Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4 selectively activates Rac1 in BMDMs stimulated by IL-4 plus GM-CSF and interacts directly with Rac1. A – B , BMDMs from WT or TRPV4 mice were stimulated with IL-4 plus GM-CSF (25 ng/ml) for 0, 2, and 10 min, and whole-cell lysates were prepared for the analysis of total and activated Rac1, RhoA, and Cdc42. A , immunoblots showing expression levels of activated Rac1, RhoA, and Cdc42 in whole-cell lysates after antibody-mediated pull down. Total RhoA, Rac1, and Cdc42, as well as GAPDH, were analyzed in whole-cell lysates. Results are representative of three independent experiments. B , quantification of results from experiment shown in A. Student’s t test; ∗∗ p < 0.01 (0 min versus 10 in WT), ### p < 0.001 (10 min in WT versus 10 min in TRPV4 KO). C , interaction between TRPV4 and Rac1 proteins was determined using proximity ligation assay in unstimulated or IL-4 plus GM-CSF–stimulated (10 min) WT and TRPV4 KO BMDMs. WT and TRPV4 KO BMDMs with no primary antibodies were used as a negative control for the assay. Images (original magnification: 63× oil) are representative of five different fields per condition. D – E , quantification of data from the experiment shown in C. D , bar graph shows the TRPV4–Rac1 interaction efficiency as the number of red puncta per cell in different conditions. E , histograms show quantification of red signal intensity per field under different conditions. Student’s t test, n ≥ 3 independent experiments, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. F , co-immunoprecipitation (IP) followed by immunoblot (IB) analysis shows interaction of TRPV4 with Rac1 in IL-4 plus GM-CSF–stimulated macrophages at 10 and 30 min. Isotype control IgG was used as a control. G , quantification of results from experiment shown in F . Student’s t test; n = 3 independent experiments, ∗∗ p < 0.01 (UT versus 10 min in WT macrophages). BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Western Blot, Expressing, Proximity Ligation Assay, Negative Control, Immunoprecipitation, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4 directly regulates IL-4 plus GM-CSF–induced activation of Rac1 in macrophages . A , BMDMs from WT and TRPV4 KO mice were transduced with Ad(RGD)-GFP (1 × 10 8 pfu/ml). The expression and retention time of Ad(RGD)-GFP vector was confirmed in BMDMs by fluorescence microscopy at different time points. Representative images are shown; 20× magnification. B – C , WT BMDMs were treated with/without IL-4 plus GM-CSF, and TRPV4 KO BMDMs were transduced with either Ad-Vec or Ad-TRPV4 and were untreated or treated with IL-4 plus GM-CSF for 10 min on day 5 of transduction. B , Rac1-GTP levels were determined by G-LISA assay. The expression level was normalized to the total Rac1 level in each sample. Data are expressed as mean ± SEM, n = 3 independent experiments, Student’s t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns , not significant. C , TRPV4 and total Rac1 expression levels in both transduced and untransduced cells were analyzed by Western blotting. D , FlexStation 3 recording of GSK1016790A-induced Ca 2+ influx in TRPV4 KO BMDMs transfected with Ad-TRPV4 or Ad-Vec constructs. E , bar graph shows quantification of Ca 2+ influx from the experiment. The experiment was repeated three times in quadruplicate. Student’s t test, ∗∗∗ p < 0.001. BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; RFU, relative fluorescence unit; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Activation Assay, Transduction, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Transfection, Construct, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4-Rac1 signaling axis plays a crucial role in the augmentation of intracellular stiffness and regulation of cytoskeletal remodeling in BMDMs . A , schematic diagram of atomic force microscopy (AFM) setup to determine the stiffness (Young’s modulus) of BMDMs. A detector records deflection of a laser beam by deformation of the cantilever attached to a circular symmetric quartz probe with a radius of 30 nm. Force curves generated by this process are fitted to the Hertz model to achieve Young’s modulus value (kPa). B , representative high-resolution AFM micrographs show distribution of lamellipodia/filopodial areas of indicated cell groups. Scale bars: 2 μm; n = 10 cells/group; 2 scanned areas/cell. Quantification of data from experiment shown in B: Histograms show percent area of filopodia ( C ), the number of filopodia ( D ), and size of filopodia ( E ). Student’s t test; $$ p < 0.01 (KO, UT versus KO+Ad-TRPV4), ∗∗ p < 0.01 (WT, UT versus WT+IL-4+GM-CSF), ∗∗∗ p < 0.001, and ### p < 0.001 (KO+Ad-TRPV4 versus KO+Ad-TRPV4+Rac1-I). F , quantification of Young’s modulus (kPa) of the dataset, and G , upper quartile data points acquired from the experiment shown in B. n = 70 data points/group; One-way ANOVA followed by Bonferroni test; ∗∗∗ p < 0.001. BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Microscopy, Generated, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4-dependent Rac1 activation regulates fusogenic cytokine–induced FBGC formation. A , Giemsa-stained images showing FBGC formation by WT or TRPV4 KO BMDMs transduced with Ad-Vec or Ad-TRPV4 construct with or without Rac1-I (2, 10, and 50 μM) treatment after 8 days of fusogenic cytokine stimulation. Quantification of the number of FBGC/high power field ( B ), percent fusion ( C ), and average size of FBGCs ( D ) from experiment shown in ( A ). Scale bars: 50 μm; Student’s t test for B – D ; ∗∗ p < 0.01 (Ad-Vec versus Ad-TRPV4), # p < 0.05 (Ad-TRPV4 versus Ad-TRPV4+Rac1-I), ## p < 0.01, ### p < 0.001, $$ p < 0.01, and $$$ p < 0.001 (WT, IL-4+GM-CSF versus WT+ No IL-4+GM-CSF or WT+Rac1-I+IL-4+GM-CSF). BMDM, bone marrow–derived macrophage; FBGC, Foreign body giant cell; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Activation Assay, Staining, Transduction, Construct, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4 selectively activates Rac1 in BMDMs stimulated by IL-4 plus GM-CSF and interacts directly with Rac1. A – B , BMDMs from WT or TRPV4 mice were stimulated with IL-4 plus GM-CSF (25 ng/ml) for 0, 2, and 10 min, and whole-cell lysates were prepared for the analysis of total and activated Rac1, RhoA, and Cdc42. A , immunoblots showing expression levels of activated Rac1, RhoA, and Cdc42 in whole-cell lysates after antibody-mediated pull down. Total RhoA, Rac1, and Cdc42, as well as GAPDH, were analyzed in whole-cell lysates. Results are representative of three independent experiments. B , quantification of results from experiment shown in A. Student’s t test; ∗∗ p < 0.01 (0 min versus 10 in WT), ### p < 0.001 (10 min in WT versus 10 min in TRPV4 KO). C , interaction between TRPV4 and Rac1 proteins was determined using proximity ligation assay in unstimulated or IL-4 plus GM-CSF–stimulated (10 min) WT and TRPV4 KO BMDMs. WT and TRPV4 KO BMDMs with no primary antibodies were used as a negative control for the assay. Images (original magnification: 63× oil) are representative of five different fields per condition. D – E , quantification of data from the experiment shown in C. D , bar graph shows the TRPV4–Rac1 interaction efficiency as the number of red puncta per cell in different conditions. E , histograms show quantification of red signal intensity per field under different conditions. Student’s t test, n ≥ 3 independent experiments, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. F , co-immunoprecipitation (IP) followed by immunoblot (IB) analysis shows interaction of TRPV4 with Rac1 in IL-4 plus GM-CSF–stimulated macrophages at 10 and 30 min. Isotype control IgG was used as a control. G , quantification of results from experiment shown in F . Student’s t test; n = 3 independent experiments, ∗∗ p < 0.01 (UT versus 10 min in WT macrophages). BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Western Blot, Expressing, Proximity Ligation Assay, Negative Control, Immunoprecipitation, Control, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4 directly regulates IL-4 plus GM-CSF–induced activation of Rac1 in macrophages . A , BMDMs from WT and TRPV4 KO mice were transduced with Ad(RGD)-GFP (1 × 10 8 pfu/ml). The expression and retention time of Ad(RGD)-GFP vector was confirmed in BMDMs by fluorescence microscopy at different time points. Representative images are shown; 20× magnification. B – C , WT BMDMs were treated with/without IL-4 plus GM-CSF, and TRPV4 KO BMDMs were transduced with either Ad-Vec or Ad-TRPV4 and were untreated or treated with IL-4 plus GM-CSF for 10 min on day 5 of transduction. B , Rac1-GTP levels were determined by G-LISA assay. The expression level was normalized to the total Rac1 level in each sample. Data are expressed as mean ± SEM, n = 3 independent experiments, Student’s t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns , not significant. C , TRPV4 and total Rac1 expression levels in both transduced and untransduced cells were analyzed by Western blotting. D , FlexStation 3 recording of GSK1016790A-induced Ca 2+ influx in TRPV4 KO BMDMs transfected with Ad-TRPV4 or Ad-Vec constructs. E , bar graph shows quantification of Ca 2+ influx from the experiment. The experiment was repeated three times in quadruplicate. Student’s t test, ∗∗∗ p < 0.001. BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; RFU, relative fluorescence unit; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Activation Assay, Transduction, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Transfection, Construct, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4-Rac1 signaling axis plays a crucial role in the augmentation of intracellular stiffness and regulation of cytoskeletal remodeling in BMDMs . A , schematic diagram of atomic force microscopy (AFM) setup to determine the stiffness (Young’s modulus) of BMDMs. A detector records deflection of a laser beam by deformation of the cantilever attached to a circular symmetric quartz probe with a radius of 30 nm. Force curves generated by this process are fitted to the Hertz model to achieve Young’s modulus value (kPa). B , representative high-resolution AFM micrographs show distribution of lamellipodia/filopodial areas of indicated cell groups. Scale bars: 2 μm; n = 10 cells/group; 2 scanned areas/cell. Quantification of data from experiment shown in B: Histograms show percent area of filopodia ( C ), the number of filopodia ( D ), and size of filopodia ( E ). Student’s t test; $$ p < 0.01 (KO, UT versus KO+Ad-TRPV4), ∗∗ p < 0.01 (WT, UT versus WT+IL-4+GM-CSF), ∗∗∗ p < 0.001, and ### p < 0.001 (KO+Ad-TRPV4 versus KO+Ad-TRPV4+Rac1-I). F , quantification of Young’s modulus (kPa) of the dataset, and G , upper quartile data points acquired from the experiment shown in B. n = 70 data points/group; One-way ANOVA followed by Bonferroni test; ∗∗∗ p < 0.001. BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Microscopy, Generated, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4-dependent Rac1 activation regulates fusogenic cytokine–induced FBGC formation. A , Giemsa-stained images showing FBGC formation by WT or TRPV4 KO BMDMs transduced with Ad-Vec or Ad-TRPV4 construct with or without Rac1-I (2, 10, and 50 μM) treatment after 8 days of fusogenic cytokine stimulation. Quantification of the number of FBGC/high power field ( B ), percent fusion ( C ), and average size of FBGCs ( D ) from experiment shown in ( A ). Scale bars: 50 μm; Student’s t test for B – D ; ∗∗ p < 0.01 (Ad-Vec versus Ad-TRPV4), # p < 0.05 (Ad-TRPV4 versus Ad-TRPV4+Rac1-I), ## p < 0.01, ### p < 0.001, $$ p < 0.01, and $$$ p < 0.001 (WT, IL-4+GM-CSF versus WT+ No IL-4+GM-CSF or WT+Rac1-I+IL-4+GM-CSF). BMDM, bone marrow–derived macrophage; FBGC, Foreign body giant cell; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Activation Assay, Staining, Transduction, Construct, Derivative Assay

Journal: BMC Cancer

Article Title: Rac1 and Cdc42 are regulators of HRas V12 -transformation and angiogenic factors in human fibroblasts

doi: 10.1186/1471-2407-10-13

Figure Lengend Snippet: Evidence that Rac1 activity is essential to maintain HRas V12 -induced tumor formation . Solid lines represent mice that were not administered tetracycline (Tet). Dotted lines represent mice injected with the same cell strain, but mice were given Tet in order to suppress dominant-negative expression. Tumors were measured weekly. When tumors reached a volume of approximately 0.5 cm 3 , mice were sacrificed and the tumors were removed for further analysis. The data are plotted using Kaplan-Meier analyses. (a) PH3MT-tTak-C1 (parent); N = 4 (- Tet), N = 4 (+ Tet) p > 0.1. (b) PH3MT-VC-C2 (vector control); N = 5 (- Tet), N = 6 (+ Tet) p > 0.1. (c) PH3MT-Rac1 N17 -C1; N = 7 (- Tet), N = 9 (+ Tet), p < 0.001. (d) PH3MT-Rac1 N17 -C2; N = 4 (- Tet), N = 6 (+ Tet), p > 0.1. (e & f) Western blots probed with myc (9E10) antibody to detect dominant-negative protein expression in two tumor-derived cell lines (Tumor 1 and Tumor 2) Both blots were probed with β-actin to verify loading.

Article Snippet: To generate MSU-1.1 cell strains expressing GFP-Rac1 V12 or GFP-Cdc42 V12 fusion proteins, the GFP nucleotide sequence from the pCRUZ-GFP vector (Santa Cruz Biotechnology, Santa Cruz, CA) was isolated and ligated into the pcDNA6-V5-HisA vector (Invitrogen, Carlsbad, CA), which confers blasticidin resistance.

Techniques: Activity Assay, Injection, Dominant Negative Mutation, Expressing, Plasmid Preparation, Control, Western Blot, Derivative Assay

Journal: BMC Cancer

Article Title: Rac1 and Cdc42 are regulators of HRas V12 -transformation and angiogenic factors in human fibroblasts

doi: 10.1186/1471-2407-10-13

Figure Lengend Snippet: Evidence that Rac1 and/or Cdc42 activity is essential to maintain HRas V12 -induced tumor formation . Solid lines represent mice that were not administered tetracycline (Tet). Dotted lines represent mice injected with the same cell strain, but mice were given Tet in order to suppress dominant-negative expression. Tumors were measured weekly. When tumors reached a volume of approximately 0.5 cm 3 , mice were sacrificed and the tumors were removed for study. The data are plotted using Kaplan-Meier analyses. (a) PH3MT-Cdc42 N17 -C1; N = 8 (- Tet), N = 8 (+ Tet), p > 0.1. (b) PH3MT-Cdc42 N17 -C2; N = 6 (- Tet), N = 6 (+ Tet), p > 0.1. (c & d) Western blots probed with FLAG antibody to detect dominant-negative protein expression in two tumor derived cell lines (Tumor 1 and Tumor 2). (e) PH3MT-Rac1 N17 /Cdc42 N17 ; N = 9 (- Tet), N = 9 (+ Tet) p < 0.001. (f) Western blots probed with either myc (9E10) or FLAG antibody to detect dominant-negative protein expression in two tumor derived cell lines (Tumor 1 and Tumor 2). All blots were probed with β-actin to verify loading.

Article Snippet: To generate MSU-1.1 cell strains expressing GFP-Rac1 V12 or GFP-Cdc42 V12 fusion proteins, the GFP nucleotide sequence from the pCRUZ-GFP vector (Santa Cruz Biotechnology, Santa Cruz, CA) was isolated and ligated into the pcDNA6-V5-HisA vector (Invitrogen, Carlsbad, CA), which confers blasticidin resistance.

Techniques: Activity Assay, Injection, Dominant Negative Mutation, Expressing, Western Blot, Derivative Assay

Journal: BMC Cancer

Article Title: Rac1 and Cdc42 are regulators of HRas V12 -transformation and angiogenic factors in human fibroblasts

doi: 10.1186/1471-2407-10-13

Figure Lengend Snippet: Transformed phenotypes elicited by expression of Rac1 V12 or Cdc42 V12 in human fibroblasts . (a) MSU-1.1 fibroblasts expressing either GFP alone (MSU-1.1-GFP-VC), or GFP-tagged constitutively-activated proteins. (b) The indicated cell strains were grown in medium with reduced serum (0.5% SCS). Growth curves were plotted. Doubling time was calculated based on an equation derived from a best-fit exponential curve when cells were in log-phase growth. Error bars represent the SD of triplicate experiments. (c) The indicated cell strains were plated in 0.33% agarose and grown for three weeks. Each picture represents one representative field from each cell line. This experiment was completed in triplicate, each with similar results.

Article Snippet: To generate MSU-1.1 cell strains expressing GFP-Rac1 V12 or GFP-Cdc42 V12 fusion proteins, the GFP nucleotide sequence from the pCRUZ-GFP vector (Santa Cruz Biotechnology, Santa Cruz, CA) was isolated and ligated into the pcDNA6-V5-HisA vector (Invitrogen, Carlsbad, CA), which confers blasticidin resistance.

Techniques: Transformation Assay, Expressing, Derivative Assay

Journal: BMC Cancer

Article Title: Rac1 and Cdc42 are regulators of HRas V12 -transformation and angiogenic factors in human fibroblasts

doi: 10.1186/1471-2407-10-13

Figure Lengend Snippet: Activated Rac1 and Cdc42 independently regulate uPA expression . The indicated cell strains were serum starved for 24 hours then stimulated with medium containing 10% SCS. (a) Grown in the absence of tetracycline, PH3MT cell strains expressing a vector control (PH3MT-VC-C2), Rac1 N17 (PH3MT- Rac1 N17 -C1), Cdc42 N17 (PH3MT-Cdc42 N17 -C2) or both Rac1 N17 and Cdc42 N17 (PH3MT-Rac1 N17 /Cdc42 N17 ) were tested for uPA expression levels using ELISA. Data is presented as percent of control. Error bars indicate the SD from triplicate experiments. * indicates significant difference, p < 0.05. ** indicates a significant difference compared to PH3MT-Rac1 N17 -C1 and PH3MT-Cdc42 N17 -C2, p < 0.05. (b) conditioned medium was collected from MSU-1.1 cells expressing GFP alone (MSU-1.1-GFP-VC), GFP-tagged Rac1 V12 (MSU-1.1-GFP-Rac1 V12 ) or GFP-tagged Cdc42 V12 (MSU-1.1-GFP-Cdc42 V12 ), and uPA expression was analyzed. Data is presented as fold-induction of uPA expression. Error bars represent the SD from triplicate experiments. * indicates significant difference, p < 0.05.

Article Snippet: To generate MSU-1.1 cell strains expressing GFP-Rac1 V12 or GFP-Cdc42 V12 fusion proteins, the GFP nucleotide sequence from the pCRUZ-GFP vector (Santa Cruz Biotechnology, Santa Cruz, CA) was isolated and ligated into the pcDNA6-V5-HisA vector (Invitrogen, Carlsbad, CA), which confers blasticidin resistance.

Techniques: Expressing, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay

Journal: BMC Cancer

Article Title: Rac1 and Cdc42 are regulators of HRas V12 -transformation and angiogenic factors in human fibroblasts

doi: 10.1186/1471-2407-10-13

Figure Lengend Snippet: Both Rac1 and Cdc42 regulate VEGF expression in non-hypoxic and hypoxic conditions . All cell strains were serum starved for 24 hours prior to exposure to the indicated agent. Conditioned media were collected after 24 hr and ELISA analyses were completed. The data is presented as either percent of control, or fold induction as indicated in the figure. Error bars represent the SD from triplicate experiments. (a) PH3MT cell strains expressing a vector control (PH3MT-VC-C2), Rac1 N17 (PH3MT- Rac1 N17 -C1), Cdc42 N17 (PH3MT-Cdc42 N17 -C2), or both (PH3MT-Rac1 N17 /Cdc42 N17 ), were stimulated with either medium containing 10% SCS, 100 μM DFO, 100 μM CoCl 2 or Hypoxia (1% O 2 ). (b) MSU-1.1 cells expressing GFP alone (MSU-1.1-GFP-VC-C2), or GFP-tagged Rac1 V12 (MSU-1.1-GFP-Rac1 V12 ), or GFP-tagged Cdc42 V12 (MSU-1.1-GFP-Cdc42 V12 ). Cells were stimulated with medium containing 10% SCS. * denotes a significant difference (p < 0.01).

Article Snippet: To generate MSU-1.1 cell strains expressing GFP-Rac1 V12 or GFP-Cdc42 V12 fusion proteins, the GFP nucleotide sequence from the pCRUZ-GFP vector (Santa Cruz Biotechnology, Santa Cruz, CA) was isolated and ligated into the pcDNA6-V5-HisA vector (Invitrogen, Carlsbad, CA), which confers blasticidin resistance.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4 selectively activates Rac1 in BMDMs stimulated by IL-4 plus GM-CSF and interacts directly with Rac1. A – B , BMDMs from WT or TRPV4 mice were stimulated with IL-4 plus GM-CSF (25 ng/ml) for 0, 2, and 10 min, and whole-cell lysates were prepared for the analysis of total and activated Rac1, RhoA, and Cdc42. A , immunoblots showing expression levels of activated Rac1, RhoA, and Cdc42 in whole-cell lysates after antibody-mediated pull down. Total RhoA, Rac1, and Cdc42, as well as GAPDH, were analyzed in whole-cell lysates. Results are representative of three independent experiments. B , quantification of results from experiment shown in A. Student’s t test; ∗∗ p < 0.01 (0 min versus 10 in WT), ### p < 0.001 (10 min in WT versus 10 min in TRPV4 KO). C , interaction between TRPV4 and Rac1 proteins was determined using proximity ligation assay in unstimulated or IL-4 plus GM-CSF–stimulated (10 min) WT and TRPV4 KO BMDMs. WT and TRPV4 KO BMDMs with no primary antibodies were used as a negative control for the assay. Images (original magnification: 63× oil) are representative of five different fields per condition. D – E , quantification of data from the experiment shown in C. D , bar graph shows the TRPV4–Rac1 interaction efficiency as the number of red puncta per cell in different conditions. E , histograms show quantification of red signal intensity per field under different conditions. Student’s t test, n ≥ 3 independent experiments, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. F , co-immunoprecipitation (IP) followed by immunoblot (IB) analysis shows interaction of TRPV4 with Rac1 in IL-4 plus GM-CSF–stimulated macrophages at 10 and 30 min. Isotype control IgG was used as a control. G , quantification of results from experiment shown in F . Student’s t test; n = 3 independent experiments, ∗∗ p < 0.01 (UT versus 10 min in WT macrophages). BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Western Blot, Expressing, Proximity Ligation Assay, Negative Control, Immunoprecipitation, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4 directly regulates IL-4 plus GM-CSF–induced activation of Rac1 in macrophages . A , BMDMs from WT and TRPV4 KO mice were transduced with Ad(RGD)-GFP (1 × 10 8 pfu/ml). The expression and retention time of Ad(RGD)-GFP vector was confirmed in BMDMs by fluorescence microscopy at different time points. Representative images are shown; 20× magnification. B – C , WT BMDMs were treated with/without IL-4 plus GM-CSF, and TRPV4 KO BMDMs were transduced with either Ad-Vec or Ad-TRPV4 and were untreated or treated with IL-4 plus GM-CSF for 10 min on day 5 of transduction. B , Rac1-GTP levels were determined by G-LISA assay. The expression level was normalized to the total Rac1 level in each sample. Data are expressed as mean ± SEM, n = 3 independent experiments, Student’s t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns , not significant. C , TRPV4 and total Rac1 expression levels in both transduced and untransduced cells were analyzed by Western blotting. D , FlexStation 3 recording of GSK1016790A-induced Ca 2+ influx in TRPV4 KO BMDMs transfected with Ad-TRPV4 or Ad-Vec constructs. E , bar graph shows quantification of Ca 2+ influx from the experiment. The experiment was repeated three times in quadruplicate. Student’s t test, ∗∗∗ p < 0.001. BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; RFU, relative fluorescence unit; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Activation Assay, Transduction, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Transfection, Construct, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4-Rac1 signaling axis plays a crucial role in the augmentation of intracellular stiffness and regulation of cytoskeletal remodeling in BMDMs . A , schematic diagram of atomic force microscopy (AFM) setup to determine the stiffness (Young’s modulus) of BMDMs. A detector records deflection of a laser beam by deformation of the cantilever attached to a circular symmetric quartz probe with a radius of 30 nm. Force curves generated by this process are fitted to the Hertz model to achieve Young’s modulus value (kPa). B , representative high-resolution AFM micrographs show distribution of lamellipodia/filopodial areas of indicated cell groups. Scale bars: 2 μm; n = 10 cells/group; 2 scanned areas/cell. Quantification of data from experiment shown in B: Histograms show percent area of filopodia ( C ), the number of filopodia ( D ), and size of filopodia ( E ). Student’s t test; $$ p < 0.01 (KO, UT versus KO+Ad-TRPV4), ∗∗ p < 0.01 (WT, UT versus WT+IL-4+GM-CSF), ∗∗∗ p < 0.001, and ### p < 0.001 (KO+Ad-TRPV4 versus KO+Ad-TRPV4+Rac1-I). F , quantification of Young’s modulus (kPa) of the dataset, and G , upper quartile data points acquired from the experiment shown in B. n = 70 data points/group; One-way ANOVA followed by Bonferroni test; ∗∗∗ p < 0.001. BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Microscopy, Generated, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

doi: 10.1074/jbc.RA120.014597

Figure Lengend Snippet: TRPV4-dependent Rac1 activation regulates fusogenic cytokine–induced FBGC formation. A , Giemsa-stained images showing FBGC formation by WT or TRPV4 KO BMDMs transduced with Ad-Vec or Ad-TRPV4 construct with or without Rac1-I (2, 10, and 50 μM) treatment after 8 days of fusogenic cytokine stimulation. Quantification of the number of FBGC/high power field ( B ), percent fusion ( C ), and average size of FBGCs ( D ) from experiment shown in ( A ). Scale bars: 50 μm; Student’s t test for B – D ; ∗∗ p < 0.01 (Ad-Vec versus Ad-TRPV4), # p < 0.05 (Ad-TRPV4 versus Ad-TRPV4+Rac1-I), ## p < 0.01, ### p < 0.001, $$ p < 0.01, and $$$ p < 0.001 (WT, IL-4+GM-CSF versus WT+ No IL-4+GM-CSF or WT+Rac1-I+IL-4+GM-CSF). BMDM, bone marrow–derived macrophage; FBGC, Foreign body giant cell; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: Rac1, Cdc42, and RhoA Pull-Down Activation Assay Biochem Kits (Bead Pull-Down Format) (which included antibodies against Rac1, Cdc42, and RhoA) and Rac1 G-LISA Activation Assay Kit (Colorimetric Based) were procured from Cytoskeleton (Denver, CO, USA).

Techniques: Activation Assay, Staining, Transduction, Construct, Derivative Assay

Journal: Journal of Leukocyte Biology

Article Title: The trafficking protein JFC1 regulates Rac1‐GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration

doi: 10.1002/jlb.1vma0818-320r

Figure Lengend Snippet: FIGURE 5 JFC1 interacts with active Rac1 in a Rab27a-independent manner. (A) Co-immunoprecipitation analysis of the JFC1-Rac1 inter- action. Cells were transfected with myc-JFC1 and with either WT Rac1-GFP, the constitutively active Rac1 Q61L-GFP or the dominant negative Rac1 T17N-GFP. Cell lysates were used in pulldown assays, carried out using anti-myc antibodies and magnetic beads. Western blots are represen- tative of at least three experiments with similar results. (B) Densitometric quantification of the immunoprecipitated bands from three independent experiments using the ImageJ software. The data is represented as mean±SEM. **P < 0.01. (C) Pulldown experiments were performed in cells trans- fected with GFP-Rac1Q61L with either wild type myc-JFC1 or with the point mutant myc-JFC1-W83S, which lacks binding to Rab27a. (D) Pulldown experiments were performed in cells transfected with EGFP-Rab27a with either myc-JFC1 WT or with the myc-JFC1-W83S mutant

Article Snippet: GFP-Rac1WT, GFP-Rac1 Q61L, GFP-Rac1 T17N were obtained from Addgene.Myc-JFC1, DsRed-JFC1,myc-JFC1-W83S, andGFP-Rab27a were generated as described before.20,57

Techniques: Immunoprecipitation, Transfection, Dominant Negative Mutation, Magnetic Beads, Western Blot, Software, Mutagenesis, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: CD93 Signaling via Rho Proteins Drives Cytoskeletal Remodeling in Spreading Endothelial Cells

doi: 10.3390/ijms222212417

Figure Lengend Snippet: CD93 signaling activates Rac1 at the cell edge of spreading cells. ( a ) FRET analysis on control (sh-unr) or CD93-silenced (sh-CD93) HUVECs transduced with a lentiviral construct expressing the Rac1 biosensor. Cells were fixed at the early phase of adhesion to the ECM. Representative confocal images of transduced cells before photobleaching (acceptor pre) are shown. Rectangles indicate the photobleached cell area. Magnifications of the photobleached area are shown (2.5×). The colored scale represents the color range of FRET efficiency. Scale bars, 20 µm. ( b ) Plot showing the fluorescence increase (% FRET efficiency) upon photobleaching at the cell edge of early spreading ECs ( n = 10 cells for sh-unr and n = 9 cells for sh-CD93). Data are presented as scatter plot. ** p < 0.01; Student t -test.

Article Snippet: The following plasmids were purchased from Addgene (Watertown, MA, USA): a lentiviral negative control vector containing scrambled shRNA (#1864, sh-unr) [ ]; Rac1 generation fluorescence resonance energy transfer (FRET) biosensor for lentivirus production (#66111, pLenti-Rac1-2G) [ ]; FRET-based biosensor reporting on Cdc42 activation (#68813, pLenti-Cdc42-2G) [ ]; RhoA second-generation FRET biosensor for lentivirus production (#40179, pLenti-RhoA-2G) [ ]. shRNA-mediated knockdown of CD93 was performed as previously outlined [ ] by using a pLKO.1 retroviral vector from the Mission shRNA Library (Merck KGaA, Darmstadt, Germany), which expresses a shRNA (clone TRCN0000029085) specific for the silencing of the human protein.

Techniques: Transduction, Construct, Expressing, Fluorescence