Journal: The Journal of Cell Biology

Article Title: Polarity of varicosity initiation in central neuron mechanosensation

doi: 10.1083/jcb.201606065

Figure Lengend Snippet: TRPV4 channel plays a major role in axonal varicosity initiation in hippocampal neuron mechanosensation. (A and B) When expressed in HEK293 cells, TRPV4 but not YFP was activated by TRPV4 agonist GSK101 (GSK; 0.5 µM; A) and puffing (B). (C) Summary of the effects of their respective agonist (top) or puffing (bottom) on HEK293 cells expressing YFP, TRPV4, or TRPV1. (D) An axonal segment loaded with calcien AM (inverted signals) before (left) and after (right) puffing. (E) A distal axonal segment loaded with calcien AM (green) without puffing and stained for endogenous TRPV4 (red). (F) Post hoc staining of the axon in D for endogenous TRPV4 (red). (G) Effects of puffing on axons transfected with control (top) or TRPV4 (bottom) siRNA (siR). (H) The onset and degree of varicosity formation along axons with TRPV4 siRNA markedly reduced. (I and J) Under the same puffing condition, axons with TRPV4 siRNA had significantly longer onset time (I) and smaller varicosities (J). (K) Young axons (7 DIV) expressing YFP (green) and control siRNA were stained for endogenous TRPV4 (red in merged image and inverted in grayscale image). (L) Young axons (7 DIV) expressing YFP (green) and TRPV4 siRNA were stained for endogenous TRPV4 (red in merged image and inverted in grayscale image). Arrows indicate coexpressing YFP and double-stranded small RNA. Bars, 20 µm. (M) TRPV4 siRNA significantly reduced TRPV4 staining intensity in both axons and soma of cultured neurons. Error bars indicate means ± SEM. Unpaired t test: **, P < 0.01.

Article Snippet: In particular, the anti-TRPV4 antibody (LS-C94498; Lifespan Biosciences) was extensively validated by using Western blots and immunostaining of wild-type and TRPV4-null mice (as well as TRPV4 cDNA; ).

Techniques: Expressing, Staining, Transfection, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Polarity of varicosity initiation in central neuron mechanosensation

doi: 10.1083/jcb.201606065

Figure Lengend Snippet: The level of MT-binding protein NSTOP correlates with the resistance of puffing-induced varicosity formation. (A and B) Axons expressing different MT-binding proteins were puffed, and expressing EB1-YFP (A) or YFP-MAP2 (B) in axons did not change puffing-induced varicosity formation. (C) The axon expressing NSTOP-GFP became more resistant to puffing. (D) Summary of the effects of three MT-binding proteins on axonal varicosity formation. Error bars indicate means ± SEM. One-way ANOVA followed by Dunnett’s test: **, P < 0.01. (E) Expression levels of STOP and TRPV4 in the brain during development. Western blotting for STOP and TRPV4 from mouse brain lysate was performed, and β-tubulin was used as a control. Molecular masses are indicated in kilodaltons. (F) Costaining for endogenous STOP (green) and TRPV4 (red) in the hippocampal slice of an adult mouse. (G and H) Costaining for endogenous STOP (green in merged) and MAP2 (red in merged) was performed in cultured hippocampal neurons at 12 DIV (G) and 21 DIV (H). Arrows indicate MAP2-negative axons containing anti-STOP staining signals. Bars: (A–C) 25 µm; (F–H) 100 µm.

Article Snippet: In particular, the anti-TRPV4 antibody (LS-C94498; Lifespan Biosciences) was extensively validated by using Western blots and immunostaining of wild-type and TRPV4-null mice (as well as TRPV4 cDNA; ).

Techniques: Binding Assay, Expressing, Western Blot, Cell Culture, Staining

Journal: British journal of pharmacology

Article Title: Glucosylsphingosine evokes pruritus via activation of 5-HT 2A receptor and TRPV4 in sensory neurons

doi: 10.1111/bph.15733

Figure Lengend Snippet: GS can directly activate TRPV4. Representative calcium imaging results before and after 100 μM GS treatment upon HEK293T cells transiently expressing either mock vector (pcDNA; a) or TRPV4 cDNA (b). (c) Summary of dose-dependent intracellular calcium responses induced by GS in HEK293T cells expressing TRPV4 (‘TRPV4-HEK293T’). A total of 100 μM GS was treated on pcDNA-transfected group (n = 110 cells). Various concentrations of GS were applied on TRPV4-expressing groups (GS in μM: 0.1 [n = 109 cells], 0.5 [n = 112 cells], 1 [n = 108 cells], 10 [n = 82 cells], 50 [n = 127 cells], 100 [n = 134 cells], 500 [n = 122 cells] and 1,000 [n = 120 cells]). Red horizontal bar indicates the average value of each group. (d) A TRPV4-specific inhibitor HC067047 (1 μM [n = 118 cells] and 10 μM [n = 89 cells]) significantly inhibited the GS-induced calcium influx in the TRPV4-HEK293T compared to control (‘GS 100’, n = 62 cells). Red horizontal bar indicates the average value of each group. (e) Comparison of 50 μM GS responses in HEK293T cells transfected with either pcDNA (n = 160 cells), TRPV1 (n = 170 cells), Trpv3 (n = 127 cells), TRPA1 (n = 139 cells), Trpm3 (n = 149 cells) or TRPV4 (n = 188 cells). Notice the significantly higher maximum response in TRPV4 than in others. (f–h) A whole-cell patch clamp experiment was performed in HEK293T cells transfected with either pIRES-eFGP-TRPV4 (TRPV4) or mock vector (control). The holding potential was −80 mV. (f) Cells transfected with a mock vector did not show any inward currents to the GS treatment. (g) An inward current was observed in cells expressing TRPV4. (h) Summary of the peak inward currents between control versus TRPV4-expressing cells (n = 6 per group). *P < 0.05

Article Snippet: Additionally, TRPV4 cDNA was also subcloned into pIRES-EFGP (Invitrogen), allowing TRPV4 and EGFP to be translated from a single bicistronic mRNA, and further used in whole-cell patch-clamp experiments.

Techniques: Imaging, Expressing, Plasmid Preparation, Transfection, Patch Clamp