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