Journal: Materials Today Bio

Article Title: High-viscosity driven modulation of biomechanical properties of human mesenchymal stem cells promotes osteogenic lineage

doi: 10.1016/j.mtbio.2024.101058

Figure Lengend Snippet: Higher extracellular viscosity enhanced the translocation of TRPV4 to the cell membrane, thereby promoting calcium influx and osteogenic lineage in hMSCs. (A) The time course of intracellular Ca 2+ levels, quantified in terms of Fluo 8-am, in hMSCs with and without a 3-h pre-treatment of the 10 μM TRPV4 inhibitor (GSK205), followed by stimulation with a high-viscosity medium (68.14 cP). Cell culture in DMEM (0.98 cP) serves as control. (B) Time-dependent relative changes in hMSCs' area, corresponding to A. Data represent mean ± SD (n = 10). (C – E) The surface labeling of TRPV4 (in green) on hMSCs, with and without prior treatment with 10 μM of GSK205 (a TRPV4 antagonist), followed by stimulation with a highly viscous medium (68.14 cP) for 1 h. The surface labeling procedure included the blocking of non-permeabilized fixed hMSCs with BSA for 1 h, followed by a 1-h incubation with anti-TRPV4 and two PBS washes. Finally, the cells were incubated with an Alexa Fluor 488-conjugated antibody for 1 h. Scale bars = 50 μm. (F) Quantification of the number and the intensity of TRPV4 fluorescence spots on the cell membrane, as depicted in (C–E). Data represent mean ± SD (n = 15). (G – I) Representative micrographs of hMSCs, cultured in differentiation medium with and without treatment with 10 μM of GSK205 followed by stimulation with a highly viscous medium (68.14 cP) for 14 days. The cells were stained to visualize the presence of ALP activity (in purple) and lipids (Oil Red O, red). Scale bars = 50 μm. (J) The percentage of osteogenesis (ALP-positive cells) and adipogenesis (Oil Red O-positive cells) in hMSCs, as depicted in (I). Data represent mean ± SD (n = 3; i.e., three replicated independent experiments). * for p < 0.05 and ** for p < 0.01. ; N.S.: Not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used in this experiment were 1:100 dilution of anti-TRPV4 rabbit antibody (65893, Cell Signaling), 1:50 dilution of anti-NFATc1 mouse monoclonal antibody (sc-7294, Santa Cruz Biotechnology), 1:200 dilution of anti-paxillin rabbit polyclonal antibody (GTX125891, GeneTex), and 1:200 dilution of anti-YAP rabbit polyclonal antibody (GTX129151, GeneTex).

Techniques: Viscosity, Translocation Assay, Membrane, Cell Culture, Control, Labeling, Blocking Assay, Incubation, Fluorescence, Staining, Activity Assay

Journal: Materials Today Bio

Article Title: High-viscosity driven modulation of biomechanical properties of human mesenchymal stem cells promotes osteogenic lineage

doi: 10.1016/j.mtbio.2024.101058

Figure Lengend Snippet: Higher extracellular viscosity promoted membrane translocation of TRPV4 and the nuclear translocation of NFATc1. (A) Immunofluorescence micrographs displaying NFATc (in red), TRPV4 on membrane (in green), and the nucleus (in blue), in hMSCs with and without prior treatment with GSK205 (10 μM), and subsequently stimulated with a highly viscous medium (68.14 cP) for 1 h. Scale bars = 50 μm. (B) Cell area, (C) mean fluorescence intensity of TRPV4, and (D) the ratio of NFATc in the nucleus to that in the cytoplasm of hMSC, as indicated in (A). Data represent mean ± SD (n = 15). * for p < 0.05 and ** for p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used in this experiment were 1:100 dilution of anti-TRPV4 rabbit antibody (65893, Cell Signaling), 1:50 dilution of anti-NFATc1 mouse monoclonal antibody (sc-7294, Santa Cruz Biotechnology), 1:200 dilution of anti-paxillin rabbit polyclonal antibody (GTX125891, GeneTex), and 1:200 dilution of anti-YAP rabbit polyclonal antibody (GTX129151, GeneTex).

Techniques: Viscosity, Membrane, Translocation Assay, Immunofluorescence, Fluorescence

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: 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: 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: 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: 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: 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: 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: 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: 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: 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

Journal: Biochemistry and Biophysics Reports

Article Title: Basal cells express functional TRPV4 channels in the mouse nasal epithelium

doi: 10.1016/j.bbrep.2015.09.008

Figure Lengend Snippet: (A) RT-PCR analysis of TRPV4 transcripts in adult mouse nasal mucosa. Controls, carbonic anhydrase II (CAII) and β-actin fragments are also shown. (B) Examination of the expression of TRPV4 transcripts in the mouse nasal mucosa at embryonic day 18 (E18) by in situ hybridization. Some cells located in the basal layer demonstrated intense TRPV4 expression when the TRPV4 anti-sense probe was used. Epithelial cells in the choroid plexus, which is known to express TRPV4 mRNA, also exhibited positive reactions when the anti-sense probe was employed. Scale bars: 30 μm.

Article Snippet: After the sections were air-dried, they were fixed with Bouin's fixative for 15 min at 4 °C and incubated with a rabbit anti-TRPV4 antibody (1:400) (Alomone Lab, Jerusalem, Israel) in phosphate-buffered saline (PBS) containing 5% normal donkey serum and 0.3% Triton X-100 overnight at 4 °C followed by an Alexa 594-conjugated donkey anti-rabbit IgG secondary antibody (Invitrogen).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, In Situ Hybridization

Journal: Biochemistry and Biophysics Reports

Article Title: Basal cells express functional TRPV4 channels in the mouse nasal epithelium

doi: 10.1016/j.bbrep.2015.09.008

Figure Lengend Snippet: Immunohistochemical analysis of TRPV4 in the mouse nasal mucosa. (A, B) Cellular localization of the TRPV4 protein in wild-type mice. TRPV4 was expressed in the basal layers of the olfactory epithelium (OE) (arrow heads) and the airway epithelium (AE) (arrows). TRPV4 immunoreactivity (IR) was also observed in the cartilage (C) and basal layers of the squamous epithelium (SE) (arrow heads in B). NC; nasal cavity (C, D). These structures were not stained in the TRPV4-KO mice (arrow heads and arrows). (E, F) Double-staining immunohistochemistry analysis of TRPV4 with keratin pan. TRPV4-IR is co-localized with keratin pan-positive (K14-positive) basal cells in the AE and OE.

Article Snippet: After the sections were air-dried, they were fixed with Bouin's fixative for 15 min at 4 °C and incubated with a rabbit anti-TRPV4 antibody (1:400) (Alomone Lab, Jerusalem, Israel) in phosphate-buffered saline (PBS) containing 5% normal donkey serum and 0.3% Triton X-100 overnight at 4 °C followed by an Alexa 594-conjugated donkey anti-rabbit IgG secondary antibody (Invitrogen).

Techniques: Immunohistochemical staining, Staining, Double Staining Immunohistochemistry

Journal: Biochemistry and Biophysics Reports

Article Title: Basal cells express functional TRPV4 channels in the mouse nasal epithelium

doi: 10.1016/j.bbrep.2015.09.008

Figure Lengend Snippet: Intracellular Ca 2+ ([Ca 2+ ] i ) response in primary cultured mouse nasal epithelial cells. (A, B) Representative traces demonstrating that some cells responded to hypo-osmotic stimulation for 5 min (magenta and blue traces in A) and 4α-PDD for 10 min (magenta trace in B) and that the simultaneous application of ruthenium red (RuR) for 10–14 min suppressed the stimuli-induced Ca 2+ increase (A and B) in wild-type mice. A summary of peak changes in the Ca 2+ fluorescent ratio evoked by the TRPV4 activator (Δratio) is shown as a graph on the right in each panel. The number in parentheses indicates the number of cells analyzed. ** p <0.0001 vs . stimulus-insensitive (hypo- or 4αPDD-) cells, unpaired Student's t -test). Bar=SEM. (C) Representative traces demonstrating that the brief application of 10 μM 4α-PDD (90 s) also caused an increase in [Ca 2+ ] i in a subset of the epithelial cells isolated from wild-type mice (yellow and black traces) (WT), whereas 4α-PDD-induced responses were rarely observed in cells obtained from TRPV4 KO mice (TRPV4-KO). (D) A significant difference between the results of wild-type (WT) and TRPV4-KO mice was observed (** p =0.0027 vs . WT mice, unpaired Student's t -test). The number in parentheses under the bar graph indicates the number of assays performed. Bar=SEM.

Article Snippet: After the sections were air-dried, they were fixed with Bouin's fixative for 15 min at 4 °C and incubated with a rabbit anti-TRPV4 antibody (1:400) (Alomone Lab, Jerusalem, Israel) in phosphate-buffered saline (PBS) containing 5% normal donkey serum and 0.3% Triton X-100 overnight at 4 °C followed by an Alexa 594-conjugated donkey anti-rabbit IgG secondary antibody (Invitrogen).

Techniques: Cell Culture, Isolation

Journal: Biochemistry and Biophysics Reports

Article Title: Basal cells express functional TRPV4 channels in the mouse nasal epithelium

doi: 10.1016/j.bbrep.2015.09.008

Figure Lengend Snippet: In vivo BrdU proliferation assay in the olfactory epithelium (OE) of P8 mice. (A) Representative images of staining patterns with an anti-BrdU antibody in OEs isolated from wild-type and TRPV4-KO mice 30 min after an intraperitoneal BrdU injection. Note that BrdU-positive cells were observed in the apical (arrows) and basal (arrow heads) regions of the OEs in both wild-type and TRPV4-KO mice. NC; nasal cavity (B) BrdU-immunoreactive cells in the apical and basal portions of the OE, as shown in (A), were counted in 10 fields of view per animal, and each mean±SEM is indicated ( n =3 each). In the OE, there was no significant difference in BrdU labeling between wild-type and TRPV4 KO mice under normal developmental conditions.

Article Snippet: After the sections were air-dried, they were fixed with Bouin's fixative for 15 min at 4 °C and incubated with a rabbit anti-TRPV4 antibody (1:400) (Alomone Lab, Jerusalem, Israel) in phosphate-buffered saline (PBS) containing 5% normal donkey serum and 0.3% Triton X-100 overnight at 4 °C followed by an Alexa 594-conjugated donkey anti-rabbit IgG secondary antibody (Invitrogen).

Techniques: In Vivo, Proliferation Assay, Staining, Isolation, Injection, Labeling