Journal: bioRxiv

Article Title: Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

doi: 10.1101/2021.09.14.460172

Figure Lengend Snippet: A) Immunofluorescent images of ATDC5 cells on normal gels (upper panel) treated with 30nM GSK101 for 28 hr and severe OA gels with control treatment (lower panel) stained for the nucleus (Hoechst, blue), f-actin (purple), and TRPV4 (white). The left panel shows the composite images of all stains (scale bar =10 μm). The middle panel shows nuclear and f-actin staining, and the right panel shows TRPV4 and nuclear staining. B) TRPV4 fluorescence of ATDC5 cells on normal, OA, and severe OA gels during control (dark blue) and 28 hr of 30 nM GSK101 (light blue) treatments. f-actin was used to draw ROI around the cells to measure TRPV4 fluorescence from max projections of z-stack images. Error Bars are ± SEM, * indicates p-value < 0.05.

Article Snippet: A subset of cells received 30 nM GSK101 for 28 h. Cells were fixed in 4% paraformaldehyde, blocked for 1 hr in PBS containing 1% BSA/0.2% Fish Gelatin (Fisher Scientific), and probed for membrane TRPV4 using 1:200 rabbit anti-TRPV4 (Alomone Labs, ACC-124) overnight at 4°C.

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

doi: 10.1101/2021.09.14.460172

Figure Lengend Snippet: A) mRNA expression of anabolic factors (Sox-9, aggrecan, col2 and TRPV4) in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. B) mRNA expression of catabolic chondrocyte factors, collagen I and MMP-13 in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. Fold change was normalized to rplp0 expression for each sample and then normalized to the normal control condition. C) Western blotting shows TRPV4 protein levels for ATDC5 cells on normal, OA, and severe OA gels during control treatment. The bar graph represents the normalized TRPV4 protein level to GAPDH for each sample. Error bars are ± SEM, * indicates significance with a p-value < 0.05.

Article Snippet: A subset of cells received 30 nM GSK101 for 28 h. Cells were fixed in 4% paraformaldehyde, blocked for 1 hr in PBS containing 1% BSA/0.2% Fish Gelatin (Fisher Scientific), and probed for membrane TRPV4 using 1:200 rabbit anti-TRPV4 (Alomone Labs, ACC-124) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

doi: 10.1101/2021.09.14.460172

Figure Lengend Snippet: A) Immunofluorescent images of ATDC5 cells on normal gels (upper panel) treated with 30nM GSK101 for 28 hr and severe OA gels with control treatment (lower panel) stained for the nucleus (Hoechst, blue), f-actin (purple), and TRPV4 (white). The left panel shows the composite images of all stains (scale bar =10 μm). The middle panel shows nuclear and f-actin staining, and the right panel shows TRPV4 and nuclear staining. B) TRPV4 fluorescence of ATDC5 cells on normal, OA, and severe OA gels during control (dark blue) and 28 hr of 30 nM GSK101 (light blue) treatments. f-actin was used to draw ROI around the cells to measure TRPV4 fluorescence from max projections of z-stack images. Error Bars are ± SEM, * indicates p-value < 0.05.

Article Snippet: A subset of cells received 30 nM GSK101 for 28 h. Cells were fixed in 4% paraformaldehyde, blocked for 1 hr in PBS containing 1% BSA/0.2% Fish Gelatin (Fisher Scientific), and probed for membrane TRPV4 using 1:200 rabbit anti-TRPV4 (Alomone Labs, ACC-124) overnight at 4°C.

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

doi: 10.1101/2021.09.14.460172

Figure Lengend Snippet: A) mRNA expression of anabolic factors (Sox-9, aggrecan, col2 and TRPV4) in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. B) mRNA expression of catabolic chondrocyte factors, collagen I and MMP-13 in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. Fold change was normalized to rplp0 expression for each sample and then normalized to the normal control condition. C) Western blotting shows TRPV4 protein levels for ATDC5 cells on normal, OA, and severe OA gels during control treatment. The bar graph represents the normalized TRPV4 protein level to GAPDH for each sample. Error bars are ± SEM, * indicates significance with a p-value < 0.05.

Article Snippet: A subset of cells received 30 nM GSK101 for 28 h. Cells were fixed in 4% paraformaldehyde, blocked for 1 hr in PBS containing 1% BSA/0.2% Fish Gelatin (Fisher Scientific), and probed for membrane TRPV4 using 1:200 rabbit anti-TRPV4 (Alomone Labs, ACC-124) overnight at 4°C.

Techniques: Expressing, Western Blot

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

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: Cells were then incubated with TRPV4 antibody (1:200, Cat#ACC-124, Alomone Labs, Jerusalem, Israel) or Rabbit IgG control (1:200, Cell Signalling Technologies, London, UK) overnight.

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: Cells were then incubated with TRPV4 antibody (1:200, Cat#ACC-124, Alomone Labs, Jerusalem, Israel) or Rabbit IgG control (1:200, Cell Signalling Technologies, London, UK) overnight.

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: Cells were then incubated with TRPV4 antibody (1:200, Cat#ACC-124, Alomone Labs, Jerusalem, Israel) or Rabbit IgG control (1:200, Cell Signalling Technologies, London, UK) overnight.

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: Cells were then incubated with TRPV4 antibody (1:200, Cat#ACC-124, Alomone Labs, Jerusalem, Israel) or Rabbit IgG control (1:200, Cell Signalling Technologies, London, UK) overnight.

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: Cells were then incubated with TRPV4 antibody (1:200, Cat#ACC-124, Alomone Labs, Jerusalem, Israel) or Rabbit IgG control (1:200, Cell Signalling Technologies, London, UK) overnight.

Techniques: Activation Assay, Binding Assay, Sequencing, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

doi: 10.4049/jimmunol.1901033

Figure Lengend Snippet: Sterile beads or P. aeruginosa (PA, PAM57–15) were instilled intratracheally in TRPV4 KO and age-matched female congenic WT mice with BAL and tissue harvest performed at Day 3 (injury phase). TRPV4 deleted mice (TRPV4 KO) have greater A. inflammatory cell infiltration and B. BAL total protein compared to WT (*p < 0.05). C. TRPV4 KO mice have decreased bacterial clearance as measured by retained bacterial CFU in the combined BAL/lung homogenate as compared to WT (*p = 0.012). TRPV4 KO mice have greater BAL content of D. IL-6; *p = 0.028, E. CXCL2 (MIP-2); *p = 0.049, and F. CXCL1 (KC); *p = 0.009 than WT control by ELISA. G. TRPV4 KO hematoxylin and eosin (H&E) lung sections show greater parenchymal inflammatory cell infiltration (quantified as % lung consolidation) as compared with WT. n ≥ 5 per sterile bead group and n = 20 per P. aeruginosa group on Day 2–3. The box plots (B-F) indicate the 25th-75th percentile for each measure. The error bars denotes maximum and minimum values (5–95th percentile). The horizontal white line denotes the median value. * denotes WT vs TRPV4 KO.

Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

doi: 10.4049/jimmunol.1901033

Figure Lengend Snippet: WT and TRPV4 KO mice were intratracheally administered ±GFP P. aeruginosa for 3 days. Representative confocal images of whole lung lavage cytospins of macrophages (open arrowhead) and neutrophils (filled arrowhead) in WT mice given IT sterile beads or GFP-P. aeruginosa after immunofluorescence with A. TRPV4 extracellular antibody (green, TRPV4) and C. anti-GFP (green, GFP P. aeruginosa; anti-CD45, red; dapi, blue). B, D. Quantification of A, C. *, #p < 0.05; % WT vs KO. E. Flow cytometry of macrophage populations (+F4/80, CD64) from collagenase digested lung ± GFP-P. aeruginosa from WT and TRPV4 KO mice. Cell debris was excluded on a FSC-A/SSC-A plot and cell aggregates were excluded on a FSC-A/FSC-H plot. Viable cells were selected on a DAPI/SSC-A plot. Pseudocolor plots for CD45, neutrophil, and macrophage gating thresholds are shown. Gate boundaries for CD45 positive leukocytes and F4/80 positive macrophages were set using fluorescence minus one (FMO) controls. Immunofluorescence with anti-GFP (green) performed on cytospins. F. Quantification of % cell phagocytosis (*p = 0.035). n=20 per group. All images 63X original magnification, 10μm scale bars. *,# denotes WT vs TRPV4 KO. macs: macrophages and PMNs: neutrophils.

Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

Techniques: Immunofluorescence, Flow Cytometry, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

doi: 10.4049/jimmunol.1901033

Figure Lengend Snippet: BMDMs were incubated ± LPS as above for indicated time cultured on tissue culture-treated plastic, and cells were lysed and analyzed by immunoblot for A. phosphorylated and total p38, ERK, and JNK compared to WT BMDMs (whole cell lysate). Band density quantified from immunoblot (n = 3–6) for B. p-p38/total p38 (*p < 0.001), C. p-ERK/total ERK, and D. p-JNK/total JNK (*p = 0.027). E. Representative immunoblot for phosphorylated and total MK2 in WT vs TRPV4 KO BMDMs. F. Band density quantified from immunoblot (n = 5, *p < 0.05). G. Representative immunoblot for phosphorylated and total MKK3/MKK6. H. Band density quantified from immunoblot (n = 3). I. Representative immunoblot for phosphorylated and total p38 in homogenized mouse lung after sterile or P. aeruginosa beads (3 days). J. Band density quantified from p38 immunoblot (n = 6) (*p < 0.001). * denotes WT vs TRPV4 KO.

Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

Techniques: Incubation, Cell Culture, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

doi: 10.4049/jimmunol.1901033

Figure Lengend Snippet: WT and TRPV4 KO BMDMs were incubated ± LPS as above for indicated time, and cells were lysed and analyzed by A. immunoblot for DUSP1 and B. band density quantified as DUSP1/GAPDH from immunoblot (n = 4) (*p < 0.05). C. Representative immunoblot ± DUSP1 pharmacologic inhibitor, BCI 5μM, for p-p38/total p38 and p-JNK/total JNK in WT BMDMs. Band density quantified for D. p-p38/total p38 or E. p-JNK/total JNK from immunoblot (n = 4) (*p = 0.004). * denotes WT vs TRPV4 KO, + denotes ± pharmacologic inhibitor.

Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

Techniques: Incubation, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

doi: 10.4049/jimmunol.1901033

Figure Lengend Snippet: Phosphorylated and total A. p38 and B. JNK on various matrix stiffnesses in the physiologic range (1kPa, 8kPa, and 25kPa) from WT vs TRPV4 KO BMDMs quantified for LPS 15 minutes (*p = 0.031). C. Macrophage phagocytosis of E. coli particles ± p38 inhibition (SB, BIRB) on various matrix stiffnesses (*p < 0.05). n = 3–5 for all experiments. * denotes difference in LPS response ± inhibition of p38.

Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

Techniques: Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

doi: 10.4049/jimmunol.1901033

Figure Lengend Snippet: BMDMs were incubated ± LPS (100ng/mL, 24h) ± JNK inhibitor, SP600125 (20μM, 25h) ± p38 inhibitor, SB203580 (10μM, 25h), cultured on cell culture-treated plastic, and cytokines measured via ELISA. IL-6, CXCL2, and CXCL1 secretion ± LPS in A. WT and TRPV4 KO BMDMs and B. WT BMDMs ± SP600125 ± SB203580 (*,#p < 0.05). n = 3–5, one-way ANOVA followed by Dunnett’s test or Student-Newman-Keuls used for statistical analysis. * denotes WT vs TRPV4 KO, # denotes difference in LPS response ± inhibitor.

Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

doi: 10.4049/jimmunol.1901033

Figure Lengend Snippet: Monocyte derived and alveolar macrophages from healthy (n = 6) control subjects were incubated ± LPS ± TRPV4 inhibitor, HC, and phagocytosis of E. coli particles was measured in A. monocyte-derived and B. alveolar macrophages in healthy controls. HC alone had no effect. Representative immunoblot for phosphorylated and total p38 in C. healthy monocyte derived macrophages ± LPS 15 minutes and D. band density quantified (*p < 0.05). One-way ANOVA followed by Dunnett’s test or Student-Newman-Keuls used for statistical analysis, * denotes ± LPS, # denotes difference in LPS response ± inhibitor.

Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

Techniques: Derivative Assay, Incubation, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

doi: 10.4049/jimmunol.1901033

Figure Lengend Snippet: A. In the presence of a sub-threshold mechanical signal, as seen in normal lung, TRPV4 does not influence the LPS/TLR4 signal, which results in limiting the phagocytic response to LPS, thereby maintaining lung homeostasis. B. In the presence of an above threshold mechanical signal, as seen with lung stiffening during injury, TRPV4 influences the LPS/TLR4 signal. We have previously published that TRPV4 regulates the stiffness-dependent responses of increased macrophage phagocytosis, and cytokine secretion in response to LPS (23). We now show a molecular switch from JNK activation to predominantly p38 activation, which results in abrogation of enhanced DUSP1 expression. DUSP1 regulates the MAPK molecular switch by deactivating JNK resulting in enhanced bacterial clearance, inhibiting pro-inflammatory cytokine secretion, and thereby ameliorating lung injury/ARDS. This defines a novel molecular mechanism linking inflammation-induced changes in the mechanical properties of the extracellular matrix with innate immunity.

Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

Techniques: Activation Assay, Expressing