Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, Schematic of the domain arrangement for a TRPV2 monomer with the approximate epitope sites for indicated TRPV2 antibodies. ARD, ankryin repeat domain; TM, transmembrane domain. B, Western blots with indicated TRPV2 antibodies against purified full-length rat TRPV2, purified rat TRPV2 ankryin repeat domain and purified rat TRPV2 C-terminus. C, Western blots with indicated TRPV2 antibodies against extracts from HeLa cells transiently transfected with empty vector, TRPV1-1D4 and TRPV2-1D4.

Article Snippet: 2D6 and 17A11 were used at 1 µg/ml, TRPV2 ACC-032 (Alomone) at 2 µg/ml, VRL-1 PC421 (Calbiochem) at 5 µg/ml and VRL-1 SC-22520 (Santa Cruz) at 10 µg/ml.

Techniques: Western Blot, Purification, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, Western blot analysis with anti-TRPV2 2D6 of F11 cells treated with control siRNA or TRPV2 siRNA (100 nM, 48 h). Quantification of the band corresponding to the molecular weight of TRPV2 was measured using LiCor Odyssey software. TRPV2 band intensity was normalized to actin. Error bars represent S.E.M. from 3 separate experiments. B, Immunoprecipitation of TRPV2 with 10 µg of indicated antibodies from 2.5 mg mouse brain lysate and 2.5 mg mouse heart lysate. TRPV2 was detected by western blot with IR dye-labeled 2D6 antibody. Input represents 100 µg of total protein. Membranes from yeast overexpressing recombinant rat TRPV2 were loaded as a control.

Article Snippet: 2D6 and 17A11 were used at 1 µg/ml, TRPV2 ACC-032 (Alomone) at 2 µg/ml, VRL-1 PC421 (Calbiochem) at 5 µg/ml and VRL-1 SC-22520 (Santa Cruz) at 10 µg/ml.

Techniques: Western Blot, Molecular Weight, Software, Immunoprecipitation, Labeling, Recombinant

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, HeLa cells transiently expressing TRPV2-1D4 immunolabeled with indicated TRPV2 antibodies (green) and 1D4 antibody (red). Scale bar represents 10 µm. B, HeLa cells transiently expressing TRPV1-1D4 immunolabeled with TRPV2 17A11 (green) and 1D4 antibody (red). Scale bar represents 10 µm.

Article Snippet: 2D6 and 17A11 were used at 1 µg/ml, TRPV2 ACC-032 (Alomone) at 2 µg/ml, VRL-1 PC421 (Calbiochem) at 5 µg/ml and VRL-1 SC-22520 (Santa Cruz) at 10 µg/ml.

Techniques: Expressing, Immunolabeling

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, CHO-K1 cells were treated with IGF-1 (20 ng/ml) for the indicated times and immunoblotted with a phospho-Akt specific antibody. Membranes were then stripped and re-probed with a pan-Akt antibody. B, CHO-K1 cells transiently expressing TRPV2 were treated with IGF-1 (20 ng/ml) for indicated times. Surface proteins were biotinylated in intact cells at 37°C. Cells were then lysed and biotinylated proteins were captured with streptavidin agarose. Captured proteins were resolved by SDS-PAGE and detected by western blot with the indicated antibodies. Surface proteins represent the biotinylated fraction and the total lysate represents 5% of total protein. C, TRPV2 band intensity of the biotinylated fraction was measured using LiCor Odyssey software. Intensities were normalized to biotinylated Na/K ATPase band intensities. Error bars represent S.E.M. from 3 separate experiments. Differences are not statistically significant. D, CHO-K1 cells transiently expressing TRPV2 treated with vehicle (PBS) or IGF-1 (20 ng/ml) for 20 min were fixed and immunolabeled for TRPV2 (17A11 antibody). Images are representative of 3 separate experiments. Scale bar represents 10 µm.

Article Snippet: 2D6 and 17A11 were used at 1 µg/ml, TRPV2 ACC-032 (Alomone) at 2 µg/ml, VRL-1 PC421 (Calbiochem) at 5 µg/ml and VRL-1 SC-22520 (Santa Cruz) at 10 µg/ml.

Techniques: Expressing, SDS Page, Western Blot, Software, Immunolabeling

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, F11 cells were treated with IGF-1 (20 ng/ml) for the indicated times and immunoblotted with a phospho-Akt specific antibody. Membranes were then stripped and re-probed with a pan-Akt antibody. B, Biotinylation of surface proteins from F11 cells was performed following the procedure from Figure 5B. C, F11 cells treated with vehicle (PBS) or IGF-1 (20 ng/ml) for 20 min were fixed and immunolabeled for TRPV2 (17A11 antibody). Images are representative of 3 separate experiments. Scale bar represents 10 µm.

Article Snippet: 2D6 and 17A11 were used at 1 µg/ml, TRPV2 ACC-032 (Alomone) at 2 µg/ml, VRL-1 PC421 (Calbiochem) at 5 µg/ml and VRL-1 SC-22520 (Santa Cruz) at 10 µg/ml.

Techniques: Immunolabeling

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, Schematic of the domain arrangement for a TRPV2 monomer with the approximate epitope sites for indicated TRPV2 antibodies. ARD, ankryin repeat domain; TM, transmembrane domain. B, Western blots with indicated TRPV2 antibodies against purified full-length rat TRPV2, purified rat TRPV2 ankryin repeat domain and purified rat TRPV2 C-terminus. C, Western blots with indicated TRPV2 antibodies against extracts from HeLa cells transiently transfected with empty vector, TRPV1-1D4 and TRPV2-1D4.

Article Snippet: The following commercially available antibodies were used: α-VRL-1 SC-22520 and α-phospho-Akt (Santa Cruz), α-VRL-1 PC421 (Calbiochem), α-TRPV2 ACC-032 (Alomone Labs), α-Na,K-ATPase α1, α-pan-Akt and α-β-Actin (Cell Signaling).

Techniques: Western Blot, Purification, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, Western blot analysis with anti-TRPV2 2D6 of F11 cells treated with control siRNA or TRPV2 siRNA (100 nM, 48 h). Quantification of the band corresponding to the molecular weight of TRPV2 was measured using LiCor Odyssey software. TRPV2 band intensity was normalized to actin. Error bars represent S.E.M. from 3 separate experiments. B, Immunoprecipitation of TRPV2 with 10 µg of indicated antibodies from 2.5 mg mouse brain lysate and 2.5 mg mouse heart lysate. TRPV2 was detected by western blot with IR dye-labeled 2D6 antibody. Input represents 100 µg of total protein. Membranes from yeast overexpressing recombinant rat TRPV2 were loaded as a control.

Article Snippet: The following commercially available antibodies were used: α-VRL-1 SC-22520 and α-phospho-Akt (Santa Cruz), α-VRL-1 PC421 (Calbiochem), α-TRPV2 ACC-032 (Alomone Labs), α-Na,K-ATPase α1, α-pan-Akt and α-β-Actin (Cell Signaling).

Techniques: Western Blot, Molecular Weight, Software, Immunoprecipitation, Labeling, Recombinant

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, HeLa cells transiently expressing TRPV2-1D4 immunolabeled with indicated TRPV2 antibodies (green) and 1D4 antibody (red). Scale bar represents 10 µm. B, HeLa cells transiently expressing TRPV1-1D4 immunolabeled with TRPV2 17A11 (green) and 1D4 antibody (red). Scale bar represents 10 µm.

Article Snippet: The following commercially available antibodies were used: α-VRL-1 SC-22520 and α-phospho-Akt (Santa Cruz), α-VRL-1 PC421 (Calbiochem), α-TRPV2 ACC-032 (Alomone Labs), α-Na,K-ATPase α1, α-pan-Akt and α-β-Actin (Cell Signaling).

Techniques: Expressing, Immunolabeling

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, CHO-K1 cells were treated with IGF-1 (20 ng/ml) for the indicated times and immunoblotted with a phospho-Akt specific antibody. Membranes were then stripped and re-probed with a pan-Akt antibody. B, CHO-K1 cells transiently expressing TRPV2 were treated with IGF-1 (20 ng/ml) for indicated times. Surface proteins were biotinylated in intact cells at 37°C. Cells were then lysed and biotinylated proteins were captured with streptavidin agarose. Captured proteins were resolved by SDS-PAGE and detected by western blot with the indicated antibodies. Surface proteins represent the biotinylated fraction and the total lysate represents 5% of total protein. C, TRPV2 band intensity of the biotinylated fraction was measured using LiCor Odyssey software. Intensities were normalized to biotinylated Na/K ATPase band intensities. Error bars represent S.E.M. from 3 separate experiments. Differences are not statistically significant. D, CHO-K1 cells transiently expressing TRPV2 treated with vehicle (PBS) or IGF-1 (20 ng/ml) for 20 min were fixed and immunolabeled for TRPV2 (17A11 antibody). Images are representative of 3 separate experiments. Scale bar represents 10 µm.

Article Snippet: The following commercially available antibodies were used: α-VRL-1 SC-22520 and α-phospho-Akt (Santa Cruz), α-VRL-1 PC421 (Calbiochem), α-TRPV2 ACC-032 (Alomone Labs), α-Na,K-ATPase α1, α-pan-Akt and α-β-Actin (Cell Signaling).

Techniques: Expressing, SDS Page, Western Blot, Software, Immunolabeling

Journal: PLoS ONE

Article Title: Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

doi: 10.1371/journal.pone.0085392

Figure Lengend Snippet: A, F11 cells were treated with IGF-1 (20 ng/ml) for the indicated times and immunoblotted with a phospho-Akt specific antibody. Membranes were then stripped and re-probed with a pan-Akt antibody. B, Biotinylation of surface proteins from F11 cells was performed following the procedure from Figure 5B. C, F11 cells treated with vehicle (PBS) or IGF-1 (20 ng/ml) for 20 min were fixed and immunolabeled for TRPV2 (17A11 antibody). Images are representative of 3 separate experiments. Scale bar represents 10 µm.

Article Snippet: The following commercially available antibodies were used: α-VRL-1 SC-22520 and α-phospho-Akt (Santa Cruz), α-VRL-1 PC421 (Calbiochem), α-TRPV2 ACC-032 (Alomone Labs), α-Na,K-ATPase α1, α-pan-Akt and α-β-Actin (Cell Signaling).

Techniques: Immunolabeling

Journal: Advanced Science

Article Title: The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca 2+ ‐LRMDA Axis in Myeloid Cells

doi: 10.1002/advs.202202857

Figure Lengend Snippet: Knockout of TRPV2 protects mice from lethal HSV‐1 and VSV infection. A) qRT‐PCR and Immunoblot analysis of Trpv2 mRNA (left) and TRPV2 protein (right) in Trpv2 fl/fl and Lyz2‐ Cre; Trpv2 fl/fl BMDCs. B) A representative whole‐cell recording of the Trpv2 fl/fl (left, upper) and Lyz2 ‐Cre; Trpv2 fl/fl (left, lower) BMDCs. The cell was exposed to 4 m m 2‐APB in neutral condition (pH 7.4). Summary data (right graph) of current densities evoked by 4 m M 2‐APB at a holding potential of −60 mV. C) qRT‐PCR analysis of HSV‐1 UL30 gene or VSV N gene in (left) and HSV‐1 and VSV titers in the supernatants of (right) Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs infected with HSV‐1 or VSV for 12 h. D) Transmission electron microscopy analysis of Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were infected with HSV‐1 for 12 h. E) Flow cytometry analysis (left) and fluorescent microscopy imaging (right) of Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were left uninfected or infected with H129‐G4 or VSV‐GFP for 1 h followed by PBS wash twice and cultured in full medium for 12 h. Numbers adjacent to the outlined areas indicate percentages of GFP + cells. F) A scheme of viral infection and analysis of Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl mice. G) Survival (Kaplan–Meier curve) of Trpv2 fl/fl ( n = 8) and Lyz2 ‐Cre; Trpv2 fl/fl ( n = 8) mice that were intraperitoneally injected with HSV‐1 (upper, 5 × 10 6 PFU per mouse) or VSV (lower, 2 × 10 7 PFU per mouse) and monitored for 10 days. H) qRT‐PCR analysis of HSV‐1 UL30 gene (upper) or VSV N gene (lower) in the heart, liver, brain, or kidney of Trpv2 fl/fl ( n = 3) and Lyz2 ‐Cre; Trpv2 fl/fl mice ( n = 3) that were intraperitoneally injected with HSV‐1 (2.5 × 10 6 PFU per mouse) or VSV (1 × 10 7 PFU) for 4 days. I) Plaque assays analyzing HSV‐1 (upper) in the brain or VSV (lower) titers in the brain and the heart from Trpv2 fl/fl ( n = 3) and Lyz2 ‐Cre; Trpv2 fl/fl ( n = 3) mice that were intraperitoneally injected with HSV‐1 (2.5 × 10 6 PFU per mouse) or VSV (1 × 10 7 PFU) for 4 days. * p < 0.05; ** p < 0.01; *** p < 0.001 (two‐tailed student's t ‐test in A–C,H–I, and Log‐Rank analysis in G). Graphs show mean ± S.D. in A–C,G–I). Scale bars represent 200 µm in E). Data are combined two G) independent experiments or representative of two A–E,H–I) independent experiments.

Article Snippet: [ ] HRP‐conjugated goat‐anti mouse or rabbit IgG (Thermo Scientific, PA1‐86717 and SA1‐9510), HRP‐conjugated mouse anti‐FLAG (Sigma, A8592), mouse anti‐FLAG (Sungene, KM8002), anti‐ β ‐Actin (Sungene, KM9001), anti‐GAPDH (Sungene, KM9002), anti‐Tubulin (Sungene, KM9003), Rabbit anti‐TRPV2 (Alomone labs, ACC‐032), anti‐HSV‐1/2 gB (SANTA, sc‐56987), SKF96365 (MCE, 130495‐35‐1), 2‐APB (Sigma, D9754), ionomycin (Aladdin, 56092‐81‐0), GM‐CSF (Peprotech, 315‐03), M‐SCF(Peprotech, 315‐02), Flt3L (PeproTech, 250‐31L), and CellMask Green plasma membrane stain (Invitrogen, C37608) were purchased from the indicated manufactures.

Techniques: Knock-Out, Infection, Quantitative RT-PCR, Western Blot, Transmission Assay, Electron Microscopy, Flow Cytometry, Microscopy, Imaging, Cell Culture, Injection, Two Tailed Test

Journal: Advanced Science

Article Title: The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca 2+ ‐LRMDA Axis in Myeloid Cells

doi: 10.1002/advs.202202857

Figure Lengend Snippet: Inhibition of TRPV2 channel activity by SKF96365 inhibits viral infection. A) qRT‐PCR analysis of HSV‐1 UL30 gene in Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were pretreated with DMSO or SKF96365 (100 µ m ) for 2 h followed by HSV‐1 infection for 12 h. B) Flow cytometry analysis of Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were pretreated with DMSO or SKF96365 (100 µ m ) for 2 h followed by H129‐G4 infection for 12 h. Numbers adjacent to the outlined areas indicate percentages of GFP + cells. C) A scheme of SKF96365 treatment and viral infection of Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl mice. The Trpv2 fl/fl and Lyz2‐Cre Trpv2 fl/fl mice were intraperitoneally injected with DMSO or SKF96365 (20 mg kg −1 per mouse) for 3 successive days followed by intraperitoneal injection of HSV‐1 or VSV. D–E) Survival (Kaplan–Meier curve) of Trpv2 fl/fl ( n = 5) and Lyz2 ‐Cre; Trpv2 fl/fl mice ( n = 5) that were treated as in C) and intraperitoneally injected with either HSV‐1 (D, 5 × 10 6 PFU per mouse) or VSV (E, 2 × 10 7 PFU per mouse) monitored survival for 8 days. F) qRT‐PCR analysis of HSV‐1 UL30 gene (in the heart, liver, and brain) and HSV‐1 titers (in the brain) from Trpv2 fl/fl ( n = 3) and Lyz2‐Cre; Trpv2 fl/fl ( n = 3) mice similarly treated as in D) except for that HSV‐1 (2.5 × 10 6 PFU per mouse) was intraperitoneally injected for 4 days. G) qRT‐PCR analysis of VSV N gene (in the heart, liver, and brain) and VSV titers (in the brain) from Trpv2 fl/fl ( n = 3) and Lyz2‐Cre Trpv2 fl/fl ( n = 3) mice treated as in E) except for that VSV (1 × 10 7 PFU per mouse) was intraperitoneally injected for 4 days. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (two‐tailed student's t ‐test in A,F,G), and Log‐Rank analysis in D,E). Graphs show mean ± S.D. in A,F,G). Data are representative of two D,E) or three independent experiments or representative of three A,B, F,G) independent experiments.

Article Snippet: [ ] HRP‐conjugated goat‐anti mouse or rabbit IgG (Thermo Scientific, PA1‐86717 and SA1‐9510), HRP‐conjugated mouse anti‐FLAG (Sigma, A8592), mouse anti‐FLAG (Sungene, KM8002), anti‐ β ‐Actin (Sungene, KM9001), anti‐GAPDH (Sungene, KM9002), anti‐Tubulin (Sungene, KM9003), Rabbit anti‐TRPV2 (Alomone labs, ACC‐032), anti‐HSV‐1/2 gB (SANTA, sc‐56987), SKF96365 (MCE, 130495‐35‐1), 2‐APB (Sigma, D9754), ionomycin (Aladdin, 56092‐81‐0), GM‐CSF (Peprotech, 315‐03), M‐SCF(Peprotech, 315‐02), Flt3L (PeproTech, 250‐31L), and CellMask Green plasma membrane stain (Invitrogen, C37608) were purchased from the indicated manufactures.

Techniques: Inhibition, Activity Assay, Infection, Quantitative RT-PCR, Flow Cytometry, Injection, Two Tailed Test

Journal: Advanced Science

Article Title: The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca 2+ ‐LRMDA Axis in Myeloid Cells

doi: 10.1002/advs.202202857

Figure Lengend Snippet: E572 of TRPV2 is required for the Ca 2+ permeability. A) A scheme of experiments (left) and immunoblot analysis (right) of GCaMP6m in Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs for B,C). B) Fluorescent microscopy analysis of Ca 2+ imaging in Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs infected with HSV‐1 in the presence of EGTA (5 m m ) or SKF96365 (1 m m ). C) Quantitative analysis of the GFP signals in Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs treated as in B). D) Representative whole‐cell recordings of HEK293 cells transfected with wild‐type TRPV2, TRPV2 E572Q , or TRPV2 E594/604Q that were stimulated with 2‐APB (0.5–5 m m ) under the holding potential of −60 mV. E) Dose‐response curves to 2‐APB of HEK293 cells treated as in D). Fitting by Hill's equation resulted in the following: EC 50 = 2.25 ± 0.02 m m , n H = 5.37 ± 0.28 for TRPV2 ( n = 5); EC 50 = 2.28 ± 0.02 m m , n H = 5.47 ± 0.22 for TRPV2 E572Q ( n = 5). F) Summary plot of current density. The current densities evoked by 2‐APB were determined by normalizing the membrane peak current by membrane capacitance ( n = 5). G) Fluorescent microscopy analysis of Ca 2+ imaging in GCaMP6m‐expression HEK293 cells transfected with TRPV2, TRPV2 E572Q , or TRPV2 E594/604Q followed by treatment with DMSO (Con), CBD (30 µ m ), or ionomycin (1 µ m ). H) Averaged responses of HEK293 cells treated as in G). GCaMP6m fluorescence changes were computed as (Fi–F0)/F0, where Fi represented fluorescence intensity at any frame and F0 was the baseline fluorescence calculated from the averaged fluorescence of the first 10 frames. *** p < 0.001; ns, not significant (two‐tailed student's t ‐test in F). Graphs show mean ± S.D. in C,E,F,H). Scale bars represent 100 µm in B) and 200 µm in G). Data are representative of three B–H) independent experiments.

Article Snippet: [ ] HRP‐conjugated goat‐anti mouse or rabbit IgG (Thermo Scientific, PA1‐86717 and SA1‐9510), HRP‐conjugated mouse anti‐FLAG (Sigma, A8592), mouse anti‐FLAG (Sungene, KM8002), anti‐ β ‐Actin (Sungene, KM9001), anti‐GAPDH (Sungene, KM9002), anti‐Tubulin (Sungene, KM9003), Rabbit anti‐TRPV2 (Alomone labs, ACC‐032), anti‐HSV‐1/2 gB (SANTA, sc‐56987), SKF96365 (MCE, 130495‐35‐1), 2‐APB (Sigma, D9754), ionomycin (Aladdin, 56092‐81‐0), GM‐CSF (Peprotech, 315‐03), M‐SCF(Peprotech, 315‐02), Flt3L (PeproTech, 250‐31L), and CellMask Green plasma membrane stain (Invitrogen, C37608) were purchased from the indicated manufactures.

Techniques: Permeability, Western Blot, Microscopy, Imaging, Infection, Transfection, Expressing, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca 2+ ‐LRMDA Axis in Myeloid Cells

doi: 10.1002/advs.202202857

Figure Lengend Snippet: The Ca 2+ permeability of TRPV2 is required for viral infection. A) A scheme of experiments (left) and immunoblot analysis (right) of GCaMP6m and TRPV2, TRPV2 E572Q , or TRPV2 E594/604Q in Lyz2 ‐Cre; Trpv2 fl/fl BMDCs for B,C). B) Fluorescent microscopy analysis of Ca 2+ imaging in Lyz2 ‐Cre; Trpv2 fl/fl BMDCs transfected with GCaMP6m and wild‐type TRPV2, TRPV2 E572Q , or TRPV2 E594/604Q followed by infection of HSV‐1. C) Quantitative analysis of the GFP signals in Lyz2 ‐Cre; Trpv2 fl/fl BMDCs treated as in B). D) Fluorescent microscopy imaging (left images) of Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were transfected with an empty vector, wild‐type TRPV2, TRPV2 E572Q , or TRPV2 E594/604Q followed by infection with H129‐G4 or VSV‐GFP for 12 h. The levels of TRPV2 or TRPV2 mutants in Lyz2 ‐Cre; Trpv2 fl/fl BMDCs were analyzed by immunoblot assays (right panels). E) qRT‐PCR analysis of HSV‐1 UL30 gene or VSV N gene in Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were transfected with an empty vector, TRPV2, TRPV2 E572Q , or TRPV2 E594/604Q and infected with HSV‐1 or VSV for 1 h followed by twice PBS wash and cultured in full medium for 12 h. F) Plaque assays analyzing HSV‐1 or VSV titers in the supernatants of Lyz2 ‐Cre; Trpv2 fl/fl BMDCs treated as in E). * p < 0.05; ** p < 0.01; *** p < 0.001 (two‐tailed student's t ‐test in E,F). Graphs show mean ± S.D. in C, E,F). Scale bars represent 100 µm in B) and 200 µm in D). Data are representative of two B–F) independent experiments.

Article Snippet: [ ] HRP‐conjugated goat‐anti mouse or rabbit IgG (Thermo Scientific, PA1‐86717 and SA1‐9510), HRP‐conjugated mouse anti‐FLAG (Sigma, A8592), mouse anti‐FLAG (Sungene, KM8002), anti‐ β ‐Actin (Sungene, KM9001), anti‐GAPDH (Sungene, KM9002), anti‐Tubulin (Sungene, KM9003), Rabbit anti‐TRPV2 (Alomone labs, ACC‐032), anti‐HSV‐1/2 gB (SANTA, sc‐56987), SKF96365 (MCE, 130495‐35‐1), 2‐APB (Sigma, D9754), ionomycin (Aladdin, 56092‐81‐0), GM‐CSF (Peprotech, 315‐03), M‐SCF(Peprotech, 315‐02), Flt3L (PeproTech, 250‐31L), and CellMask Green plasma membrane stain (Invitrogen, C37608) were purchased from the indicated manufactures.

Techniques: Permeability, Infection, Western Blot, Microscopy, Imaging, Transfection, Plasmid Preparation, Quantitative RT-PCR, Cell Culture, Two Tailed Test

Journal: Advanced Science

Article Title: The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca 2+ ‐LRMDA Axis in Myeloid Cells

doi: 10.1002/advs.202202857

Figure Lengend Snippet: TRPV2 facilitates virus penetration dependently on its Ca 2+ permeability. A) A scheme of experiments analyzing the attachment and the penetration of viruses. B) qPCR analysis of HSV‐1 genome of the attached and the penetrated HSV‐1 in Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs treated as in A). C) Fluorescent microscopy imaging (left) and quantification analysis (right) of the fluorescent signals of Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs treated as in A) followed by immunofluorescent staining with anti‐HSV‐1 gB protein (red) and CellMask Green (green). The arrowheads indicated gB signals on plasma membrane and the arrows indicated gB signals in the cytosol. D) Representative images captured from the a movie recording Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were infected with near‐infrared quantum dots‐encapsulated SV40 viruses. The arrows indicated SV40 viral particles. E,F) Fluorescent microscopy imaging E) and quantification analysis F) of the fluorescent signals of Lyz2 ‐Cre; Trpv2 fl/fl BMDCs transfected with an empty vector, TRPV2, TRPV2 E572Q , or TRPV2 E594/604Q followed by treatment as in C). The arrowheads indicated gB signals on plasma membrane and the arrows indicated gB signals in the cytosol. G) qPCR analysis of HSV‐1 genome in Lyz2 ‐Cre; Trpv2 fl/fl BMDCs transfected with an empty vector, TRPV2, TRPV2 E572Q , or TRPV2 E594/604Q followed by treatment as in A). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (two‐tailed student's t ‐test in B,C, F,G). Graphs show mean ± S.D. in B,C, F,G). Scale bars represent 5 µm in C–E). PM, gB signals on plasma membrane; Intra, intracellular gB signals. Data are representative of three B–G) independent experiments.

Article Snippet: [ ] HRP‐conjugated goat‐anti mouse or rabbit IgG (Thermo Scientific, PA1‐86717 and SA1‐9510), HRP‐conjugated mouse anti‐FLAG (Sigma, A8592), mouse anti‐FLAG (Sungene, KM8002), anti‐ β ‐Actin (Sungene, KM9001), anti‐GAPDH (Sungene, KM9002), anti‐Tubulin (Sungene, KM9003), Rabbit anti‐TRPV2 (Alomone labs, ACC‐032), anti‐HSV‐1/2 gB (SANTA, sc‐56987), SKF96365 (MCE, 130495‐35‐1), 2‐APB (Sigma, D9754), ionomycin (Aladdin, 56092‐81‐0), GM‐CSF (Peprotech, 315‐03), M‐SCF(Peprotech, 315‐02), Flt3L (PeproTech, 250‐31L), and CellMask Green plasma membrane stain (Invitrogen, C37608) were purchased from the indicated manufactures.

Techniques: Permeability, Microscopy, Imaging, Staining, Infection, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Advanced Science

Article Title: The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca 2+ ‐LRMDA Axis in Myeloid Cells

doi: 10.1002/advs.202202857

Figure Lengend Snippet: TRPV2 facilitates the tension and the mobility of cell membrane dependently on its Ca 2+ permeability. A) Schematic illustrating the interaction between cells and the probe‐labeled surface. Detection of the fluorescence reflected the mechanic force of cell membrane exerted by the actin filaments and integrin. B) Representative images of differential interference contrast microscopy (DIC), reflection interference contrast microscopy (RICM, which can reflect the cell adherent area), and total internal reflection fluorescence (TIRF) microscopy (left) and statistic total fluorescent intensities (right) of Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were seeded on a 17 pN DNA probe surface. C) Representative images (left) and quantitative analysis (right) of FRAP in the cell membranes of Trpv2 fl/fl and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs. D,E) Representative images of DIC, RICM, and TIRF microscopy D) and statistic total fluorescent intensities E) of Lyz2 ‐Cre Trpv2 fl/fl reconstituted with Vector, TRPV2, TRPV2 E572Q , TRPV1, and TRPV1 NQQ BMDCs that were seeded on a 17 pN DNA probe surface. F,G) Representative images F) and quantitative analysis G) of FRAP in the cell membranesof Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were reconstituted with Vector, TRPV2, TRPV2 E572Q , TRPV1, or TRPV1 D646N/E648/651Q . * p < 0.05; ** p < 0.01; *** p < 0.001 (two‐tailed student's t ‐test in B,C,E,G). Graphs show mean ± S.D. in B,C,E,G). Scale bars represent 10 µm in B,D) and 5 µm in C,F). Data are representative of three B–F) independent experiments.

Article Snippet: [ ] HRP‐conjugated goat‐anti mouse or rabbit IgG (Thermo Scientific, PA1‐86717 and SA1‐9510), HRP‐conjugated mouse anti‐FLAG (Sigma, A8592), mouse anti‐FLAG (Sungene, KM8002), anti‐ β ‐Actin (Sungene, KM9001), anti‐GAPDH (Sungene, KM9002), anti‐Tubulin (Sungene, KM9003), Rabbit anti‐TRPV2 (Alomone labs, ACC‐032), anti‐HSV‐1/2 gB (SANTA, sc‐56987), SKF96365 (MCE, 130495‐35‐1), 2‐APB (Sigma, D9754), ionomycin (Aladdin, 56092‐81‐0), GM‐CSF (Peprotech, 315‐03), M‐SCF(Peprotech, 315‐02), Flt3L (PeproTech, 250‐31L), and CellMask Green plasma membrane stain (Invitrogen, C37608) were purchased from the indicated manufactures.

Techniques: Permeability, Labeling, Fluorescence, Microscopy, Plasmid Preparation, Two Tailed Test

Journal: Advanced Science

Article Title: The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca 2+ ‐LRMDA Axis in Myeloid Cells

doi: 10.1002/advs.202202857

Figure Lengend Snippet: LRMDA functions downstream of TRPV2 for the mobility of cell membrane. A,B) Representative images A) and quantitative analysis B) of FRAP in the cell membranes of Trpv2 fl/fl BMDCs and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were reconstituted with Vector or LRMDA. C) Representative images of DIC, RICM, and TIRF microscopy (left) and statistic total fluorescent intensities (right) of Trpv2 fl/fl BMDCs and Lyz2 ‐Cre Trpv2 fl/fl BMDCs reconstituted with LRMDA‐GFP or vector, associated tension signals reported by 17 pN tension probe. D,E) Representative images of DIC, RICM, and TIRF microscopy D) and statistic total fluorescent intensities E) of Lyz2 ‐Cre Trpv2 fl/fl BMDCs and Trpv2 fl/fl BMDCs transfected with siCon, si Lrmda #1, or si Lrmda #2. F) Representative images (left) and quantitative analysis (right) of FRAP in the cell membranes of Lyz2 ‐Cre Trpv2 fl/fl BMDCs and Trpv2 fl/fl BMDCs transfected with siCon, si Lrmda #1, or si Lrmda #2. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (two‐tailed student's t ‐test in B,C, E,F). Graphs show mean ± S.D. in B,C, E,F). Scale bars represent 5 µm in A,F) and 10 µm in C,D). Data are representative of three A–F) independent experiments.

Article Snippet: [ ] HRP‐conjugated goat‐anti mouse or rabbit IgG (Thermo Scientific, PA1‐86717 and SA1‐9510), HRP‐conjugated mouse anti‐FLAG (Sigma, A8592), mouse anti‐FLAG (Sungene, KM8002), anti‐ β ‐Actin (Sungene, KM9001), anti‐GAPDH (Sungene, KM9002), anti‐Tubulin (Sungene, KM9003), Rabbit anti‐TRPV2 (Alomone labs, ACC‐032), anti‐HSV‐1/2 gB (SANTA, sc‐56987), SKF96365 (MCE, 130495‐35‐1), 2‐APB (Sigma, D9754), ionomycin (Aladdin, 56092‐81‐0), GM‐CSF (Peprotech, 315‐03), M‐SCF(Peprotech, 315‐02), Flt3L (PeproTech, 250‐31L), and CellMask Green plasma membrane stain (Invitrogen, C37608) were purchased from the indicated manufactures.

Techniques: Plasmid Preparation, Microscopy, Transfection, Two Tailed Test

Journal: Advanced Science

Article Title: The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca 2+ ‐LRMDA Axis in Myeloid Cells

doi: 10.1002/advs.202202857

Figure Lengend Snippet: LRMDA facilitates viral infections downstream of TRPV2. A) qRT‐PCR analysis of genomic RNA of the attached VSV and the penetrated VSV in Trpv2 fl/fl and Lyz2 ‐Cre Trpv2 fl/fl BMDCs transfected with siCon, si Lrmda #1, or si Lrmda #2. B) Fluorescent microscopy imaging of GFP signals in Trpv2 fl/fl and Lyz2 ‐Cre Trpv2 fl/fl BMDCs that were transfected with siCon, si Lrmda #1, or si Lrmda #2 followed by infection with H129‐G4 or VSV‐GFP for 12 h. C) qRT‐PCR analysis of HSV‐1 UL30 gene or VSV N gene in Trpv2 fl/fl and Lyz2 ‐Cre Trpv2 fl/fl BMDCs that were transfected with siCon, si Lrmda #1, or si Lrmda #2 followed by infection with HSV‐1 or VSV for 12 h. D) Plaque assays analyzing HSV‐1 or VSV titers in the supernatants of Trpv2 fl/fl and Lyz2 ‐Cre Trpv2 fl/fl BMDCs treated as in C). E) qPCR analysis of HSV‐1 genome (upper) or qRT‐PCR analysis of VSV genome (lower) of the attached and the penetrated HSV‐1 or VSV in Trpv2 fl/fl BMDCs and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were transfected with LRMDA or an empty vector. F) Fluorescent microscopy imaging of Trpv2 fl/fl BMDCs and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were transfected with LRMDA or an empty vector followed by infection with H129‐G4 or VSV‐GFP for 12 h. G,H) qRT‐PCR analysis of HSV‐1 UL30 gene, or VSV N gene in G) and plaque assays of HSV‐1 and VSV titers in the supernatants of H) Trpv2 fl/fl BMDCs and Lyz2 ‐Cre; Trpv2 fl/fl BMDCs that were transfected with LRMDA or an empty vector followed by infection with HSV‐1 or VSV for 12 h. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (two‐tailed student's t ‐test in A,C–E,G). Graphs show mean ± S.D. in A,C–E,G). Scale bars represent 200 µm in B,F). Data are representative of three A–G) independent experiments.

Article Snippet: [ ] HRP‐conjugated goat‐anti mouse or rabbit IgG (Thermo Scientific, PA1‐86717 and SA1‐9510), HRP‐conjugated mouse anti‐FLAG (Sigma, A8592), mouse anti‐FLAG (Sungene, KM8002), anti‐ β ‐Actin (Sungene, KM9001), anti‐GAPDH (Sungene, KM9002), anti‐Tubulin (Sungene, KM9003), Rabbit anti‐TRPV2 (Alomone labs, ACC‐032), anti‐HSV‐1/2 gB (SANTA, sc‐56987), SKF96365 (MCE, 130495‐35‐1), 2‐APB (Sigma, D9754), ionomycin (Aladdin, 56092‐81‐0), GM‐CSF (Peprotech, 315‐03), M‐SCF(Peprotech, 315‐02), Flt3L (PeproTech, 250‐31L), and CellMask Green plasma membrane stain (Invitrogen, C37608) were purchased from the indicated manufactures.

Techniques: Quantitative RT-PCR, Transfection, Microscopy, Imaging, Infection, Plasmid Preparation, Two Tailed Test