Geldanamycin

hESC-derived NCC were exposed for 48 h to non-cytotoxic concentration of different NCC migration-inhibiting substances in N2 medium containing EGF (20 ng/ml) and FGF2 (20 ng/ml). Six compounds were used: geldanamycin (16 nM, Selleckchem), arsenic trioxide (1 µM, Sigma-Aldrich), thricostatin A (TSA, 10 nM, Sigma-Aldrich), valproic acid sodium salt (VPA, 250 µM, Sigma-Aldrich), triadimefon (100 µM, Bayer Crop Science) and pentabromodiphenyl ether (PBDE-99, 15 µM, Clickchem). Two different solvent control groups were also produced: NCC were exposed to 0.04 % DMSO or simply to N2 medium for 48 h. Finally, a third control group (exposed to N2 medium only) was added specifically as control of arsenic trioxide, since the testing of this substance was performed not in parallel with the other compounds.

Cycloheximide (CHX), 17-(allylamino)-17-demethoxyGeldanamycin (17-AAG), and Lubrol-PX were purchased from Sigma-Aldrich (St. Louis, MO). MG-132 was obtained from Boston Biochem (Cambridge, MA). Geldanamycin was purchased from Selleck Chemicals (Houston, TX). Lipofectamine 2000 was purchased from Invitrogen (St. Louis, MO). Antibodies were purchased from Sigma (FLAG, Clone M2, F3165); ZYMED (HSP90, 37–9400); Torrey Pines Biolabs (GFP, TP401); Santa Cruz (HSP70, sc-24; anti-HA, Clone 12CA5, sc-57592); Novus (β-actin, NB600-501); Invitrogen (anti-Myc, Clone 9E10, 13–2500); Abcam (anti-CHIP, ab2917) and Dako (anti-ubiquitin, Z045801). Alexa Fluor® 594- and Alexa Fluor® 488-conjugated secondary antibodies were purchased from Invitrogen (Carlsbad, CA), and HRP-conjugated antibodies were purchased from Amersham Biosciences.