FIG. 2.

Characterization of GMX1778 cytotoxicity. (A) HeLa cells are protected from GMX1778 cytotoxicity by NA. HeLa cells were exposed to GMX1778 in the presence or absence of 10 μM NA. Viability was measured after 72 h and is presented as means ± standard deviations. The inset shows cellular NAD⁺ levels from HeLa cells treated with 100 nM GMX1778. Cellular NAD⁺ levels were measured by LC/MS and are presented as means ± standard deviations. (B) NA (10 μM) rescue of GMX1778 cytotoxicity also rescues TNF-α-induced NF-κB activity. HeLa cells were exposed to GMX1778 in the presence or absence of NA. NFκB transcriptional activity was measured by luminescence generated from expression of a reporter gene and is presented as means ± standard deviations. (C) NA rescues GMX1778-mediated inhibition of IκBα phosphorylation. IκBα phosphorylation was induced by a 5-min stimulation with TNF-α following treatment of cells with bortezomib (1 h) and 100 nM GMX1778 (30 h) with or without 10 μM NA. (D) GMX1778 cytotoxicity is rescued by NMN. HeLa cells were exposed to GMX1778 in the presence or absence of 100 μM NMN. Viability was measured after 72 h and is presented as means ± standard deviations. The inset shows cellular NAD⁺ levels from HeLa cells treated with 100 nM GMX1778. Cellular NAD⁺ levels were measured by LC/MS and are presented as means ± standard deviations. (E) Yeast nicotinamidase (PNC1) expression rescues mammalian cells from GMX1778 cytotoxicity. HeLa cells were transiently transfected with vector carrying FLAG-PNC1 or empty vector and challenged by exposure to GMX1778. Viability after 72 h is presented as means ± standard deviations. Left inset, Western blot of 25 μg of cell extract from cells transfected with either vector or PNC1 as indicated and probed with anti-FLAG. Right inset, cellular NAD⁺ levels from transfected cells treated with 100 nM GMX1778. Cellular NAD⁺ levels were measured by LC/MS and are presented as means ± standard deviations.