Journal: bioRxiv

Article Title: Essential role of an acidic endosomal environment in JAK3-mediated signal transduction

doi: 10.1101/2023.09.07.556780

Figure Lengend Snippet: (A–C) TPA-MAT cells were pre-incubated with endocytosis inhibitors Dynasore (80 μM), IPA3 (20 μM), or EHT1864 (10 μM), for 30 minutes in serum-free medium and stimulated with 50 ng/mL IL-2 for 10 minutes. The phosphorylation levels of (A) STAT5 and (B) all components of the IL-2 signal complex were assessed by western blotting. (C) The cell lysates were immunoprecipitated with an anti-γc antibody and coprecipitates were analyzed by western blotting. All data are representative of at least three independent experiments.

Article Snippet: After a five-minute incubation, the media were replaced with 3 mL of RPMI-1640 containing 5 μM mercaptoethanol and 80 μM Dynasore (Cayman Chemical, Cat. # 14062), 20 μM IPA3 (Cayman Chemical, Cat. # 14759), 10 μM EHT1864 (Cayman Chemical, Cat. # 17258), or 0.1% DMSO and pre-incubated for 30 minutes at 37°C.

Techniques: Incubation, Western Blot, 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) 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