Rocaglamide

Dichloromethane (DCM), 4-methylmorpholine N-oxide (NMO), tetrapropylammonium perruthenate (TPAP), 4-(dimethylamino)pyridine (DMAP), ethanol, NaBH_4, NH₄Cl, MgSO_4, daunomycin, vitamin C, hydrogen peroxide and HCl were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rocaglamide was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Bradford protein assay kit, Supersignal Femto LumiGLO kit and human recombinant tumor necrosis factor α (TNF-α) were obtained from Thermo Scientific (Rockford, IL, USA). Lithium dodecyl sulfate sample loading buffer (LDS), Nu-PAGE 10% SDS-PAGE Bis-Tris gel, SeeBlue® Plus2 Pre-Stained Standard Ladder, and Purelink RNase A were obtained from Invitrogen (Carlsbad, CA, USA). Primary antibodies (anti-NF-κBp65 and p50, anti-IKKα, and anti-IKKβ) were purchased from Cell Signaling Technologies (Beverly, MA, USA). Anti-rabbit horseradish peroxidase (HRP)-conjugated antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). FeSO₄ was obtained from Fischer Scientific Company (Fair Lawn, NJ). Daunorubicin was purchased from Tocris, Bristol, UK. Hydrogen peroxide was obtained from Fluka Biochemika, Steinhiem, Switzerland. Tris-buffered saline with tween-20 buffer (TBS-T), vitamin C, propidium iodine (PI), fluorescent probe 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) were obtained from Sigma Aldrich (St. Louis, MO, USA).

The NF-κB p65 assay was performed according to a previously established protocol^{15 (link)}. Nuclear extracts were prepared from HeLa cells and the Transcription Assay System (Pierce Biotechnology, Rockford, IL) was used to evaluate the binding affinity of the NF-κB subunit p65 to the biotinylated consensus sequence. Luminescence was detected using a Fluostar Optima plate reader (BMG Labtech Inc, Durham, NC). Rocaglamide (Enzo Life Sciences, Inc., Farmingdale, NY, USA) was used as a positive control.

The indole alkaloid Hap H was isolated from Fischerella ambigua as previously described (11 (link)). Reference compounds were obtained from different sources. Rocaglamide was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Daunorubicin was purchased from Tocris, Bristol, UK. Staurosporine was obtained from Cayman Chemical (Ann Arbour, MI, USA). Taxol was obtained from Tocris Bioscience, Bristol, UK. Hydrogen peroxide was obtained from Fluka Biochemika, Steinhiem, Switzerland.