eht1864 rac inhibitor  (Tocris)

Journal: bioRxiv

Article Title: Survivin is a mechanosensitive cell cycle regulator in vascular smooth muscle cells

doi: 10.1101/2022.11.09.515885

Figure Lengend Snippet: G0-synchronized human vascular smooth muscle cells (hVSMCs) treated with FAK inhibitor PF573228 (PF) or Rac inhibitor EHT1864 (EHT) in dimethyl sulfoxide (DMSO) were seeded on fibronectin-coated soft or stiff hydrogels for 24 h. Survivin mRNA ( A ) and protein ( B and C ) expression was analyzed by RT-qPCR and immunoblotting, respectively; levels were normalized to those in hVSMCs treated with DMSO on stiff hydrogels; n = 3 (A) and n = 5 (B and C) independent experiments. ( D ) hVSMCs infected with adenoviruses encoding LacZ, FAK 397F , or Rac N17 were seeded on soft or stiff hydrogels with 10% FBS for 24 h. Survivin mRNA expression was analyzed by RT-qPCR; levels were normalized to those in hVSMCs infected with LacZ on stiff hydrogels. n = 8 independent experiments. ( E and F ) G 0 -synchronized hVSMCs were transfected with control siRNA or siRNAs to FAK [1 and #2], serum-starved, and plated on stiff hydrogels with 10% FBS for 24 h. FAK and survivin levels in total cell lysates were analyzed by immunoblotting; levels were normalized to those in hVSMCs treated with control siRNA on stiff hydrogels. n = 3 independent experiments. ( G and H ) hVSMCs infected with adenoviruses encoding LacZ or FRNK were plated on stiff hydrogels for 24 h. Protein expression levels were analyzed by immunoblotting. n = 4 independent experiments. ( I ) Model of signal transduction for stiffness-mediated cell cycle progression. Data are means + SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Serum-starved hVSMCs were plated on fibronectin-coated soft or stiff hydrogels with medium containing 10% FBS and were treated with 0.1, 0.5, or 2 μM YM155 (survivin inhibitor; cat. no. 11490, Cayman Chemical), 10 μM PF573228 (FAK inhibitor; cat. no. 14924, Cayman Chemical), 10 μM EHT1864 (Rac inhibitor; cat. no. 17258, Cayman Chemical), or dimethyl sulfoxide (vehicle control).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Transfection, Control, Transduction

Journal: Scientific Reports

Article Title: A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation

doi: 10.1038/s41598-021-02683-4

Figure Lengend Snippet: Inhibition of Rac, Rho, or Cdc42 activation differentially affects VSMC spheroid formation and morphology. Human VSMCs treated with Rac (EHT1864), Rho (Rhosin), and Cdc42 (ML141) inhibitors or DMSO (vehicle control) were used to generate human VSMC spheroids. VSMC spheroids were imaged after 24 h of incubation using an upright phase-contrast microscope.

Article Snippet: For the FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK-specific inhibitor PF573228 (Sigma), 5–20 μM Rac-specific inhibitor EHT1864 (Tocris), 5–20 μM RhoA-specific inhibitor Rhosin (Tocris), or 5–10 μM Cdc42-specific inhibitor ML 141 (Tocris) reconstituted in dimethyl sulfoxide (DMSO) for selected times up to 24 h.

Techniques: Inhibition, Activation Assay, Control, Incubation, Microscopy

Journal: Scientific Reports

Article Title: A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation

doi: 10.1038/s41598-021-02683-4

Figure Lengend Snippet: Initial morphological clustering analysis identifying distinct clusters in response to inhibitors of FAK, Rac, Rho, and Cdc42. ( A ) Four morphological features extracted from human VSMC spheroid images are displayed on a UMAP plot. The different colored markers are used to represent the various drug treatments: FAK (PF573228), Rac (EHT1864), Rho (Rhosin), and Cdc42 (ML141). ( B ) The silhouette values and the number of clusters on the training set were evaluated by varying the number of nearest samples. ( C ) Silhouette plots for the clustering results on the testing set. ( D ) Four morphological clusters plotted on the UMAP plot were observed based on the “roundness of the spheroid”. The different colored markers are used to represent the various morphological clusters with the drug treatments. The most representative images were overlayed on each cluster (2 images per cluster) in the UMAP plot. Clusters #1 and #3 showed spheroids with circular morphologies, and the spheroids in Clusters #2 and #4 showed noncircular and dispersed/disrupted morphologies. Only the samples in the testing set were used. ( E ) Average proportionality plot of the distribution of VSMC spheroids in each morphological cluster with the drug treatments from the repeated random splitting of training and testing sets. Only testing sets were used.

Article Snippet: For the FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK-specific inhibitor PF573228 (Sigma), 5–20 μM Rac-specific inhibitor EHT1864 (Tocris), 5–20 μM RhoA-specific inhibitor Rhosin (Tocris), or 5–10 μM Cdc42-specific inhibitor ML 141 (Tocris) reconstituted in dimethyl sulfoxide (DMSO) for selected times up to 24 h.

Techniques:

Journal: Cancers

Article Title: Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

doi: 10.3390/cancers13215460

Figure Lengend Snippet: Effects of IQGAP, COPA, RAC1 and EZRIN depletion on the NIS abundance at the PM. ( A ) HA-NIS-TPC1 cells were transfected with siRNAs targeting IQGAP, COPA, EZRIN and RAC1, and were analyzed by cell-surface protein biotinylation. The HA-NIS protein in either the surface fraction or the correspondent whole-cell lysates (WCL) was detected by WB. The ‘no Biotin’ condition, corresponding to cells that were not incubated with biotin, was used to control the specificity of the biotinylated protein pull-down. WCL were further probed for IQGAP, COPA, RAC1 and EZRIN protein levels, in order to ascertain siRNA efficiency. PCNA detection served as the loading (WCL) and intracellular protein contamination control (Surface pool). GLUT-1 served as the positive loading control for the cell surface protein extracts. ( B ) Surface HA-NIS expression was quantified by the densitometric analysis of the WB bands, using ImageJ software. The plotted values are means ± SEM of three independent assays. The one-way ANOVA analysis detected significant differences between the treatments (F = 33.17; p < 0.001). Post-hoc Dunnett’s tests were used to identify significant variations relative to ‘siCtrl’ (transfection with a siRNA against luciferase firefly–see Materials and Methods) (*** p ≤ 0.001). ( C ) HA-NIS-TPC1 cells were treated with 50 µM EHT1864 for 1 h and analyzed by cell-surface protein biotinylation followed by WB, as in ( A ). The surface HA-NIS expression was quantified by the densitometric analysis of the WB bands, as before. A Student T test was used to evaluate significant variations relative to the ‘Ctrl’ sample (*** p ≤ 0.001). ( D ) HA-NIS-TPC1 cells stably co-expressing the HS-YFP iodide sensor were transfected with either siCtrl or siEZRIN, or treated or not with 50 µM EHT1864 for 1 h, or 50 mM of ClO 4 − for 10 min, and the YFP fluorescence was recorded continuously, as described in the legend to . The data are the means ± SEM of five independent assays. One-way ANOVA analysis detected significant differences between the treatments (F = 33.39 and p < 0.001). Post-hoc Dunnett’s tests were used to identify significant variations relative to the control (Ctrl) conditions (*** p ≤ 0.001). ( E ) PCCL3 thyroid cells were serum-starved for 24 h, followed by stimulation or not with TSH (1 mU/mL for 48 h). The cells were then treated with either vehicle or 50 µM EHT1864 for 1 h, and were analyzed by surface protein biotinylation and WB, as in ( A ). The plotted values are the means ± SEM of three independent assays. Significant variations were assessed by Student T test (** p ≤ 0.01). ( F ) PCCL3 cells stably expressing the HS-YFP iodide sensor were stimulated as in ( E ), and were treated or not with 50 µM EHT1864 for 1 h, or 1 mM of ClO 4 − for 10 min, and their YFP fluorescence was recorded continuously, as described in the legend to . The data are the means ± SEM of five independent assays. One-way ANOVA analysis detected significant differences between the treatments (F = 18.13 and p < 0.001). Post-hoc Dunnett’s tests were used to identify significant variations relative to the control (Ctrl) conditions (* p ≤ 0.05; *** p ≤ 0.001).

Article Snippet: The treatment of the cells with the RAC1 selective inhibitor EHT1864 (50 μM, SCTB, Santa Cruz, CA, USA), ARP2/3 Complex Inhibitor CK666 (100 μM, Sigma-Aldrich, St. Louis, MO, USA), JNK inhibitor SP600125 (30 μM, Sigma-Aldrich, St. Louis, MO, USA), P38 inhibitor SB203580 (10 μM, SCTB, Santa Cruz, CA, USA), PAK inhibitor IPA3 (10 μM, Calbiochem), SRC Inhibitors PP2 (2 μM, Sigma-Aldrich, St. Louis, MO, USA) or Dasatinib (150 nM, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h in the appropriate medium, using cells treated with the same volume of solvent (vehicle) as a control.

Techniques: Transfection, Incubation, Control, Expressing, Software, Luciferase, Stable Transfection, Fluorescence

Journal: Cancers

Article Title: Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

doi: 10.3390/cancers13215460

Figure Lengend Snippet: Upregulation of RAC1 and EZRIN increases the NIS residency at the PM. ( A ) HA-NIS-TPC1 cells were transiently transfected with either empty vector (Ctrl), GFP-RAC1-L61, MYC-RAC1-V12 or CFP-ERZIN constructs, and were analyzed by cell surface protein biotinylation. The surface fractions and the corresponding whole-cell lysates (WCL) were analyzed by WB, as indicated. PCNA detection served as the loading (WCL) and intracellular protein contamination control (Surface pool). GLUT-1 served as the positive loading control for the cell-surface protein extracts. ( B ) WB bands were quantified by densitometric analysis using ImageJ software. The plotted values are the means ± SEM of five independent assays. A one-way ANOVA analysis detected significant differences between the treatments (F = 17.82; p < 0.001). Post-hoc Dunnett’s tests were used to identify significant variations relative to the control (Ctrl) conditions (** p ≤ 0.01; *** p ≤ 0.001). ( C ) HA-NIS-TPC1 cells stably co-expressing the HS-YFP iodide sensor were transfected with either empty vector (Ctrl), MYC-RAC1-V12 or CFP-ERZIN, or were treated or not with 50 mM of ClO 4 − for 10 min, and YFP fluorescence was recorded continuously (as described in the legend to ). The data are the means ± SEM of three independent assays. One-way ANOVA analysis detected significant differences between the treatments (F = 59.51 and p < 0.001). Post-hoc Dunnett’s tests were used to identify significant variations relative to the control (Ctrl) conditions (** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: The treatment of the cells with the RAC1 selective inhibitor EHT1864 (50 μM, SCTB, Santa Cruz, CA, USA), ARP2/3 Complex Inhibitor CK666 (100 μM, Sigma-Aldrich, St. Louis, MO, USA), JNK inhibitor SP600125 (30 μM, Sigma-Aldrich, St. Louis, MO, USA), P38 inhibitor SB203580 (10 μM, SCTB, Santa Cruz, CA, USA), PAK inhibitor IPA3 (10 μM, Calbiochem), SRC Inhibitors PP2 (2 μM, Sigma-Aldrich, St. Louis, MO, USA) or Dasatinib (150 nM, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h in the appropriate medium, using cells treated with the same volume of solvent (vehicle) as a control.

Techniques: Transfection, Plasmid Preparation, Construct, Control, Software, Stable Transfection, Expressing, Fluorescence

Journal: Cancers

Article Title: Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

doi: 10.3390/cancers13215460

Figure Lengend Snippet: EZRIN and ARP2/3 activity are required downstream of RAC1 to sustain HA-NIS PM residency. HA-NIS-TPC1 cells were transfected with either ( A ) GFP-RAC1-L61 or a siRNA against EZRIN, or both; ( B ) with CFP-EZRIN in the presence or absence of the RAC1 inhibitor EHT1864 (50 µM for 1 h); ( C ) with either mock (siLUC control) or siARP3, and treated or not with the ARP2/3 complex inhibitor CK666 (100 µM for 1 h); or ( D ) with either empty vector (Empty) or GFP-RAC1-L61, and either mock (siLUC control) or the siARP3, as indicated, and analyzed by surface protein biotinylation. Surface fractions and whole-cell lysates (WCL) were then analyzed by WB, as indicated. The ‘no Biotin’ condition corresponds to cells not incubated with biotin, and was used to control the specificity of biotinylated protein pull-downs. PCNA expression served as both the loading (WCL) and intracellular protein contamination control (Surface pool). GLUT-1 served as the positive loading control for the cell-surface protein extracts. The WB bands were quantified by densitometric analysis using ImageJ software. The plotted values correspond to the means ± SEM of at least three independent assays. One-way ANOVA analysis detected the significant differences between the treatments [F = 23.65 and p < 0.001 for ( A ); F = 17.86 and p < 0.001 for ( B ); F = 14.79 and p < 0.001 for ( C ); F = 18.26 and p < 0.001 for ( D )]. Post-hoc Tukey’s tests were used to identify significant variations relative to the control conditions or among the different treatments (the latter are indicated by horizontal lines) (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: The treatment of the cells with the RAC1 selective inhibitor EHT1864 (50 μM, SCTB, Santa Cruz, CA, USA), ARP2/3 Complex Inhibitor CK666 (100 μM, Sigma-Aldrich, St. Louis, MO, USA), JNK inhibitor SP600125 (30 μM, Sigma-Aldrich, St. Louis, MO, USA), P38 inhibitor SB203580 (10 μM, SCTB, Santa Cruz, CA, USA), PAK inhibitor IPA3 (10 μM, Calbiochem), SRC Inhibitors PP2 (2 μM, Sigma-Aldrich, St. Louis, MO, USA) or Dasatinib (150 nM, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h in the appropriate medium, using cells treated with the same volume of solvent (vehicle) as a control.

Techniques: Activity Assay, Transfection, Control, Plasmid Preparation, Incubation, Expressing, Software

Journal: Cancers

Article Title: Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

doi: 10.3390/cancers13215460

Figure Lengend Snippet: PAK1 participates in the RAC1-stimulated upregulation of NIS PM residency. HA-NIS-TPC1 cells were ( A ) transfected with either mock (siLUC control) or a specific siRNA against PAK1 (siPAK1), or were treated or not with PAK1 chemical inhibitor IPA3 (10 µM for 1 h); or ( B ) transfected with either empty vector (Empty) or GFP-RAC1-L61, and either mock (siLUC control) or the siPAK1, as indicated, and were analyzed by surface protein biotinylation. The surface fractions and whole-cell lysates (WCL) were then analyzed by WB, as indicated. The ‘no Biotin’ condition, which corresponds to cells which were not incubated with biotin, was used to control the specificity of biotinylated protein pull-downs. PCNA expression served both as the loading (WCL) and intracellular protein contamination control (Surface pool). GLUT-1 served as the positive loading control for the cell-surface protein extracts. The WB bands were quantified by densitometric analysis using ImageJ software. The plotted values correspond to the means ± SEM of at least three independent assays. One-way ANOVA analysis detected significant differences between the treatments [F = 9.31 and p = 0.0012 for ( A ); F = 12.73 and p < 0.001 for ( B )]. Post-hoc Tukey’s tests were used to identify significant variations relative to the control conditions or among the different treatments (the latter are indicated by horizontal lines) (* p ≤ 0.05; ** p ≤ 0.01). ( C ) Representative traces of the iodide-induced YFP fluorescence decay of HA-NIS/HS-YFP-TPC1 cells treated as in ( A ), or treated with 50 mM ClO 4 − for 10 min, continuously recorded as described in the legend to . The data are the means ± SEM of five independent assays. The significant variations were assessed by one-way ANOVA (F = 30.50; p < 0.001) followed by Dunnett’s post- hoc test, as compared with the control conditions (Ctrl) (*** p < 0.001).

Article Snippet: The treatment of the cells with the RAC1 selective inhibitor EHT1864 (50 μM, SCTB, Santa Cruz, CA, USA), ARP2/3 Complex Inhibitor CK666 (100 μM, Sigma-Aldrich, St. Louis, MO, USA), JNK inhibitor SP600125 (30 μM, Sigma-Aldrich, St. Louis, MO, USA), P38 inhibitor SB203580 (10 μM, SCTB, Santa Cruz, CA, USA), PAK inhibitor IPA3 (10 μM, Calbiochem), SRC Inhibitors PP2 (2 μM, Sigma-Aldrich, St. Louis, MO, USA) or Dasatinib (150 nM, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h in the appropriate medium, using cells treated with the same volume of solvent (vehicle) as a control.

Techniques: Transfection, Control, Plasmid Preparation, Incubation, Expressing, Software, Fluorescence

Journal: Cancers

Article Title: Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

doi: 10.3390/cancers13215460

Figure Lengend Snippet: RAC1 signaling through PIP5K is required for NIS PM residency. HA-NIS-TPC1 cells were ( A ) transfected with either mock (siLUC control) or a specific siRNA against PIP5K (siPIP5K), and were analyzed by surface protein biotinylation. The surface fractions and whole-cell lysates (WCL) were then analyzed by WB, as indicated. The ‘no Biotin’ condition, corresponding to cells which were not incubated with biotin, was used to control the specificity of the biotinylated protein pull-downs. PCNA expression served as both the loading (WCL) and intracellular protein contamination control (Surface pool). GLUT-1 served as the positive loading control for cell-surface protein extracts. The WB bands were quantified by densitometric analysis using ImageJ software. The plotted values correspond to the means ± SEM of at least three independent assays. Significant variations were assessed by Student’s T test (* p ≤ 0.05). ( B ) Representative traces of the iodide-induced YFP fluorescence decay of HA-NIS/HS-YFP-TPC1 cells treated as in ( A ), or treated with or 50 mM of ClO 4 − for 10 min, which were continuously recorded for 500 s after exposure to 50 mM iodide (upper graph). The iodide influx rates (lower graph) were calculated by fitting the curves to the exponential decay function. The data are means ± SEM of five independent assays. Significant variations between the treatments were assessed by one-way ANOVA (F = 120.8; p < 0.001) followed by Dunnett’s post- hoc test, as compared with the control conditions (Ctrl) (*** p ≤ 0.001). ( C ) HA-NIS-TPC1 cells transfected with either empty vector (Empty) or GFP-RAC1-L61, and either mock (siLUC control) or the siPIP5K, as indicated, were analyzed by cell surface protein biotinylation followed by WB, as in ( A ). The plotted values correspond to the means ± SEM of at least three independent assays. One-way ANOVA analysis detected significant differences between the treatments (F = 18.49; p < 0.001). Post-hoc Tukey’s tests were used to identify significant variations relative to the control conditions or among the different treatments (the latter are indicated by horizontal lines) (* p ≤ 0.05).

Article Snippet: The treatment of the cells with the RAC1 selective inhibitor EHT1864 (50 μM, SCTB, Santa Cruz, CA, USA), ARP2/3 Complex Inhibitor CK666 (100 μM, Sigma-Aldrich, St. Louis, MO, USA), JNK inhibitor SP600125 (30 μM, Sigma-Aldrich, St. Louis, MO, USA), P38 inhibitor SB203580 (10 μM, SCTB, Santa Cruz, CA, USA), PAK inhibitor IPA3 (10 μM, Calbiochem), SRC Inhibitors PP2 (2 μM, Sigma-Aldrich, St. Louis, MO, USA) or Dasatinib (150 nM, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h in the appropriate medium, using cells treated with the same volume of solvent (vehicle) as a control.

Techniques: Transfection, Control, Incubation, Expressing, Software, Fluorescence, Plasmid Preparation

Journal: Cancers

Article Title: Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

doi: 10.3390/cancers13215460

Figure Lengend Snippet: SRC functions upstream of RAC1 to promote NIS residency at the PM. ( A ) HA-NIS-TPC1 were treated with either the vehicle or one of two SRC inhibitors, PP2 (2 µM for 1 h) or Dasatinib (150 nM for 1 h), and the endogenous RAC1 activation status was assessed by monitoring the levels of active, GTP-bound RAC1 with CRIB-domain pull-down assays. The endogenous levels of the total (input) and GTP-bound RAC1 in the pulled-down fraction were assessed by WB using an anti-RAC1 antibody. The total lysates were further probed for tubulin as the loading control. Plotted is the densitometry analysis of the WB bands (means ± SEM of three independent assays), using ImageJ software. One-way ANOVA analysis detected significant differences between the treatments (F = 19.86; p = 0.0186). Post-hoc Dunnett’s tests were used to identify significant variations relative to the control (Ctrl) conditions (* p ≤ 0.05; ** p ≤ 0.01). ( B ) HA-NIS-TPC1 cells treated as in ( A ) were analyzed by surface protein biotinylation. The surface fraction or correspondent whole-cell lysates (WCL) were analyzed by WB, as indicated. The ‘no Biotin’ condition, corresponding to cells that were not incubated with biotin, was used to control the specificity of the biotinylated protein pull-down. PCNA expression served as the loading (WCL) and intracellular protein contamination control (Surface pool). GLUT-1 served as the positive loading control for cell surface protein extracts. The WB bands were quantified as in ( A ), and were plotted as the means ± SEM of at least three independent assays. A one-way ANOVA analysis detected significant differences between the treatments (F = 14.22; p = 0.0086). Post-hoc Dunnett’s tests were used to identify significant variations relative to the control (Ctrl) (* p ≤ 0.05; ** p ≤ 0.01). ( C ) Representative traces of the iodide-induced YFP fluorescence decay of the HA-NIS/HS-YFP-TPC1 cells treated with either the vehicle, PP2 (2 µM for 1 h), or ClO 4 − (50 mM for 10 min), which were continuously recorded for 500 s after exposure to 50 mM iodide (upper graph). The iodide influx rates (lower graph) were calculated by fitting the curves to the exponential decay function. The data are means ± SEM of four independent assays. The significant variations between the treatments were assessed by one-way ANOVA (F = 43.61; p < 0.001) followed by Dunnett’s post- hoc test, as compared with the control conditions (Ctrl) (** p ≤ 0.01; *** p < 0.001). ( D ) PCCL3 cells stably expressing the HS-YFP iodide sensor were serum-starved for 24 h, followed by stimulation or not with TSH (1 mU/mL for 48 h). The cells were then treated for 1 h with either vehicle, PP2 (2 µM), IPA3 (10 µM), or CK666 (100 µM), or for 10 min with 1 mM of ClO 4 − , and the YFP fluorescence was recorded continuously, as described in the legend to . The data are the means ± SEM of at least three independent assays. A one-way ANOVA analysis detected significant differences between the treatments (F = 10.61 and p < 0.001). Post-hoc Dunnett’s tests were used to identify significant variations relative to the control (Ctrl) conditions (** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: The treatment of the cells with the RAC1 selective inhibitor EHT1864 (50 μM, SCTB, Santa Cruz, CA, USA), ARP2/3 Complex Inhibitor CK666 (100 μM, Sigma-Aldrich, St. Louis, MO, USA), JNK inhibitor SP600125 (30 μM, Sigma-Aldrich, St. Louis, MO, USA), P38 inhibitor SB203580 (10 μM, SCTB, Santa Cruz, CA, USA), PAK inhibitor IPA3 (10 μM, Calbiochem), SRC Inhibitors PP2 (2 μM, Sigma-Aldrich, St. Louis, MO, USA) or Dasatinib (150 nM, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h in the appropriate medium, using cells treated with the same volume of solvent (vehicle) as a control.

Techniques: Activation Assay, Control, Software, Incubation, Expressing, Fluorescence, Stable Transfection

Journal: Cancers

Article Title: Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

doi: 10.3390/cancers13215460

Figure Lengend Snippet: Diagram depicting the current model for the regulation of NIS functional residency at the PM by the SRC/RAC1/PIP5K/PAK/EZRIN/NIS pathway. In brief, NIS localization and function at the PM depends on its binding to SRC kinase, the activity of which, triggered by yet-unknown factors, leads to the recruitment and activation of the small GTPase RAC1. RAC1 signals not through p38 nor through JNK (two kinases downstream of RAC1 known to participate in the transcriptional regulation of NIS [ , ]), but through PAK1 and PIP5K. The combined activity of these kinases promotes ARP2/3-mediated actin polymerization, and the recruitment and binding of the actin anchoring protein EZRIN to NIS, promoting its residency and function at the PM of normal and TC cells. The figure was created with BioRender.com , accessed on 26 July 2021.

Article Snippet: The treatment of the cells with the RAC1 selective inhibitor EHT1864 (50 μM, SCTB, Santa Cruz, CA, USA), ARP2/3 Complex Inhibitor CK666 (100 μM, Sigma-Aldrich, St. Louis, MO, USA), JNK inhibitor SP600125 (30 μM, Sigma-Aldrich, St. Louis, MO, USA), P38 inhibitor SB203580 (10 μM, SCTB, Santa Cruz, CA, USA), PAK inhibitor IPA3 (10 μM, Calbiochem), SRC Inhibitors PP2 (2 μM, Sigma-Aldrich, St. Louis, MO, USA) or Dasatinib (150 nM, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h in the appropriate medium, using cells treated with the same volume of solvent (vehicle) as a control.

Techniques: Functional Assay, Binding Assay, Activity Assay, Activation Assay