FIGURE 5.

NAMPT promotes EMT independent of its enzymatic activity. A, 10⁵ NAMPT-MCF10A or vector cells/well were incubated in 6-well plates for 48 h in the presence or absence of 300 nm FK866. Thereafter, cells were lysed in perchloric acid, and intracellular NAD⁺ content was determined. B, NAMPT-MCF10A or vector cells were cultured in 6-well plates for 14 days in the presence or absence of 300 nm FK866 or CHS 828. Thereafter, cells were used for RNA extraction and E-cadherin, N-cadherin, vimentin, and ZEB1 levels were quantified by QPCR. C, NAMPT-MCF10A or vector cells were cultured in 6-well plates for 14 days with or without 100 μm nicotinic acid (NA), nicotinamide (NAM), or nicotinamide mononucleotide (NMN). Thereafter, RNA was isolated, and E-cadherin, vimentin, and ZEB1 mRNA levels were quantified by QPCR. D, 10⁶ cells transduced with NAMPT, NAMPT H247A, or a control vector were plated in 10-cm Petri dishes. 48 h later, cells were used for RNA extraction, and E-cadherin, vimentin, and NAMPT mRNA levels were determined by QPCR. E, MDA-MB-231 cells were cultured in 6-well plates for 2 weeks in the presence or absence of 300 nm FK866. Thereafter, cells were used for RNA extraction, and N-cadherin, vimentin, and ZEB1 levels were quantified by QPCR. A—E, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not statistically significant. One representative experiment of three is presented.