17 dimethylaminoethylamino 17 demethoxygeldanamycin 17 dmag

In these studies, the HER2-specific Affibody® molecule with albumin binding domain, ABD-(Z_HER2:342)₂-Cys, was used which was kindly provided by Affibody Co. through our Cooperative Research and Development Agreement (CRADA). The ABD-HER2-Affibody was conjugated with AlexaFluor 750 with malemide linker (Invitrogen, Eugene, OR). Detail of the conjugation process can be found in (26 (link)). 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) (InvivoGen, San Diego, CA), which is known as a heat shock protein 90 (HSP90) inhibitor, was used as therapeutic agent. It is known from the literature (34 (link)-36 (link)) that this drug (like a similar inhibitor 17-AAG) results in HER2 degradation due to HSP90 inhibition.

Coelenterazine was purchased from Nanolight Technology, dissolved in ethanol as 5 mg/mL stock. All of the animal cell culture media, FBS, the antibiotics streptomycin and penicillin (P/S), 4% to 12% gradient SDS-PAGE gels were purchased from Invitrogen. EnduRen Live Cell Substrate was purchased from Promega. 17-(Dimethylaminoethylamino)-17-demethoxy-geldanamycin (17-DMAG) and puromycin hydrochloride (100 mg/mL) were purchased from Invivogen. The purine-scaffold Hsp90i PU-H71 was synthesized and dissolved previously reported (26 -28 (link)). Valproic acid sodium salt (VPA) was dissolved in distilled water as 2 mol/L stock. APHA compound 8 and trichostatin A were dissolved in DMSO (1 mg/mL). Tubacin, WT161, and MS-275 were synthesized at Broad Institute as previously described (3 (link), 29 (link), 30 (link)) and dissolved in DMSO as 20 mmol/L stock. All chemical reagents and solvents were purchased from Sigma-Aldrich, Acros, Novabiochem. The carrier controls that were used matched that of the highest concentration used for dilution of the compounds (100% PBS, 0.3% DMSO, and 90% β-cyclodextran: 10% DMSO for 1A12, 0.01% of PBS for PU-H71; 0.1% for 17-DMAG; 0.1% for WT161; 0.1% for Tubacin; 0.1% for APHA8; 0.1% MS275) to account for their biologic effects.

FITC conjugated geldanamycin (GA-FITC), Rhodamine, biotin-conjugated geldanamycin (Biotin-GA), 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), and 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) were purchases from Invivogen (San Diego, CA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO). The anti-6x His antibody, anti-HSP90 antibody, anti-AKT antibody, anti-pAKT antibody, streptavidin-magnetic beads, and all cell culture supplies were purchased from Life Technologies (Invitrogen, Grand Island, NY). All samples for flow cytometry (FACSCanto; BD bioscience, Heidelberg, Germany) and confocal microcopy (CLSM; Nikon A1R confocal microscope) were analyzed in a core facility at the University of Utah (Salt Lake City, UT).