rac1 inhibitor eht1864  (Tocris)

Journal: Journal of Leukocyte Biology

Article Title: Nexinhib20 inhibits JFC1-mediated mobilization of a subset of CD11b/CD18 + vesicles decreasing integrin avidity, but does not inhibit Rac1

doi: 10.1093/jleuko/qiaf012

Figure Lengend Snippet: Nexinhib20 inhibits CD11b/CD18 mobilization and exocytosis but only mildly affects integrin activation. A) Schematic representation of the analysis of granule mobilization and integrin activation. B) Mobilization of the adhesion molecule CD11b from intracellular stores to the plasma membrane in human neutrophils. CD11b was detected using anti-CD11b clone M1/70 (conformation unspecific). Where indicated, neutrophils were primed with GM-CSF and stimulated with fMLF in the presence of Nexinhib20 (10 µM), the Rac1 activator ML099 (10 µM), the Rac1 inhibitor EHT1864 (10 µM) or vehicle (DMSO). C and D) Effect of Nexinhib20 or Rac1 modulators on integrin activation in human neutrophils. Neutrophils were treated with inhibitors or vehicle and stimulated as in B) and integrins were detected using either the anti-CD18 monoclonal antibody, clone m24 C), or the anti-CD11b antibody clone CBRM1/5 D), which detects their respective active conformations, by flow cytometry. B to D), Neutrophils from healthy donors were treated with GM-CSF (10 ng/ml for 30 min) and fMLF (1 µM for 10 min) or vehicle (unstimulated), in 3 independent experiments. E to G) Mobilization of CD11b E) and integrin activation F and G) in response to IL-8. H) Mobilization of CD11b from intracellular stores to the plasma membrane in Jfc1 -KO neutrophils. I and J) Effects of Nexinhib20 on CD11b mobilization I) and azurophilic granule secretion (CD63) J) in murine neutrophils. B to G) Mean ± SEM, n = 6 to 9 independent donors. Relative MFI represents MFI in human or mouse samples related to the nonstimulated DMSO control or nonstimulated WT control. B to J) One-way ANOVA or Wilcoxon signed rank test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant.

Article Snippet: The Rac1 inhibitor EHT1864 was purchased from MedChemExpress (cat# HY-16659).

Techniques: Activation Assay, Membrane, Flow Cytometry, Control

Journal: Journal of Leukocyte Biology

Article Title: Nexinhib20 inhibits JFC1-mediated mobilization of a subset of CD11b/CD18 + vesicles decreasing integrin avidity, but does not inhibit Rac1

doi: 10.1093/jleuko/qiaf012

Figure Lengend Snippet: Nexinhib20 inhibits JFC1 recruitment to CD11b + vesicles in human neutrophils. A) 3D enhanced resolution microscopy analysis of endogenous JFC1, CD11b, and Rac1-GTP in human neutrophils. Where indicated, the cells were treated with Nexinhib20 (10 µM) or vehicle (DMSO) and subsequently primed with GM-CSF (10 ng/ml for 30 min) followed by stimulation with formylated peptide fMLF (1 µM for 10 min). Scale bar = 3 µm. B and C) Super-plot analyses of the colocalization of JFC1 B) or Rac1 C) with CD11b. Large dots represent the average value from each independent donor ( n = 5 to 6), and individual cells are represented as small circles, color-coded for each donor. B) Analysis of the colocalization of JFC1 at CD11b + intracellular organelles. NEI20, Nexinhib20. C) Analysis of the localization of Rac1 at CD11b + vesicles under the experimental conditions described in A and B). D) Quantitative analysis of Rac1-GTP expression by fluorescence intensity, showing that the endogenous levels of Rac1-GTP do not change upon Nexinhib20 treatment. Large dots represent the average value from each independent donor, and individual cells are represented as small circles. Mean ± SEM; ns, not significant. * P < 0.05; ** P < 0.01 by 1-way ANOVA followed by Tukey's multiple comparison test; ns, not significant.

Article Snippet: The Rac1 inhibitor EHT1864 was purchased from MedChemExpress (cat# HY-16659).

Techniques: Microscopy, Expressing, Fluorescence, Comparison

Journal: Journal of Leukocyte Biology

Article Title: Nexinhib20 inhibits JFC1-mediated mobilization of a subset of CD11b/CD18 + vesicles decreasing integrin avidity, but does not inhibit Rac1

doi: 10.1093/jleuko/qiaf012

Figure Lengend Snippet: Nexinhib20 inhibits Rab27a-JFC1 binding but not Rac1-GTP-PAK1 interaction. A) Schematic representation of the TR-FRET binding reaction between Rac1 and PAK1. Cell lysates expressing myc-PAK1 and DN-EGFP-Rac1 (T17N) or CA-EGFP-Rac1 (Q61L) were incubated in the presence of terbium-conjugated anti-myc antibody. The samples were excited at 340 nm, and TR-FRET was measured by detecting GFP emission at 520 nm. Results are expressed as the emission ratio of the acceptor (GFP, 520 nm) to the donor (terbium, 490 nm, used as internal control). B) Specific signal of the myc-PAK1/EGFP-Rac1CA was inhibited by EDTA (50 mM) but not by Nexinhib20 (10 µM). Mean ± SEM, n = 3 independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001 by 1-way ANOVA followed by Tukey's multiple comparison test; ns, not significant. C) Schematic representation of the TR-FRET binding reaction of Rab27a and JFC1. Cell lysates expressing myc-JFC1 or EGFP-Rab27a were mixed and incubated as described in A). D) Specific signal of the myc-JFC1/EGFP-Rab27a was inhibited by Nexinhib20 (10 µM). Mean ± SEM from 3 independent experiments. ** P < 0.01, unpaired Student's t- test. E) Dose–response analysis of the effect of Nexinhib20 on Rac1-PAK1 binding by TR-FRET. CA, constitutively active; DN, dominant negative; ns, not significant (1-way ANOVA). F) AlphaFold2-multimer generated 3D complex structure of JFC1-Rab27a. The complex shows JFC1 in green and Rab27a in yellow. Nexihib20 is depicted using a “ball-and-stick” model, binding at the interface of JFC-Rab27a (red arrow). The AlphaFold JFC1-Rab27a complex is superimposed with the experimental crystal structure of SLP2a-Rab27a (PDB:3BC1), where SLP2a is colored orange and Rab27a crystal structure is shown in cyan. SLP2a-Rab27a bound GNP is represented using a “ball-and-stick” model (black arrow).

Article Snippet: The Rac1 inhibitor EHT1864 was purchased from MedChemExpress (cat# HY-16659).

Techniques: Binding Assay, Expressing, Incubation, Control, Comparison, Dominant Negative Mutation, Generated

Journal: EMBO Molecular Medicine

Article Title: Pyrin inflammasome-driven erosive arthritis caused by unprenylated RHO GTPase signaling

doi: 10.1038/s44321-025-00298-0

Figure Lengend Snippet: ( A , B ) Immunoblots showing levels of total RAC1 and RAC1-GTP ( A ), and total RHOA and RHOA-GTP ( B ) in LPS-stimulated BMDMs isolated from Pggt1b +/+ and Pggt1b Δ/Δ mice either treated or not with EHT1864 for 8 h. ( C ) Immunoblots showing levels of total RHOA and RHOA-GTP in lysates of Pggt1b +/+ and Pggt1b Δ/Δ BMDMs after treatment with LPS for 3 h. Actin was used as a loading control.

Article Snippet: RAC1 inhibitor-EHT1864 , Tocris , 3872.

Techniques: Western Blot, Isolation, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: The sequence of the primers used for gene expression analysis

Article Snippet: Depending on the experimental setup, treatment with the RAC1 inhibitor EHT1864 (cat n° SC-361175, Santa Cruz Biotechnology), was done as follows: 24 h after seeding in 12wells multiwell plates, cells were treated with 50mM of EHT1864 for 18 h; then the cells were processed for the following experiments.

Techniques: Sequencing, Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: RBFOX2, hnRNPM and lncH19 bind RAC1 mRNA. ( A - B ) RIP assay with anti-Fox2 and anti-hnRNPM antibodies to assess the binding of the RNABPs to RAC1 RNA in HCT-116 and SW620 cells, IgG was used as control. RAC1 levels were determined by qRT–PCR normalized with input and presented as fold enrichment in RBFOX2 or hnRNPM relative to IgG. (Normality test and subsequent t-test ( A ) or Wilcox test ( B ). ( C ) RNA pull-down with biotin-labeled lncH19 oligonucleotides (lncH19 RAP) in CRC cell lines (SW620 and HCT116) to analyze the interaction between lncH19 and RAC1 mRNA. RAC1 levels were determined by qRT–PCR and presented as fold enrichment in lncH19 samples respect to RNA pull-down obtained with scrambled oligonucleotides (Normality test and t-test). ( D ) Agarose electrophoresis of splice-sensitive PCR RAC1-RAC1B from lncH19 RAP in CRC cell lines (SW620 and HCT116). One representative experiment of three is shown. ( E ) Quantitative analysis of RAC1B levels determined by qRT–PCR and presented as fold enrichment in lncH19 samples relative to input. Statistical analyses were performed using one sample t-test, the p-value is shown in the graphs

Article Snippet: Depending on the experimental setup, treatment with the RAC1 inhibitor EHT1864 (cat n° SC-361175, Santa Cruz Biotechnology), was done as follows: 24 h after seeding in 12wells multiwell plates, cells were treated with 50mM of EHT1864 for 18 h; then the cells were processed for the following experiments.

Techniques: Binding Assay, Control, Quantitative RT-PCR, Labeling, Electrophoresis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: RBFOX2 is involved in RAC1 alternative splicing in CRC cells. ( A - B - D , E - F - H ) QRT-PCR for the indicated mRNA in CRC cells, silenced for RBFOX2 with two different siRNA. Graphs show 2-ΔΔct calculated in silenced cells respect to relative controls (Normality test and t-test). ( C , F ) Western Blot and densitometric analyses for RAC1B in SW620 and HCT116 silenced for RBFOX2 and relative controls. For densitometric analysis data are represented as normalized OD. ( I - L ) qRT-PCR of the indicated genes in SW620 and HCT116 silenced for H19, the graphs represent the 2^- ΔΔ ct of the indicated calculated respect the expression in control cells. ( M ) Western Blot of RAC1b protein levels in CRC cells (SW620 and HCT116) in H19 silenced cells and relative control cells. Statistical analyses were performed using normality test and t-test, p-value is shown in th e graphs

Article Snippet: Depending on the experimental setup, treatment with the RAC1 inhibitor EHT1864 (cat n° SC-361175, Santa Cruz Biotechnology), was done as follows: 24 h after seeding in 12wells multiwell plates, cells were treated with 50mM of EHT1864 for 18 h; then the cells were processed for the following experiments.

Techniques: Alternative Splicing, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: CRC tissues present higher levels of lncH19 compared to respective marginal non-tumor. ( A - C ) Gene expression levels for the indicated genes were examined by qRT-PCR in tumor and paired marginal non-tumor samples ( n = 20). ( D ) Pearson correlation between lncH19, RAC1, and RAC1B expression analyzed in 14 colorectal cancer samples with lncH19 overexpressed compared to marginal non tumor tissue. ( E - F ) qRT-PCR for the indicated genes in colorectal cancer samples with lncH19 levels. All statistical analyses were performed using two-tail paired t-test, p-value is shown in the graphs

Article Snippet: Depending on the experimental setup, treatment with the RAC1 inhibitor EHT1864 (cat n° SC-361175, Santa Cruz Biotechnology), was done as follows: 24 h after seeding in 12wells multiwell plates, cells were treated with 50mM of EHT1864 for 18 h; then the cells were processed for the following experiments.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: LncH19 is required to drive splicing factors on RAC1 mRNA. ( A - D ) QRT-PCR for RAC and RAC1b from RNA-immunoprecipitation (RIP) with anti-Fox2 ( A , C ) or anti-hnRNPM ( B , D ) antibodies in SW620 and HCT116 cells, RAC1 and RAC1B levels presented as fold enrichment in RBFOX2 or hnRNPM IP relative to IgG IP. Statistical analyses were performed using two-tail unpaired t-test to compare the binding between wt and shH19 silenced cells, p-value is shown in the graphs. ( E ) Schematic representation of the proposed model. Representation of binding sites position of the complex lncH19-hnRNPM-RBFOX2 with RAC1 unspliced mRNA

Article Snippet: Depending on the experimental setup, treatment with the RAC1 inhibitor EHT1864 (cat n° SC-361175, Santa Cruz Biotechnology), was done as follows: 24 h after seeding in 12wells multiwell plates, cells were treated with 50mM of EHT1864 for 18 h; then the cells were processed for the following experiments.

Techniques: Quantitative RT-PCR, RNA Immunoprecipitation, Binding Assay

Journal: Science advances

Article Title: Genetics of mirror movements identifies a multifunctional complex required for Netrin-1 guidance and lateralization of motor control.

doi: 10.1126/sciadv.add5501

Figure Lengend Snippet: Fig. 8. Arhgef7het mice have increased symmetrical paw placements. (A) Weight of control (n = 10) and Arhgef7het (n = 13) male mice. (B) Weight of control (n = 13) and Arhgef7het (n = 11) female mice. Mann-Whitney test. (C and D) The total number of errors made in the horizontal ladder rung test, for males and females. (E) Images of mice walking on the ladder rungs. Red arrows indicate the paw positions. Top: Image of a control mouse with typical asymmetrical (alternating) paw placements. Bottom: Image of an Arhgef7het mouse with symmetrical paw placements. (F and G) The number of symmetrical paw placements during the ladder rung test for males and females. (C, D, F, and G) Number of males: control (n = 6) and Arhgef7het (n = 11), and females: control (n = 10) and Arhgef7het (n = 7). Mann-Whitney, ***P < 0.001. (H) Arhgef7 forms a multifunctional effector complex required for Netrin-1–mediated axon guidance and lateralization of motor control. Arhgef7/Git1 directly bind to Dcc. Netrin-1 activates Rac1 and inactivates Arf1 in an Arhgef7/Git1-dependent manner. Arf1 inactivation increases cell surface Dcc. Arhgef7mut does not bind to Dcc and Git1. When Arhgef7mut is expressed, Netrin-1 fails to activate Rac1 and inactivate Arf1. Consequently, axon guidance is impaired resulting in lateralization defects.

Article Snippet: A similar methodology was used for the detection of activated Rac1 in conditions where cells were stimulated with Netrin-1 for 5 min and treated with the Rac1 inhibitor EHT1864 (Selleckchem, S7482; batch: 5748201).

Techniques: Control, MANN-WHITNEY

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