Figure 2.

Sorafenib-induced decrease of nicotinamide adenine dinucleotide (NAD) levels in hepatocarcinoma cells is not mediated by nicotinamide phosphoribosyltransferase (NAMPT) or sirtuin (SIRT1) NAMPT (A), and SIRT1 (D) protein were analyzed by Western blot analysis. NAMPT and SIRT1 protein were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (not shown). One representative blot out of three experiments is shown as an insert. Densitometric analysis was performed to semi-quantify values (n = 3). (B) Intracellular NAMPT enzyme activity was determined by measuring the conversion of ¹⁴C-labeled nicotinamide to ¹⁴C-NMN (n = 3). (C) Intracellular NAD levels were measured by gradient HPLC/UV analysis (n = 3). (E) Expression of miRNA34a was measured using miScript primer assays for miR34a-1 and normalized to Hs_RNU6_2_1 (n = 3). For all experiments, HUH7 and Hep3B cells were stimulated with sorafenib (1 µM; 2.5 µM, and 5 µM) for 24 h. The mean value of serum-free medium (0 µM) was used to normalize values of the respective datasets. Data are shown as mean ± SEM. con: solvent control (DMSO); ° p < 0.05 (HUH7); * p < 0.05 (Hep3B).