmFRET plots of WT and mutant BCL10, showing how each mutation affects polymerization as quantified in (I). (I) EC50 (left) and ẟ (right) values derived from the fits in (H). EC50 describes the median concentration at which nucleation occurs. The slope parameter ẟ is a dimensionless proxy for the conformational free energy of nucleation. The R36E and E53R mutations, which lie in separate polymer interfaces, prevent the protein from polymerizing at low concentrations. The cancer-associated R58G mutation decreases the nucleation barrier, resulting in more frequent spontaneous nucleation relative to the WT protein. Shown are means ± standard deviation (SD) of three replicates. t-Test ***p < 0.001, **p < 0.005.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) HEK293T American Type Culture Collection # CRL- 3216 Cell line (Homo sapiens) THP- 1 American Type Culture Collection # TIB- 202 Strain, strain background (Saccharomyces cerevisiae) rhy1713 Khan et al., 2018 Parent strain for DAmFRET experiments Strain, strain background (Saccharomyces cerevisiae) rhy1734 Khan et al., 2018 Parent strain for DAmFRET experiments Transfected construct (Homo sapiens) m1.0151 This paper Plasmid for expressing Hs CARD9- (G4S)mCardinal from CMV promoter Transfected construct (Homo sapiens) m1.0012 This paper Plasmid for expressing Hs BCL10- (EA3R)4- mEos3.2 from CMV promoter Antibody anti- BCL10 (Rabbit polyclonal) Cell Signaling Technology C78F1 Simple Western (1:10) Antibody anti- CARD9 (Mouse monoclonal) Santa Cruz Biotechnology B- 12: sc- 46677 Simple Western (1:150) Recombinant DNA reagent pCW57.1 Addgene #41393 Recombinant DNA reagent pLV- EF1a- IRES- Hygro Addgene #85134 Chemical compound, drug PMA BioVision 1544–5 Software, algorithm GraphPad Prism Version 9 for Windows

Techniques: Protein Concentration, Mutagenesis, Derivative Assay, Concentration Assay, Polymer, Standard Deviation

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/elife.79826

Figure Lengend Snippet: Figure 3. Pathogenic mutations disrupt BCL10 template formation within CARD-CC multimers. (A) Schematic of the DAmFRET experiment used to test for nucleating interactions between CARD-BCL10-MALT1 (CBM) components. Artificial ‘seeds’ are created by expressing the putative nucleating protein as a fusion to the homo-oligomerizing module, μNS (purple). (B) DAmFRET plots of BCL10 (top) or CARDCARD (bottom) in the absence (left) or presence (right) of CARD9CARD seeds expressed in trans. For BCL10, the presence of seeds shifted all low-AmFRET cells to high-AmFRET, indicating that the protein had been supersaturated and is nucleated by CARD9CARD. In contrast, the seeds had no effect on CARD9CARD DAmFRET, suggesting that CARD9CARD only polymerizes at high concentrations and does not become supersaturated. The vertical line denotes the phase boundary for CARD9 CARD polymerization. (C) Graph of spline fits of AmFRET values for CARD9CARD mutants. All of the pathogenic mutations within the CARD domain disrupted polymer formation except for R70W, which stabilized polymers. (D) Effect of CARD9 mutations in the full-length protein context with autorepression eliminated by the I107E mutation. 293T cells transduced with the nuclear transcription factor-κB (NF-κB) reporter were transfected with the indicated CARD9-mCardinal constructs and analyzed for T-Sapphire expression at 48-hr post-transfection. T-Sapphire expression was normalized to that of cells transfected with plasmid expressing mCardinal alone. Shown are means ± standard deviation (SD). Analysis of variance (ANOVA) with post hoc Tukey test ***p < 0.0001. (E) Weibull fits to DAmFRET plots of BCL10 coexpressed with mutant CARD9 seeds, showing that only WT CARD9 nucleates BCL10

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) HEK293T American Type Culture Collection # CRL- 3216 Cell line (Homo sapiens) THP- 1 American Type Culture Collection # TIB- 202 Strain, strain background (Saccharomyces cerevisiae) rhy1713 Khan et al., 2018 Parent strain for DAmFRET experiments Strain, strain background (Saccharomyces cerevisiae) rhy1734 Khan et al., 2018 Parent strain for DAmFRET experiments Transfected construct (Homo sapiens) m1.0151 This paper Plasmid for expressing Hs CARD9- (G4S)mCardinal from CMV promoter Transfected construct (Homo sapiens) m1.0012 This paper Plasmid for expressing Hs BCL10- (EA3R)4- mEos3.2 from CMV promoter Antibody anti- BCL10 (Rabbit polyclonal) Cell Signaling Technology C78F1 Simple Western (1:10) Antibody anti- CARD9 (Mouse monoclonal) Santa Cruz Biotechnology B- 12: sc- 46677 Simple Western (1:150) Recombinant DNA reagent pCW57.1 Addgene #41393 Recombinant DNA reagent pLV- EF1a- IRES- Hygro Addgene #85134 Chemical compound, drug PMA BioVision 1544–5 Software, algorithm GraphPad Prism Version 9 for Windows

Techniques: Expressing, Polymer, Mutagenesis, Transduction, Transfection, Construct, Plasmid Preparation, Standard Deviation

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/elife.79826

Figure Lengend Snippet: Figure 4. The BCL10 nucleation barrier causes binary activation of nuclear transcription factor-κB (NF-κB). (A) Schematic of experiments to determine if BCL10 polymerization activates NF-κB in 293T cells. In the OFF state, BCL10 is dispersed in the cell and the p65 NF-κB subunit is localized to the cytosol. In the ON state, BCL10 is polymerized and p65 relocates to the nucleus, where it induces the transcription of NF-κB response genes. (B) DAmFRET plots of the indicated proteins expressed in 293T cells containing the NF-κB reporter, 48 hr after transfection. The reporter mEos3.2 remains in the monomeric state and does not activate NF-κB. The gray and green boxes designate the regions gated for cells containing either monomer or polymerized protein, respectively, and analyzed for T-Sapphire expression in (C). (C) Quantification of T-Sapphire expression in the gated areas of the DAmFRET plots shown in (B). Shown are means ± standard deviation (SD). Only cells that contained BCL10 polymers, but not cells containing the same concentration of monomeric BCL10 nor cells containing ASC polymers, activated NF-κB. (D) Time-lapse microscopy of 293T BCL10-KO cells reconstituted with BCL10-mScarlet and EYFP-p65. Cells were stimulated with 10 ng/ml PMA and imaged every 10 min. BCL10 and p65 are distributed throughout the cytosol at 0 min, but respectively relocalize to cytosolic puncta and the nucleus over the course of the experiment. Scale bar: 10 µm. (E) Top, ratios of EYFP fluorescence intensities in the nucleus versus cytosol; and bottom, values of the coefficient of variation of mScarlet pixel intensities, over the time course of cells stimulated with PMA at the indicated concentrations. BCL10 polymerization produced visible puncta within approximately

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) HEK293T American Type Culture Collection # CRL- 3216 Cell line (Homo sapiens) THP- 1 American Type Culture Collection # TIB- 202 Strain, strain background (Saccharomyces cerevisiae) rhy1713 Khan et al., 2018 Parent strain for DAmFRET experiments Strain, strain background (Saccharomyces cerevisiae) rhy1734 Khan et al., 2018 Parent strain for DAmFRET experiments Transfected construct (Homo sapiens) m1.0151 This paper Plasmid for expressing Hs CARD9- (G4S)mCardinal from CMV promoter Transfected construct (Homo sapiens) m1.0012 This paper Plasmid for expressing Hs BCL10- (EA3R)4- mEos3.2 from CMV promoter Antibody anti- BCL10 (Rabbit polyclonal) Cell Signaling Technology C78F1 Simple Western (1:10) Antibody anti- CARD9 (Mouse monoclonal) Santa Cruz Biotechnology B- 12: sc- 46677 Simple Western (1:150) Recombinant DNA reagent pCW57.1 Addgene #41393 Recombinant DNA reagent pLV- EF1a- IRES- Hygro Addgene #85134 Chemical compound, drug PMA BioVision 1544–5 Software, algorithm GraphPad Prism Version 9 for Windows

Techniques: Activation Assay, Transfection, Expressing, Standard Deviation, Concentration Assay, Time-lapse Microscopy, Fluorescence, Produced

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/elife.79826

Figure Lengend Snippet: Figure 5. Endogenous BCL10 is constitutively supersaturated. (A) Schematic of the optogenetic nucleation experiment. CARD9CARD is expressed as a fusion to the near-infrared fluorescent protein miRFP670nano and the light-controlled homomultimerizing module Cry2clust. Upon blue light exposure, Cry2clust forms reversible multimers that constrain the fused CARD9CARD domains to a local concentration sufficient to form a polymeric template for BCL10. (B) Microscopy images of 293T BCL10-KO cells stably expressing CARD9CARD-miRFP670nano-Cry2clust and BCL10-mScarlet. Cells were maintained at 37°C with 5% CO2 and imaged every 2 min followed by a short pulse of 488-nm light stimulation. Inset number 1, at 6-min post- stimulation, shows BCL10 colocalizing with a CARD9CARD cluster. Inset number 2, at 20 min, shows that the CARD9CARD cluster has dissolved, while the BCL10 punctum has instead elongated into a filament. Scale bar: 10 µm. (C) Values of the coefficient of variation of mIRFP670nano and mScarlet pixel intensities over time. The data are from 40 cell measurements. Shown are means ± standard deviation (SD). (D) Microscopy images of cells expressing CARD9CARD seeds, BCL10-mScarlet and EYFP-p65. Images were taken every 2 min following blue light stimulation. For technical reasons, EYFP-p65 was imaged only at the first and last time points. Scale bar: 10 µm. (E) Ratios of EYFP fluorescence intensities in the nucleus versus cytosol after blue light stimulation for the indicated durations. Shown are measurements from each cell, along with the means ± standard error of the mean (SEM).

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) HEK293T American Type Culture Collection # CRL- 3216 Cell line (Homo sapiens) THP- 1 American Type Culture Collection # TIB- 202 Strain, strain background (Saccharomyces cerevisiae) rhy1713 Khan et al., 2018 Parent strain for DAmFRET experiments Strain, strain background (Saccharomyces cerevisiae) rhy1734 Khan et al., 2018 Parent strain for DAmFRET experiments Transfected construct (Homo sapiens) m1.0151 This paper Plasmid for expressing Hs CARD9- (G4S)mCardinal from CMV promoter Transfected construct (Homo sapiens) m1.0012 This paper Plasmid for expressing Hs BCL10- (EA3R)4- mEos3.2 from CMV promoter Antibody anti- BCL10 (Rabbit polyclonal) Cell Signaling Technology C78F1 Simple Western (1:10) Antibody anti- CARD9 (Mouse monoclonal) Santa Cruz Biotechnology B- 12: sc- 46677 Simple Western (1:150) Recombinant DNA reagent pCW57.1 Addgene #41393 Recombinant DNA reagent pLV- EF1a- IRES- Hygro Addgene #85134 Chemical compound, drug PMA BioVision 1544–5 Software, algorithm GraphPad Prism Version 9 for Windows

Techniques: Concentration Assay, Microscopy, Stable Transfection, Expressing, Standard Deviation, Fluorescence

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/elife.79826

Figure Lengend Snippet: Figure 6. MALT1 clustering suffices for activation and does not depend on an ordered structure of BCL10 polymers. (A) Schematic of the optogenetic MALT1 activation experiment. A fragment of MALT1 lacking its death domain (MALT1126-824) is expressed as a fusion to mIRFP670nano and Cry2clust, allowing for its oligomerization upon blue light exposure. (B) Schematic of optogenetic MALT1 dimerization experiment. As for (A), but with Cry2clust replaced with the light-controlled dimerizing module, VfAU1-LOV. (C) Microscopy images of 293T cells expressing EYFP-p65 transfected with either the construct in (A) or (B). Images were taken every 2 min following a single pulse of blue light. EYFP-p65 was imaged before blue light stimulation and again 40 min after stimulation, showing that it translocated to the nucleus only in cells in which MALT1 had multimerized. Scale bar: 10 µm. (D) Ratios of EYFP fluorescence intensities in the nucleus versus cytosol at the 40 min time point for the experiments in (C). As a control, 293T WT expressing EYFP-p65 were treated with PMA 5 ng/ml. Shown are measurements from all cells, along with means ± standard deviation (SD) of three replicates. (E) Nuclear transcription factor-κB (NF-κB) activation in 293T MALT1-KO NF-κB reporter cells transfected with the constructs in (A) and (B) and stimulated with continuous blue light for the indicated durations. Cells were treated with PMA at 5 ng/ml. Data represent medians of three independent measurements. (F) Flow cytometry histograms of T-Sapphire fluorescence for the experiment in (E).

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) HEK293T American Type Culture Collection # CRL- 3216 Cell line (Homo sapiens) THP- 1 American Type Culture Collection # TIB- 202 Strain, strain background (Saccharomyces cerevisiae) rhy1713 Khan et al., 2018 Parent strain for DAmFRET experiments Strain, strain background (Saccharomyces cerevisiae) rhy1734 Khan et al., 2018 Parent strain for DAmFRET experiments Transfected construct (Homo sapiens) m1.0151 This paper Plasmid for expressing Hs CARD9- (G4S)mCardinal from CMV promoter Transfected construct (Homo sapiens) m1.0012 This paper Plasmid for expressing Hs BCL10- (EA3R)4- mEos3.2 from CMV promoter Antibody anti- BCL10 (Rabbit polyclonal) Cell Signaling Technology C78F1 Simple Western (1:10) Antibody anti- CARD9 (Mouse monoclonal) Santa Cruz Biotechnology B- 12: sc- 46677 Simple Western (1:150) Recombinant DNA reagent pCW57.1 Addgene #41393 Recombinant DNA reagent pLV- EF1a- IRES- Hygro Addgene #85134 Chemical compound, drug PMA BioVision 1544–5 Software, algorithm GraphPad Prism Version 9 for Windows

Techniques: Activation Assay, Microscopy, Expressing, Transfection, Construct, Fluorescence, Control, Standard Deviation, Flow Cytometry

Journal: eLife

Article Title: A nucleation barrier spring-loads the CBM signalosome for binary activation

doi: 10.7554/elife.79826

Figure Lengend Snippet: Figure 7. Ancient origin of the sequence-encoded nucleation barrier in BCL10. (A) Phylogenetic tree of CARD-BCL10-MALT1 (CBM) signalosome components in the major metazoan clades. One or more CARD-CC proteins occur in all clades, as does a MALT1-like paracaspase. BCL10 was present in the common ancestor to chordates and cnidarians. (B) DAmFRET plots of 293T cells expressing CARD9CARD, BCL10, or MALT1DD from human (top) or the sea anemone Nematostella vectensis (bottom). The gray and green boxes designate the regions gated for cells containing either monomer or polymerized protein, respectively, and analyzed for T-Sapphire expression in (C). (C) Flow cytometry histograms of T-Sapphire fluorescence in 293T BCL10-KO nuclear transcription factor-κB (NF-κB) reporter cells 48 hr after transfection with either human or N. vectensis BCL10-mEos3.2. Gray and green shading represent cells containing either monomeric or polymerized BCL10, respectively, from the corresponding gates illustrated in (B). Data are representative of three independent experiments.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) HEK293T American Type Culture Collection # CRL- 3216 Cell line (Homo sapiens) THP- 1 American Type Culture Collection # TIB- 202 Strain, strain background (Saccharomyces cerevisiae) rhy1713 Khan et al., 2018 Parent strain for DAmFRET experiments Strain, strain background (Saccharomyces cerevisiae) rhy1734 Khan et al., 2018 Parent strain for DAmFRET experiments Transfected construct (Homo sapiens) m1.0151 This paper Plasmid for expressing Hs CARD9- (G4S)mCardinal from CMV promoter Transfected construct (Homo sapiens) m1.0012 This paper Plasmid for expressing Hs BCL10- (EA3R)4- mEos3.2 from CMV promoter Antibody anti- BCL10 (Rabbit polyclonal) Cell Signaling Technology C78F1 Simple Western (1:10) Antibody anti- CARD9 (Mouse monoclonal) Santa Cruz Biotechnology B- 12: sc- 46677 Simple Western (1:150) Recombinant DNA reagent pCW57.1 Addgene #41393 Recombinant DNA reagent pLV- EF1a- IRES- Hygro Addgene #85134 Chemical compound, drug PMA BioVision 1544–5 Software, algorithm GraphPad Prism Version 9 for Windows

Techniques: Sequencing, Expressing, Flow Cytometry, Fluorescence, Transfection