eht1864  (MedChemExpress)

Journal: Science Advances

Article Title: Formation control between leader and migratory follower tissues allows coordinated growth

doi: 10.1126/sciadv.ads2310

Figure Lengend Snippet: ( A ) Yap protein distribution at 26 ss. Nuclear localization in FP and HC cells (dashed lines) is indicated by filled and open arrowheads, respectively. ( B ) Scatter plot showing the Yap nuclear-to-cytoplasmic ratio in FP (red, n = 97 cells) and HC (magenta, n = 58 cells) cells along the anterior-posterior axis with corresponding regression lines. ( C ) Scatter plots showing cell length versus Yap nuclear-to-cytoplasmic ratio in FP ( n = 50 cells) and HC ( n = 28 cells). Pearson’s correlation coefficient r = 0.232 (H, P = 0.106) and 0.514 (I, P = 0.005). ( D ) EdU staining of FP cells of 26-ss embryos injected with control or yap morpholino oligos. Arrowheads indicate EdU-positive cells. ( E ) EdU-positive fractions of FP and HC cells in control ( n = 8 embryos) and yap morphants ( n = 9 embryos). ( F ) Lengths of FP and HC cells in control ( N = 4 embryos; n = 111 cells for FP, 36 cells for HC) and yap morphants ( N = 4 embryos; n = 89 cells for FP, 19 cells for HC). ( G ) Dorsal view of a sox2:sox2-2a-sfGFP yap morphant embryo showing HC cells. Arrowheads indicate cell gaps. ( H ) Yap nuclear-to-cytoplasmic ratio of FP cells when treated with DMSO ( N = 7 embryos, n = 182 cells), NSC23766 ( N = 8 embryos, n = 210 cells), EHT1864 ( N = 6 embryos, n = 159 cells), or CK666 ( N = 7 embryos, n = 167 cells). ( I ) EdU-positive fractions of FP cells in the DMSO-treated ( n = 9 embryos) and blebbistatin-treated ( n = 8) embryos. Anterior to the left. FP, floorplate; HC, hypochord; NT, neural tube. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bars, 20 μm [(A) and (D)] and 50 μm (G).

Article Snippet: Dechorionated embryos at 12 ss were soaked in 1/3× Ringer’s solution containing 400 μM NSC23766 (SML0952, Sigma-Aldrich, 50 mM stock in DMSO), 100 μM EHT1864 (S7482, Selleck, 50 mM stock in DMSO), or 500 μM CK666 (ab141231, Abcam, 100 mM stock in DMSO) and incubated at 28°C for 4 hours until fixation with 4% PFA in PBS.

Techniques: Staining, Injection, 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 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