Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Representative intracellular calcium signal of AD293 cells transfected with hTRPC5 (1-764) evoked by 256 nM EA ( A ) or 14 mM extracellular calcium ( B, C ), and inhibited by different concentrations of HC-070 ( B ) or clemizole (CMZ) ( C ), measured by FLIPR calcium assay. Arrow 1 denotes the time point for application of buffer alone or buffer with inhibitors; arrow 2 denotes the time point for application of the activators. ( D ) Fluorescence size exclusion chromatography profile of full-length hTRPC5 and hTRPC5 (1-764) . ( E ) Representative size exclusion chromatography of hTRPC5 (1-764) purified in glycol-diosgenin (GDN) micelles. ( F ) SDS-PAGE gel of hTRPC5 (1-764) eluted from size exclusion chromatography. The position of TRPC5 is labeled. Fractions that were pooled for cryo-EM analysis are denoted by asterisks.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Transfection, Calcium Assay, Fluorescence, Size-exclusion Chromatography, Purification, SDS Page, Labeling, Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A, B ) Inhibitory effect of clemizole (CMZ) ( A ) and HC-070 ( B ) on the extracellular calcium-induced intracellular calcium increase of cells with wild-type hTRPC5 over-expression measured by FLIPR calcium assay (data are shown as means ± standard error, n = 3 independent experiments). ( C, D ) Cryo-EM density maps of CMZ-bound hTRPC5 shown in side view ( C ) and top view ( D ). Subunits A, B, C, and D are colored in purple, light blue, orange, and gray, respectively. Lipids are colored in gold. The approximate boundary of the cell membrane is indicated by gray lines. TMD, transmembrane domain; ICD, intracellular cytosolic domain. ( E ) Ribbon diagram of a single subunit with secondary structure elements represented in different colors. ARD, ankyrin repeats domain; LHD, linker-helix domain; CH1, C-terminal helix 1; CH2, C-terminal helix 2. Figure 1—source data 1. Inhibition of WT hTRPC5 by clemizole or by HC-070.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Over Expression, Calcium Assay, Cryo-EM Sample Prep, Inhibition

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Representative raw micrograph recorded on K2 Summit camera. ( B ) Representative 2D class averages of apo hTRPC5. ( C ) Flowchart for cryo-EM data processing of apo hTRPC5. ( D ) Fourier shell correlation (FSC) curves of the two independently refined maps for unmasked (blue line, 3.9 Å) and corrected (purple line, 3 Å). Resolution estimation was based on the criterion of FSC 0.143 cut-off. ( E ) Angular distribution of clemizole (CMZ)-bound hTRPC5. This is a standard output from cryoSPARC. ( F ) FSC curve of the refined model versus EM map.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of apo hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as a dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 4.1 σ, gray mesh) with atomic models superimposed.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Representative raw micrograph recorded on K2 Summit camera. ( B ) Representative 2D class averages of CMZ-bound hTRPC5. ( C ) Flowchart for cryo-EM data processing of CMZ-bound hTRPC5. ( D ) Fourier shell correlation (FSC) curves of the two independently refined maps for unmasked (blue line, 3.5 Å) and corrected (purple line, 2.7 Å). Resolution estimation was based on the criterion of FSC 0.143 cut-off. ( E ) Angular distribution of CMZ-bound hTRPC5. This is a standard output from cryoSPARC. ( F ) FSC curve of the refined model versus EM map.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of HC-070-bound hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 3 σ, gray mesh) with atomic model superimposed. ( E–F ) Cryo-EM density map of HC-070 shown in different views contoured at 3 and 7 σ, respectively. ( G–J ) Cryo-EM densities of HC-070-binding pocket in ( G ) HC-070-bound, ( H ) clemizole (CMZ)-bound, ( I ) apo, and ( J ) Pico145-bound hTRPC5 maps, contoured at 3.8 σ.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of CMZ-bound hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as a dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 3.8 σ, gray mesh) with atomic models superimposed. ( E ) Cryo-EM density of CMZ shown in different views (contoured at 3.7 σ). ( F ) Cryo-EM densities of CMZ-binding pocket in CMZ-bound hTRPC5 map (contoured at 4.6 σ). ( G, H ) Cryo-EM densities in HC-070-bound hTRPC5 and apo hTRPC5 maps corresponding to the CMZ-binding pocket contoured at the same σ as in ( F ).

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Comparison of experimental density map of diacylglycerol (DAG) (gray) (contoured at 3.2 σ) and simulated density map of lysophospholipid (yellow). Please note, the density for electron-dense phosphate head group is absent in the cryo-EM maps obtained experimentally. ( B–D ) Close-up views of lipid 1 ( B ), lipid 2 ( C ), and cholesteryl hemisuccinate (CHS) ( D ) binding sites. Subunits A and B are colored in purple and light blue, respectively. Residues that interact with lipids are shown as sticks. Insets show cryo-EM densities of these lipids, contoured at 2.3, 3.1, and 3.1 σ, respectively.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Immunity

Article Title: Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals

doi: 10.1016/j.immuni.2017.06.013

Figure Lengend Snippet: Human PC-Ms Accumulate in the Gut together with ME-M B Cells and Include a Circulating Counterpart Expressing Gut-Homing Receptors (A) Flow cytometry (FCM) of IgM and IgA on CD19 + CD38 hi CD10 − PCs from human ileum and colon. (B) Frequency of PC-Ms among total PCs, assessed by FCM. (C) FCM of selected surface molecules on naive (N) B cells, PC-Ms, and switched PCs (PC-SW) from human ileum. Numbers indicate mean fluorescence intensity (MFI). (D) Immunofluorescence analysis (IFA) of IgM (green), IgA (red), and DNA (blue) in human ileum and mouse small intestine (SI) lamina propria (LP). Original magnification, 20×. Scale bars, 50 μm. (E) Number of PC-Ms (top), PC-As (center) per mm 2 of LP, and PC-M/PC-A ratio (bottom) from human or mouse SI assessed following tissue IFA. Data summarize six different tissue samples where at least four high-power microscopic fields were analyzed. (F and G) Representative FCM (F) and frequency (G) of β7 + CCR9 + cells in human circulating PC-Ms, PC-As, and PC-G/Es. (H) Representative FCM showing IgM versus IgD staining on CD19 + CD38 − CD10 − B cells from different human tissues. (I) Frequency of ME-M B cells from tissues shown in (H). (J) FCM of IgD, CD24, CD27, and CD148 on naive, ME-M, and ME-SW B cells from ileum. Data show one representative result (A, F, H) of 12 (B), 8 (G), or 52 (I) experiments or are from one experiment of at least 3 with similar results (C, D, J). Results are presented as mean ± SEM; two-tailed unpaired Student’s t test (B and E) and one-way ANOVA with Tukey’s post hoc test (I). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S1 .

Article Snippet: All ELISAs were developed using HRP-labeled goat anti-human IgM (0.2 μg/ml; cappel) or IgA Fc Ab (0.25 μg/ml; Southern Biotech) and TMB substrate reagent set (BD Bioscience).

Techniques: Expressing, Flow Cytometry, Fluorescence, Immunofluorescence, Staining, Two Tailed Test

Journal: Immunity

Article Title: Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals

doi: 10.1016/j.immuni.2017.06.013

Figure Lengend Snippet: Human Gut ME-M B Cells Inhabit Mucosal Follicles, Show Post-GC Traits, Include a Circulating Counterpart Expressing Gut-Homing Receptors, and Emerge Early in Life (A) IFA of IgM (green), IgD (red), and DNA (blue) in human ileum tissue sections. Boxes correspond to enlarged right images. Original magnification, 20× (left) or 60× (right). Scale bars, 50 μm. (B) Number of IgM + IgD − ME-M B cells from human intestine calculated by counting cells/mm 2 following tissue IFA. Data summarize results from five different tissue samples where at least four microscopic fields were analyzed. (C) FCM of CCR7 and CXCR4 on naive, ME-M, and ME-SW B cells from human ileum and colon. (D) Replication history analyzed by KREC assay. Dashed line corresponds to past cell divisions in control GC B cells from human tonsils. (E and F) Representative FCM (E) and frequency (F) of human circulating β7 + CCR9 + B cells. (G) IFA of IgM (green), IgD (red), IgA (magenta), and DNA (blue) in intestinal tissues from children. Boxes correspond to enlarged right images. Original magnification, 10× (top left), 20× (top right), 4× (mid left), 40× (mid right), 10× (bottom left), and 40× (bottom right). Scale bars, 50 μm. Data are from 1 of at least 3 experiments with similar results (A, C, G), summarize 3 experiments (D), or show 1 representative result (E) of 24 experiments (F). Results are presented as mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (two-tailed unpaired Student’s t test). See also Figure S1 .

Article Snippet: All ELISAs were developed using HRP-labeled goat anti-human IgM (0.2 μg/ml; cappel) or IgA Fc Ab (0.25 μg/ml; Southern Biotech) and TMB substrate reagent set (BD Bioscience).

Techniques: Expressing, Two Tailed Test

Journal: Immunity

Article Title: Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals

doi: 10.1016/j.immuni.2017.06.013

Figure Lengend Snippet: Human Gut ME-M B Cells Undergo Proliferation, PC Differentiation, IgM Secretion, and IgA Class Switching in Response to TD or TI Signals (A and B) FCM of CFSE dilution profiles (A) and GeoMean (% of max) of CFSE staining (B) in naive (N) and ME-M B cells from human ileum cultured for 5 days as indicated. Ctrl, medium alone. (C) FCM of CFSE and CD38 on naive (top) and ME-M (bottom) B cells from human ileum cultured as in (A) and (B). Numbers indicate percent of newly formed CD38 hi CFSE lo PCs. (D) FCM of IgM and IgA on CD38 hi CFSE lo plasmablasts emerging upon stimulation as in (A) and (B). (E) ELISA of IgM and IgA secreted by naive (N) and ME-M B cells from human ileum cultured for 5–7 days as in (A) and (B). (F) FCM of CD38, CD27, IgM, and IgA on sorted FcRL4 − (left) or FcRL4 + (right) ME-M B cells from human ileum cultured for 5 days with medium alone (ctrl) or CD40L, IL-21, and IL-10. (G) qRT-PCR analysis of mRNA encoding AID ( AICDA ) in naive, FcRL4 − ME-M, and FcRL4 + ME-M B cells from human ileum. Data represent one representative experiment of two with similar results (A–D, F) or summarize at least three different experiments (E, G). Results are presented as mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (two-tailed unpaired Student’s t test).

Article Snippet: All ELISAs were developed using HRP-labeled goat anti-human IgM (0.2 μg/ml; cappel) or IgA Fc Ab (0.25 μg/ml; Southern Biotech) and TMB substrate reagent set (BD Bioscience).

Techniques: Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Immunity

Article Title: Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals

doi: 10.1016/j.immuni.2017.06.013

Figure Lengend Snippet:

Article Snippet: All ELISAs were developed using HRP-labeled goat anti-human IgM (0.2 μg/ml; cappel) or IgA Fc Ab (0.25 μg/ml; Southern Biotech) and TMB substrate reagent set (BD Bioscience).

Techniques: Recombinant, Blocking Assay, Fluorsave, Giemsa Stain, Sample Prep, DNA Purification, SYBR Green Assay, Microarray, Transformation Assay, Software