Journal: Nature Communications

Article Title: Efficient cellular transformation via protein delivery through the protrusion-derived extracellular vesicles

doi: 10.1038/s41467-025-66351-1

Figure Lengend Snippet: A Schematic representation of the role of EVs in the FBS on cell migration. FBS was heat-inactivated as a conventional treatment for cell culture, and EVs in it were depleted by ultracentrifugation. Cell migration was measured by the wound healing assay. B Cell migration during the wound healing of WT or CIP4 KO PANC-1 cells in the presence of FBS (Top) or EV-depleted FBS (Bottom). Cell edges at 0 h (dotted lines) and 12 h (solid lines) are shown. Scale bars, 100 μm. C Cell migration in the presence of FBS, EV-depleted FBS, or EV-depleted FBS supplemented with l-EV fraction from FBS (4 × 10 6 cells were treated with 6 × 10 8 EVs/ml in 1 ml). Dynamin inhibitor dynasore was applied at 40 μM. Cell migration areas are the wounded areas occupied by migrated cells after 12 h. Data show the means ± SD from 4 independent experiments. D Illustration of Rac1-containing EVs from HEK293 cell culture medium. E Cell migration in the presence of l-EV fraction from MIM I-BAR-expressing HEK293 cells (4 × 10 6 cells were treated with 1.4 × 10 9 EVs/ml in 1 ml). Dynasore was applied at 40 μM. Data show the means ± SD from 4 independent experiments. F Western blotting of Rac1 in the l-EV fractions of GFP or MIM I-BAR-expressing HEK293 cells and EV fractions from FBS. The number of EVs per lane was indicated. Representative blots from three technical replicates using the same lot of FBS are shown. G Western blotting of MIM in the EV fractions from FBS. A representative blot from the three technical replicates using the same lot of FBS is shown. H Cell migration under the l-EV fraction from FBS treated with the Rac1 inhibitor EHT1864 at 20 μM. 4 × 10 6 cells were treated with 6 × 10 8 EVs/ml in 1 ml. Data show the means ± SD from 3 independent experiments. Statistical significance was performed by one-way ANOVA with Tukey’s honestly significant difference (HSD) test. Source data are provided as a file.

Article Snippet: To inhibit Rac1 in EVs, EHT1864 (TargetMol, #T6483) was added to the l-EV fraction from FBS at a final concentration of 20 μM, and the solution was incubated for 45 min at 37 °C.

Techniques: Migration, Cell Culture, Wound Healing Assay, Expressing, Western Blot

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: iScience

Article Title: Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

doi: 10.1016/j.isci.2024.110139

Figure Lengend Snippet: Mammalian Rac and Rac/Cdc42 inhibitors suppress conidial germination in Trichophyton rubrum (A) Structures of Rac inhibitors EHT1864 and NSC23766 and Rac/Cdc42 inhibitor AZA1. (B) Effects of mammalian small GTPase inhibitors on conidial germination in T. rubrum were observed. Conidia were stained with calcofluor white. n = 3 each. ∗∗, p < 0.01; ∗∗∗, p < 0.001. Mean ± SD. The lower panel showed representative fungal cell. (C) Effects of mammalian small GTPase inhibitors on mycelial growth in T. rubrum were observed. (D) Effects of mammalian Rac and Rac/Cdc42 inhibitors EHT1864, AZA1, and NSC23766 on conidial germination in T. rubrum were observed. Conidia were stained with calcofluor white. n = 3 each. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. Mean ± SD.

Article Snippet: EHT1864 , Cayman Chemical Company , Cat#17258.

Techniques: Staining

Journal: iScience

Article Title: Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

doi: 10.1016/j.isci.2024.110139

Figure Lengend Snippet: Effect of mammalian Rac and/or Cdc42 inhibitors against TrCdc42, TrRac, and conidial germination in T. rubrum

Article Snippet: EHT1864 , Cayman Chemical Company , Cat#17258.

Techniques:

Journal: iScience

Article Title: Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

doi: 10.1016/j.isci.2024.110139

Figure Lengend Snippet:

Article Snippet: EHT1864 , Cayman Chemical Company , Cat#17258.

Techniques: Virus, Recombinant, Software