Journal: Thorax

Article Title: Essential role of smooth muscle Rac1 in severe asthma-associated airway remodelling

doi: 10.1136/thoraxjnl-2020-216271

Figure Lengend Snippet: Rac1 inhibition reduces bFGF-induced and PDGFbb-induced aSMCs proliferation. (A) Representative images of airway smooth muscle cell (aSMC) proliferation from control and severe asthmatics induced by bFGF and PDGFbb, in the absence and in the presence of the Rac1 inhibitor, EHT1864. Nuclei are detected by 4′,6-diamidino-2-phenylindole staining (blue) and aSMC proliferation by EdU staining (green). Scale bar, 25 µm. (B, C) Quantification of aSMC proliferation by EdU staining in the absence (B) and in the presence (C) of EHT1864. The results are expressed as the percentage of cell proliferation (EdU-positive cells) (mean±SEM of n=3 independent experiments). Kruskal-Wallis test followed by Dunns’ post-test were used. *P<0.05, **p<0.01 versus untreated cells from control subjects; $ P<0.05 versus untreated cells from patients with severe asthma; # p<0.05 versus cells from control subjects.

Article Snippet: When indicated, human aSMCs were treated with the Rac inhibitor, EHT1864 (10 −5 M; Tocris Bioscience), P21-activated kinases (Pak) inhibitor IPA3 (10 −5 M; Tocris Bioscience), Akt inhibitor VIII (10 −5 M; Calbiochem), MEK inhibitor PD98059 (10 −5 M; ThermoFisher), JAK inhibitor ruxolitinib (10 −5 M; InvivoGen) added 30 min before stimulation with bFGF (25 ng/mL; Miltenyi Biotech), PDGF-bb (25 ng/mL; Miltenyi Biotech), interleukins (IL)-13 (10 ng/mL; Miltenyi Biotech), IL-33 (10 ng/mL; Miltenyi Biotech), IL-17 (20 ng/mL; Miltenyi Biotech), IL-9 (10 ng/mL; Miltenyi Biotech) or TSLP (10 ng/mL; Miltenyi Biotech) for 48 hours.

Techniques: Inhibition, Control, Staining

Journal: Thorax

Article Title: Essential role of smooth muscle Rac1 in severe asthma-associated airway remodelling

doi: 10.1136/thoraxjnl-2020-216271

Figure Lengend Snippet: Role of Rac1/P21-activated kinases (Pak1) in bFGF-induced activation of Akt-dependent signalling pathway. (A) Immunoblot analysis and corresponding quantification of Pak and STAT3 expression and phosphorylation in control airway smooth muscle cell (aSMCs) stimulated with bFGF or PDGFbb at different time points, in the absence, or in the presence of EHT1864 (n=4–5 independent experiments). (B) Control aSMC proliferation induced by bFGF and PDGFbb, in the absence and in the presence of inhibitors of PAK (IPA3), Akt (Akt Inhib VIII), P44/42 (PD98059) or JaAK2 (ruxolitinib). Nuclei are detected by 4′,6-diamidino-2-phenylindole staining (blue) and haSMC proliferation by EdU staining (green). Scale bar, 25 µm. Quantification of aSMC proliferation by EdU staining. The results are expressed as the percentage of EdU-positive cells. (n=3–4 independent experiments). Data are presented as mean±SEM. Kruskal-Wallis test followed by Dunns’ post-test were used. *P<0.05, **p<0.01, ***p<0.001 versus untreated cells; $$ p<0.01, $$$ p<0.001 versus bFGF treated cells; ## p<0.01, ### p<0.001 versus PDGFbb treated cells.

Article Snippet: When indicated, human aSMCs were treated with the Rac inhibitor, EHT1864 (10 −5 M; Tocris Bioscience), P21-activated kinases (Pak) inhibitor IPA3 (10 −5 M; Tocris Bioscience), Akt inhibitor VIII (10 −5 M; Calbiochem), MEK inhibitor PD98059 (10 −5 M; ThermoFisher), JAK inhibitor ruxolitinib (10 −5 M; InvivoGen) added 30 min before stimulation with bFGF (25 ng/mL; Miltenyi Biotech), PDGF-bb (25 ng/mL; Miltenyi Biotech), interleukins (IL)-13 (10 ng/mL; Miltenyi Biotech), IL-33 (10 ng/mL; Miltenyi Biotech), IL-17 (20 ng/mL; Miltenyi Biotech), IL-9 (10 ng/mL; Miltenyi Biotech) or TSLP (10 ng/mL; Miltenyi Biotech) for 48 hours.

Techniques: Activation Assay, Western Blot, Expressing, Phospho-proteomics, Control, Inhibition, Staining

Journal: Journal of Molecular Endocrinology

Article Title: Antagonistic effects of RAC1 and tumor-related RAC1b on NIS expression in thyroid

doi: 10.1530/jme-19-0195

Figure Lengend Snippet: Figure 3 Impact of NSC23766- and EHT1864-mediated endogenous RAC1 inhibition on NIS transcriptional expression. FRTL5 and PCCL3 were subjected to a 24-h starvation period (TSH minus) followed by stimulation with TSH (1 mU/mL for 24 h; TSH plus), in the presence or absence of either NSC23766 (100 μM for 4 h) or EHT1864 (50 μM for 4 h). (A) Effect of NSC23766- and EHT1864-mediated endogenous RAC1 inhibition on NIS transcript levels. Levels of NIS mRNA quantified by RT‐qPCR correspond to arbitrary units representing fold differences relative to a reference sample and corrected to HPRT levels used as endogenous control gene. Plotted values are the mean ± s.d. (error bars) of three independent assays, compared with the group treated with only TSH (TSH plus). Comparisons were made using a two-tailed Student’s t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Inhibition of endogenous RAC1 activation upon NSC23766 (B), EHT1864 (C) or TSH (D) treatment was assessed by monitoring the active GTP-bound fraction of RAC1 by PAK-CRIB domain pull-down assay. RAC1 levels were assessed by Western Blot using anti-RAC1 primary antibody.

Article Snippet: Treatment of cells with the RAC1 selective inhibitors NSC23766 (100μM, Santa Cruz) or EHT1864 (50 μM, Santa Cruz) was performed for 4 h in the appropriate supplemented medium.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Control, Two Tailed Test, Activation Assay, Pull Down Assay, Western Blot

Journal: Journal of Molecular Endocrinology

Article Title: Antagonistic effects of RAC1 and tumor-related RAC1b on NIS expression in thyroid

doi: 10.1530/jme-19-0195

Figure Lengend Snippet: Figure 5 Effects of RAC1 selective inhibitors NSC23766 and EHT1864 on TSH- induced iodide uptake in PCCL3 cells. PCCL3 cells stably expressing the YFP-halide sensor were subjected to a 24-h starvation period (TSH minus) and then treated with TSH (1 mU/mL) for 96 h (TSH plus), in the presence or absence of either NSC23766 (100 μM for 4 h) or EHT1864 (50 μM for 4 h). TSH-stimulated PCCL3 cells transfected with GFP-RAC1b-expressing construct were assessed also. (A) Fluorescence decay traces and (B) initial iodide influx rates. Iodide influx assays were performed both with or without TSH stimulation (TSH plus and TSH minus, respectively) and in the presence or absence of either NSC23766- or EHT1864-induced endogenous RAC1 inhibition. Additional assays were performed in the presence of both TSH and ClO4− (1 mM, 10 min; ClO4-), a competitive inhibitor of iodide uptake by NIS, and upon ectopic RAC1b overexpression (RAC1b). YFP fluorescence was recorded continuously for 600 s, acquiring an image every 10 s, after exposure to 1mM NaI (as described in Rhoden et al. 2007). Fluorescence (F) was plotted over time as percentage of fluorescence at time 0 (F0). Iodide influx rates calculated by fitting the curves to the exponential decay function to derive the maximal slope that corresponds to initial influx of I− into the cells. Data are means ± s.e.m. of three independent assays. Comparisons were made using a two-tailed Student’s t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Treatment of cells with the RAC1 selective inhibitors NSC23766 (100μM, Santa Cruz) or EHT1864 (50 μM, Santa Cruz) was performed for 4 h in the appropriate supplemented medium.

Techniques: Stable Transfection, Expressing, Transfection, Construct, Fluorescence, Inhibition, Over Expression, Two Tailed Test

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: Macrophages (10 6 /well) were plated in 24-well plates for ELISA experiments, and supernatants were isolated after treatment with or without LPS (20 ng/ml) for 8 h. For inhibitor experiments, macrophages were pre-treated for 30 min with RAC1 inhibitor (EHT1864, Tocris), RHOA inhibitor-CCG1423 (5233, Tocris), p38 Inhibitor (SB203580, Invivogen), or Colchicine (C3915, Sigma) followed by stimulation with LPS for 6 to 8 h. For IL-1β signaling inhibition experiments, macrophages were pretreated with recombinant Mouse IL-1RA (769704, BioLegend) for 3 h prior to stimulation with LPS.

Techniques: Western Blot, Isolation, Control

Journal: Oncotarget

Article Title: Therapeutic sensitivity to Rac GTPase inhibition requires consequential suppression of mTORC1, AKT, and MEK signaling in breast cancer

doi: 10.18632/oncotarget.15586

Figure Lengend Snippet: (A) Breast cancer cells were treated with 0-100 μM EHT1864 for 4-5 d. Relative viable cell numbers were assessed by SRB assay. Mutational and DNA copy number profiles were obtained from ref. . RAC3 -amp- RAC3 gene amplification. RAC1 -mut- RAC1 N39S mutation, predicted to be low-impact per mutationassessor.org . MCF-7/FR- fulvestrant-resistant MCF-7 cells maintained and treated in 1 μM fulv. (B) Cell lysates were analyzed by immunoblot. (C) Comparison of IC 50 values between cells harboring a PIK3CA mutation and/or HER2 amplification vs. PIK3CA /HER2-wild-type cells. * p =0.015 by Mann-Whitney U-test. (D) Cells were treated with EHT for 72 h before apoptosis assay. * p <0.0001 by Bonferonni post-hoc test compared to control for each cell line unless otherwise indicated with brackets.

Article Snippet: EHT1864 [ ] was generously provided by Diaxonhit (Paris, France).

Techniques: Sulforhodamine B Assay, Amplification, Mutagenesis, Western Blot, Comparison, MANN-WHITNEY, Apoptosis Assay, Control

Journal: Oncotarget

Article Title: Therapeutic sensitivity to Rac GTPase inhibition requires consequential suppression of mTORC1, AKT, and MEK signaling in breast cancer

doi: 10.18632/oncotarget.15586

Figure Lengend Snippet: (A–B) Cells were treated with 0-50 μM EHT1864 for 3 h (A), or 10 or 50 μM EHT1864 for 0-120 min (B), and lysates were analyzed by immunoblot. Cells that are relatively sensitive vs. resistant to EHT1864 (from Figure ) are indicated. (C) Mining of sensitivity data from 656 cancer cell lines treated with a panel of 138 drugs revealed that the sensitivity profile of EHT1864 most strongly correlates with the profile of the p70S6K inhibitor PF-4708671. (D–E) In (D), activated Rac was pulled down under control-, GTPγs-, GDP-, and EHT1864-treated conditions. In (E), AKT, p70S6K, ERK, and MEK1/2 were immunoprecipitated from cell lysates. Eluates and lysates were analyzed by immunoblot.

Article Snippet: EHT1864 [ ] was generously provided by Diaxonhit (Paris, France).

Techniques: Western Blot, Control, Immunoprecipitation

Journal: Oncotarget

Article Title: Therapeutic sensitivity to Rac GTPase inhibition requires consequential suppression of mTORC1, AKT, and MEK signaling in breast cancer

doi: 10.18632/oncotarget.15586

Figure Lengend Snippet: (A–B) MCF-7 and BT-474 cells were stably transfected with vectors encoding AKT1 DD or AKT1 myr constitutively active mutants or EV, and sensitivity to EHT1864 was assessed via growth assay. In (C) lysates were analyzed by immunoblot. * p <0.05 by Bonferroni multiple comparison-adjusted post-hoc test compared to EV control at each dose of EHT.

Article Snippet: EHT1864 [ ] was generously provided by Diaxonhit (Paris, France).

Techniques: Stable Transfection, Transfection, Growth Assay, Western Blot, Comparison, Control

Journal: Oncotarget

Article Title: Therapeutic sensitivity to Rac GTPase inhibition requires consequential suppression of mTORC1, AKT, and MEK signaling in breast cancer

doi: 10.18632/oncotarget.15586

Figure Lengend Snippet: Cells were treated with 12.5, 25, or 50 μM EHT1864 for 0, 1, 2, 4, 8, 24, 48, 72, or 120 hours, then drug was washed out. Relative viable cell numbers were quantified at the 120-h time point.

Article Snippet: EHT1864 [ ] was generously provided by Diaxonhit (Paris, France).

Techniques:

Journal: Oncotarget

Article Title: Therapeutic sensitivity to Rac GTPase inhibition requires consequential suppression of mTORC1, AKT, and MEK signaling in breast cancer

doi: 10.18632/oncotarget.15586

Figure Lengend Snippet: Mice were injected i.p. with a single dose of EHT1864 (100 mg/kg), and blood was collected from 3 mice per time point over the next 24 h. Plasma was separated for EHT1864 concentration measurement. The Tmax- time to maximum concentration; Cmax- maximum concentration; were the observed mean values and the terminal elimination half-life was estimated using non compartmental analysis. (mean elimination t 1/2 = 99.2 min /1.65h). The shaded region indicates estimated time that plasma EHT1864 concentration exceeded 10 μM.

Article Snippet: EHT1864 [ ] was generously provided by Diaxonhit (Paris, France).

Techniques: Injection, Clinical Proteomics, Concentration Assay

Journal: Oncotarget

Article Title: Therapeutic sensitivity to Rac GTPase inhibition requires consequential suppression of mTORC1, AKT, and MEK signaling in breast cancer

doi: 10.18632/oncotarget.15586

Figure Lengend Snippet: (A, C) Mice bearing BT-474 tumors (A) or MCF-7 tumors (C) were randomized to drug treatments as indicated. Data are presented as % tumor volume relative to baseline (mean + SEM). (B, D, E) After 6 wk of treatment, tumors were harvested at 1 h after the final dose of EHT1864. Lysates were analyzed by immunoblot (B/D), or used for active Rac ELISA (E).

Article Snippet: EHT1864 [ ] was generously provided by Diaxonhit (Paris, France).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Cas phosphorylation regulates focal adhesion assembly

doi: 10.7554/eLife.90234

Figure Lengend Snippet: ( A ) Allosteric inhibitor EHT1864 inhibits Rac1 activation during cell attachment. FRET ratio images (left) and quantification (right) of Rac1-2G MCF10A cells treated with DMSO or EHT1864 and imaged after 30 min attachment. ( B ) Cell spreading and vinculin but not Cas recruitment requires Rac1. Images and quantification of spreading Cas mSc YFP-VCL MCF10A cells treated with DMSO or EHT1864. Error bars show mean and standard error of the mean (SEM) of 8–20 cells in three biological repeats. ( C, D ) Rac1 activation requires Cas and Yes1. FRET ratio images (left) and quantification (right) of Rac1-2G MCF10A cells treated with ( C ) control or Cas, or ( D ) control or Yes1 siRNA. Error bars show mean and SEM from >30 cells from three biological replicates. ns, not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001 by Mann–Whitney U -tests.

Article Snippet: EHT1864 , ApexBio, Cat: B5487 , 10 μM.

Techniques: Activation Assay, Cell Attachment Assay, MANN-WHITNEY

Journal: eLife

Article Title: Cas phosphorylation regulates focal adhesion assembly

doi: 10.7554/eLife.90234

Figure Lengend Snippet:

Article Snippet: EHT1864 , ApexBio, Cat: B5487 , 10 μM.

Techniques: Concentration Assay