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2 protocols using mg624

1

Cell Line Characterization and Culture

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Human cancer or immortal cell lines (A549, BEAS-2B, U87MG, MCF-7, MDA-MB-231, 293T) were purchased from the ATCC, which characterizes them using cytogenetic analysis. We have not authenticated these cell lines. A549, U87MG, MCF-7, MDA-MB-231 and 293T cells were cultured in DMEM medium with 10% fetal bovine serum (FBS). BEAS-2B cells were cultured in RPMI1640 medium with 10% FBS. All media were supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. All cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C. BCX treatments were carried out at a series of concentrations (0 to 4 μM) and incubated for different times, as specified. MG624 and PNU282987 were purchased from Tocris Bioscience. Rac1 vectors were purchased from GE Healthcare and ARF6 vectors were purchased from OriGene Technologies. PDGF, PMA, LY294002, tetramethyl rhodamine isothiocyanate (TRITC)-labeled phalloidin and all other reagents and chemicals were purchased from Sigma.
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2

Synthesis and Characterization of Bis(heptyl)-Cognitin

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Bis(heptyl)-cognitin was synthesized as previously described by us11 (link). The purity of bis(heptyl)-cognitin was evaluated by using liquid chromatography-mass spectrometry. Bis(heptyl)-cognitin was dissolved in Milli-Q water at a concentration of 1 mM and stored frozen at −20 °C. Before being used, bis(heptyl)-cognitin was further diluted with Milli-Q water. Donepezil, tacrine, methyllycaconitine (MLA) and hexafluoroisopropanol (HFIP) were purchased from Sigma (St Louis, MO, USA). Curcumin, KT5720, MG624 and H89 were purchased from Tocris (Bristol, UK). Curcumin, Donepezil, KT5720, MG624 and H89 were dissolved in dimethyl sulfoxide (DMSO) with a maximum final concentration of 0.1% (DMSO). Other chemicals were prepared in Milli-Q water. All media and supplements used for cell culture were from Invitrogen (Carlsbad, CA).
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