Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitation (IP) analysis of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in mCCDcl1 cells and mouse kidney. A: immunoblots of mCCDcl1 cell homogenates show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). B: immunoblots of mouse kidney homogenates also show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously ((20), Fig. 1). CAV-1 (22 kD) protein band is demonstrated in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Immunoprecipitation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitaton (IP) analysis of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney. Immunoblots of mCCDcl1 cell homogenates show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (A). Immunoblots of mouse kidney homogenates also show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (B). Endogenous immunoprecipitaton of IK1 (C) or BKα (D) followed by blotting for CAV-1, TRPV4, and each of the KCa channels, in mCCDcl1 cells, showed enrichment of CAV-1, TRPV4, BKα, SK3, and BKα or IK1. Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously [(20) Fig. 1]. CAV-1 (22 kD) was verified in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Western Blot, Immunoprecipitation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Effect of caveolin-1 (CAV-1) knockdown on transient receptor potential cation channel subfamily V member 4 (TRPV4)-mediated (GSK101) intracellular Ca2+ ([Ca2+]i) response to blockers of TRPV4 and KCa channels. The normal peak [Ca2+]i response to TRPV4 activation (GSK101) is markedly reduced from 597 ± 45 nM (n = 70) to 231 ± 47 nM (n = 85) following CAV-1 siRNA treatment (A and B). Furthermore, the apparent initial rate of TRPV4-mediated Ca2+ influx was markedly depressed from 108 ± 27 nM/min (n = 70) in normal conditions to 42 ± 16 nM/min (n = 85) in the presence of CAV-1 siRNA (C), reflecting a markedly depressed level of TRPV4 activation. The subsequent panels demonstrate that the typical normal peak elevation in [Ca2+]i following TRPV4 activation is markedly reduced upon inhibition of each KCa channel following CAV-1 knockdown. Iberiotoxin (IbTX) treatment reduces the peak [Ca2+]i for BK inhibition from 227 ± 34 nM (n = 73) to 84 ± 33 nM (n = 59) after CAV-1 siRNA treatment (D and E). TRAM-34 treatment reduces the peak [Ca2+]i for IK1 inhibition from 312 ± 31 nM (n = 90) to 81 ± 12 nM (n = 95) after CAV-1 siRNA treatment (F and G). Apamin treatment reduces the peak [Ca2+]i for SK3 inhibition from 414 ± 60 nM (n = 72) to 196 ± 23 nM (n = 60) after CAV-1 siRNA treatment (H and I). Therefore, after CAV-1 siRNA treatment the response for each of the KCa channels to TRPV4 activation is markedly depressed. ***P < 0.001. BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Activation Assay, Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Representative immunofluorescence images show colocalization of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in collecting duct mCCDcl1 cells in mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for TRPV4 (TRPV4, green) and SK3 (SK3-ATTO-594, red) show strong colocalization as apparent in the merged image (Merged, yellow) (A). Similarly, immunostaining for TRPV4 (red) and BKα (green) (B) and for TRPV4 (red) and IK1 (green) (C) demonstrated noted colocalization in the merged images (Merged, yellow). Mouse kidney CCD immunostained for TRPV4 (TRPV4, green) and SK3 (SK3, ATTO-594, red) display noted colocalization in the merged images (Merged, yellow) (D). In a similar manner, immunostaining for TRPV4 (TRPV4, red) and BKα (BKα, green) (E) and for TRPV4 (TRPV4, red) and IK1 (IK1, green) (F) likewise demonstrated apparent colocalization in the merged images (Merged, yellow). Areas of more diffuse staining are also apparent in some cases, especially for IK1 (see F). Note that we verified the primary antibodies using blocking peptides in an earlier publication [(20) Figs. 1 and ​and4].4]. All the immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Immunofluorescence, Immunostaining, Staining, Blocking Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Immunofluorescence images demonstrates colocalization of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) showed strong colocalization in the merged image (Merged, yellow) (A). Similarly, immunostaining for CAV-1 with each of the KCa channel demonstrated robust colocalization in the merged images as shown for CAV-1 (green) and SK3 (red) (B), CAV-1 (red) and BKα (green) (C), and CAV-1 (red) and IK1 (green) (D). Diffuse localization was also apparent, especially for CAV-1 and IK1 (D). Mouse kidney CCD immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) also displayed robust colocalization as shown in the merged image (Merged, yellow) (E). Similarly, immunostaining for CAV-1 and each of the KCa channels displayed noted colocalization in the merged images (Merged, yellow) for CAV-1 (green) and SK3 (red) (F), for CAV-1 (red) and BKα (green) (G), and for CAV-1 (green) and IK1 (red) (H). Areas of more diffuse localization were also apparent, especially for BKα and IK1 (see merged images in G and H). Immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Immunofluorescence, Immunostaining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitation (IP) analysis of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in mCCDcl1 cells and mouse kidney. A: immunoblots of mCCDcl1 cell homogenates show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). B: immunoblots of mouse kidney homogenates also show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously ((20), Fig. 1). CAV-1 (22 kD) protein band is demonstrated in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunoprecipitation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitaton (IP) analysis of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney. Immunoblots of mCCDcl1 cell homogenates show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (A). Immunoblots of mouse kidney homogenates also show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (B). Endogenous immunoprecipitaton of IK1 (C) or BKα (D) followed by blotting for CAV-1, TRPV4, and each of the KCa channels, in mCCDcl1 cells, showed enrichment of CAV-1, TRPV4, BKα, SK3, and BKα or IK1. Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously [(20) Fig. 1]. CAV-1 (22 kD) was verified in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Western Blot, Immunoprecipitation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Effect of caveolin-1 (CAV-1) knockdown on transient receptor potential cation channel subfamily V member 4 (TRPV4)-mediated (GSK101) intracellular Ca2+ ([Ca2+]i) response to blockers of TRPV4 and KCa channels. The normal peak [Ca2+]i response to TRPV4 activation (GSK101) is markedly reduced from 597 ± 45 nM (n = 70) to 231 ± 47 nM (n = 85) following CAV-1 siRNA treatment (A and B). Furthermore, the apparent initial rate of TRPV4-mediated Ca2+ influx was markedly depressed from 108 ± 27 nM/min (n = 70) in normal conditions to 42 ± 16 nM/min (n = 85) in the presence of CAV-1 siRNA (C), reflecting a markedly depressed level of TRPV4 activation. The subsequent panels demonstrate that the typical normal peak elevation in [Ca2+]i following TRPV4 activation is markedly reduced upon inhibition of each KCa channel following CAV-1 knockdown. Iberiotoxin (IbTX) treatment reduces the peak [Ca2+]i for BK inhibition from 227 ± 34 nM (n = 73) to 84 ± 33 nM (n = 59) after CAV-1 siRNA treatment (D and E). TRAM-34 treatment reduces the peak [Ca2+]i for IK1 inhibition from 312 ± 31 nM (n = 90) to 81 ± 12 nM (n = 95) after CAV-1 siRNA treatment (F and G). Apamin treatment reduces the peak [Ca2+]i for SK3 inhibition from 414 ± 60 nM (n = 72) to 196 ± 23 nM (n = 60) after CAV-1 siRNA treatment (H and I). Therefore, after CAV-1 siRNA treatment the response for each of the KCa channels to TRPV4 activation is markedly depressed. ***P < 0.001. BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Activation Assay, Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Representative immunofluorescence images show colocalization of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in collecting duct mCCDcl1 cells in mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for TRPV4 (TRPV4, green) and SK3 (SK3-ATTO-594, red) show strong colocalization as apparent in the merged image (Merged, yellow) (A). Similarly, immunostaining for TRPV4 (red) and BKα (green) (B) and for TRPV4 (red) and IK1 (green) (C) demonstrated noted colocalization in the merged images (Merged, yellow). Mouse kidney CCD immunostained for TRPV4 (TRPV4, green) and SK3 (SK3, ATTO-594, red) display noted colocalization in the merged images (Merged, yellow) (D). In a similar manner, immunostaining for TRPV4 (TRPV4, red) and BKα (BKα, green) (E) and for TRPV4 (TRPV4, red) and IK1 (IK1, green) (F) likewise demonstrated apparent colocalization in the merged images (Merged, yellow). Areas of more diffuse staining are also apparent in some cases, especially for IK1 (see F). Note that we verified the primary antibodies using blocking peptides in an earlier publication [(20) Figs. 1 and ​and4].4]. All the immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunofluorescence, Immunostaining, Staining, Blocking Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Immunofluorescence images demonstrates colocalization of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) showed strong colocalization in the merged image (Merged, yellow) (A). Similarly, immunostaining for CAV-1 with each of the KCa channel demonstrated robust colocalization in the merged images as shown for CAV-1 (green) and SK3 (red) (B), CAV-1 (red) and BKα (green) (C), and CAV-1 (red) and IK1 (green) (D). Diffuse localization was also apparent, especially for CAV-1 and IK1 (D). Mouse kidney CCD immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) also displayed robust colocalization as shown in the merged image (Merged, yellow) (E). Similarly, immunostaining for CAV-1 and each of the KCa channels displayed noted colocalization in the merged images (Merged, yellow) for CAV-1 (green) and SK3 (red) (F), for CAV-1 (red) and BKα (green) (G), and for CAV-1 (green) and IK1 (red) (H). Areas of more diffuse localization were also apparent, especially for BKα and IK1 (see merged images in G and H). Immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunofluorescence, Immunostaining

Journal: Cells

Article Title: Regulation of TGFβ Signalling by TRPV4 in Chondrocytes

doi: 10.3390/cells10040726

Figure Lengend Snippet: TRPV4 is expressed and can be activated in TC28a2 chondrocytes. ( A ) Immunostaining for TRPV4 in TC28a2 chondrocytes, using anti-TRPV4 antibody and DAPI nuclear stain. Scale bar represents 100 µm. ( B ) Dose response of GSK101 on Fluo8 fluorescence 15 min post stimulation. ( C ) Fluorescence imaging of Fluo8-loaded TC28a2 cells 15 min post stimulation with 100 nM GSK101 or DMSO control. Scale bar represents 200 µm. ( D ) Representative traces of Fluo8 fluorescence following DMSO (upper), 100 nM GSK101 stimulation (middle) or 100 nM GSK101 stimulation in cells pre-incubated with 500 nM GSK219 (lower) for 15 min. Data representative of three independent experiments.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% BSA in 1x TBS-0.1%Tween-20, then probed with antibodies for SMAD2 (1:1000, CST 5339), pSMAD2 (1:1000, CST 18338), TRPV4 (1:200, Cat#ACC-124, Alomone Labs) and left overnight on a rocker at 4 °C.

Techniques: Immunostaining, Staining, Fluorescence, Imaging, Incubation

Journal: Cells

Article Title: Regulation of TGFβ Signalling by TRPV4 in Chondrocytes

doi: 10.3390/cells10040726

Figure Lengend Snippet: Activation of TRPV4 modulates TGFβ signalling in a time-dependent manor. TC28a2 cells with SBE-nLUCp reporter were used to monitor TGFβ signalling. ( A ) Cells were stimulated with 10 ng/mL TGFβ3 or medium control, incubated for 15 min then stimulated with 100 nM GSK101 (activator) or DMSO control (vehicle) and then incubated for a further 3 h 45 min before SBE-nLUCp activity was determined. TRPV4 inhibitor (500 nM GSK219) was added to cells along with TGFβ3. ( B ) and ( C ) Cells were either not transfected (NT), mock transfected (TR), transfected with siRNA to TGFB1 (siTGFB1) or transfected with siRNA to TRPV4 (siTRPV4) for 24 h and then serum starved and incubated for a further 48 h. Following incubation, cells were stimulated with TGFβ3 ( B ) or media control ( C ) and then TRPV4 activated using GSK101, SBE-nLUCp activity determined as described in ( A ). ( D ) Schematic illustrating the order of stimulation/activation for A–C. ( E ) Cells were stimulated with 10 ng/mL TGFβ3. TRPV4 was activated (100 nM GSK101/DMSO control) either before (-ve mins), with (0 min) or after (+ve mins) TGFβ3 stimulation. ( F ) Cells were stimulated with 10 ng/mL TGFβ3 or medium control, incubated for 15 min then TRPV4 activated using 100 nM GSK101 or DMSO control. SBE-nLUCp activity was determined after the indicated amount of time post TGFβ3 stimulation. ( G ) Schematic representation of conditions shown in E. ( H ) Schematic representation of conditions shown in F. FC SBE RLU; fold change in SMAD-binding element relative light units NT; no treatment. Data in A combined from four independent experiments, and data in B–F combined from three independent experiments. Raw data are shown in . GSK101 treatment was normalised to the DMSO control for each siRNA/timepoint. Statistical differences were calculated by two-way ANOVA followed by Sidak’s multiple comparisons test; p < 0.05 *, p < 0.01 **, and p < 0.001 ***.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% BSA in 1x TBS-0.1%Tween-20, then probed with antibodies for SMAD2 (1:1000, CST 5339), pSMAD2 (1:1000, CST 18338), TRPV4 (1:200, Cat#ACC-124, Alomone Labs) and left overnight on a rocker at 4 °C.

Techniques: Activation Assay, Incubation, Activity Assay, Transfection, Binding Assay

Journal: Cells

Article Title: Regulation of TGFβ Signalling by TRPV4 in Chondrocytes

doi: 10.3390/cells10040726

Figure Lengend Snippet: RNA-seq identification of TGFβ3 response genes that are enhanced by TRPV4 activation. ( A ) Experimental design for RNA-seq (triplicate). ( B ) Hierarchical clustering and ( C ) PCA analysis shows separation of DMSO, GSK101, TGFβ3+DMSO and TGFβ3+GSK101 treatment groups, the TGFβ3+GSK219 and TGFβ3+GSK219+GSK101 treatment groups both clustered with TGFβ3+DMSO. ( D ) Histogram indicating number of differentially expressed genes (DEGs) between experimental conditions according to DESeq2. ( E ) Venn diagram indicating commonality between genes significantly up regulated in GSK101 vs. DMSO, TGFβ3+DMSO vs. DMSO and TGFβ3+GSK101 vs. DMSO. ( F ) Scatter plot of significant genes comparing fold change in gene expression in GSK101 vs. DMSO and TGFβ3+DMSO vs. DMSO. ( G ) Venn diagram of genes significantly up regulated in TGFβ3+GSK101 vs. TGFβ3+DMSO or TGFβ3+DMSO vs. DMSO illustrating that GSK101 causes further enhancement of TGFβ response genes. ( H ) Scatter plot of significant genes comparing fold change in gene expression following TGFβ3+DMSO vs. DMSO and TGFβ3+GSK101 vs. TGFβ3+DMSO.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% BSA in 1x TBS-0.1%Tween-20, then probed with antibodies for SMAD2 (1:1000, CST 5339), pSMAD2 (1:1000, CST 18338), TRPV4 (1:200, Cat#ACC-124, Alomone Labs) and left overnight on a rocker at 4 °C.

Techniques: RNA Sequencing Assay, Activation Assay, Expressing

Journal: Cells

Article Title: Regulation of TGFβ Signalling by TRPV4 in Chondrocytes

doi: 10.3390/cells10040726

Figure Lengend Snippet: Reduction in extracellular calcium or calmodulin inhibition prevents GSK101 enhancement of TGFβ signalling. TC28a2 cells grown with indicated concentration of calcium ( A ) or KN93 ( B ) for ~16 h and then stimulated with TGFβ3 followed by DMSO (black) or GSK101 (red) after 15 min, and luciferase activity was determined 4 h after TGFβ3. ( A ) TRPV4 activation (using 100 nM GSK101) does not enhance TGFβ signalling at low calcium concentrations in medium. ( B ) Pre-treatment with calmodulin inhibitor (KN93) prevents TRPV4 activation (using 100 nM GSK101) of enhanced TGFβ signalling. ( C , D ) Schematics showing timing for calcium removal or calmodulin inhibition (KN93) in relation to stimulation/activation. Data were combined from three independent experiments. CFM; calcium-free medium, FC SBE RLU; fold change in SMAD-binding element relative light units. Statistical differences were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test; p < 0.05 *, p < 0.01 **, and p < 0.001 ***.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% BSA in 1x TBS-0.1%Tween-20, then probed with antibodies for SMAD2 (1:1000, CST 5339), pSMAD2 (1:1000, CST 18338), TRPV4 (1:200, Cat#ACC-124, Alomone Labs) and left overnight on a rocker at 4 °C.

Techniques: Inhibition, Concentration Assay, Luciferase, Activity Assay, Activation Assay, Binding Assay

Journal: Cells

Article Title: Regulation of TGFβ Signalling by TRPV4 in Chondrocytes

doi: 10.3390/cells10040726

Figure Lengend Snippet: TRPV4 activation enhances TGFβ signalling through the JUN and SP1 transcription factors. ( A , B ) TRRUST analysis of genes significantly increased in RNAseq for each of the indicated experimental comparisons. ( C ) siRNA knockdown of JUN and SP1 prevents TRPV4 enhancement of TGFβ signalling. Data were combined from three independent experiments. GSK101 treatment was normalised to DMSO for each siRNA. Statistical differences were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test; p < 0.05 * and p < 0.001 ***. FC SBE RLU; fold change in SMAD-binding element relative light units. ( D ) Sequence motif logos of JUN (MA0490.1; p -value 1.83 × 10 −11 ) and SP1 (MA0079.3.1; p -value 5.89 × 10 −7 ) within upregulated genes following TRPV4 activation created by TOMTOM from JASPAR2018_CORE_vertebrates_non-redundant database. ( E ) Schematic representation of possible mode of action of TGFβ and GSK101. In the presence of only TGFβ, SMAD2/3 causes transcriptional response of TGFβ target genes. In the presence of only GSK101, TRPV4 is activated, causing increased intracellular calcium, leading to activation of TRPV4 response genes. When TGFβ stimulation is followed by TRPV4 activation after 15 min, TGFβ activates SMAD3, and then TRPV4 activation causes increased calcium, enhancing the effect of TGFβ, through a mechanism involving SP1 and JUN, which are known SMAD3-binding partners. NT, no treatment; TR, transfection reagent control.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% BSA in 1x TBS-0.1%Tween-20, then probed with antibodies for SMAD2 (1:1000, CST 5339), pSMAD2 (1:1000, CST 18338), TRPV4 (1:200, Cat#ACC-124, Alomone Labs) and left overnight on a rocker at 4 °C.

Techniques: Activation Assay, Binding Assay, Sequencing, Transfection