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: Nature Communications

Article Title: Excessive mechanical loading promotes osteoarthritis through the gremlin-1–NF-κB pathway

doi: 10.1038/s41467-019-09491-5

Figure Lengend Snippet: Gremlin-1 induction by mechanical stress loading occurs through Rac1 activation. a Gremlin-1 mRNA levels in mouse primary chondrocytes treated with 10 µM inhibitors of FAK (PF-573228), ROCK (Y-27632), or RAC1 (NSC23766) 24 h after cyclic tensile strain loading. n = 3 biologically independent samples. * P < 0.05, # P < 0.005 versus stress+, vehicle (one-way ANOVA test). b Gremlin-1 mRNA levels in mouse primary chondrocytes treated with Rac1 inhibitors NSC23766 or EHT1864 at 24 h after cyclic tensile strain loading. n = 3 biologically independent samples. * P < 0.05, # P < 0.005, ## P < 0.001 versus stress + , vehicle (one-way ANOVA test). c Rac1 pull-down activation assay using mouse primary chondrocytes with or without cyclic tensile strain loading. Quantification of densitometry data are shown below, and ratios of positive Rac1 per total Rac1 are shown as fold-increase in the right graph. n = 5 biologically independent samples. * P < 0.05 versus stress– (Student’s unpaired two-tailed t -test). d Gremlin-1 mRNA levels in mouse primary chondrocytes transduced with an adenoviral vector containing Rac1 or GFP . n = 3 biologically independent samples. * P < 0.05 versus GFP (Student’s unpaired two-tailed t -test). e Gremlin-1 mRNA levels in ATDC5 cells lentivirally overexpressing Rac1 or GFP . n = 3 biologically independent samples. * P < 0.05 versus GFP (Student’s unpaired two-tailed t -test). All data are expressed as means ± SD

Article Snippet: In some experiments, cells were pretreated with recombinant human gremlin-1 (rhGREM1, #120-42, PeproTech for Figs. , , , , Supplementary Figs. , – , , and ; 5190-GR, R&D Systems for Supplementary Fig. ), recombinant human BMP-2 (rhBMP2, #120-02, PeproTech), recombinant human BMP-4 (rhBMP4, #120-05, PeproTech), recombinant human BMP-7 (rhBMP7, #120-03, PeproTech), IKK inhibitor BMS-345541 (B9935, Sigma), selective VEGFR2 inhibitor SU5416 (ab145056, Abcam), FAK inhibitor PF-573228 (S2013, Selleck Chemicals), ROCK inhibitor Y-27632 (257-00511, WAKO), Rac1 inhibitor NSC23766 (SML0952, Sigma) and/or EHT1864 (S7482, Selleck Chemicals).

Techniques: Activation Assay, Two Tailed Test, Transduction, Plasmid Preparation

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