Figure 3.

eNAMPT is a SASP factor. (A) PBS control non-senescent and bleomycin-induced senescent cells were analyzed for NAMPT RNA expression at the single cell level. [Dataset originally described in (40)]. (B–D) Cells were induced to senesce by IR, shSIRT3, or sodium butyrate (NaBu). Ten days after induction, cells were cultured in serum-free media for 24 h. Conditioned media was then analyzed by ELISA and normalized to cell number. (E) NAD levels were measured in cells from (D). (F, G) Cells were transduced with either a GSE22 expression lentivirus or an empty vector and either mock irradiated or induced to senesce with 10 Gy of IR. Conditioned media were generated as in (B), and cells were collected, counted and analyzed for (F). eNAMPT secretion by ELISA, and (G). NAD levels. (H) Conditioned media from non-senescent and senescent [SEN(IR)] cells were analyzed by western blot under non-reducing (left) and reducing (right) conditions. (I) Relative intensities of eNAMPT dimer blots were quantified by densitometry. (J) Extracellular vesicles were isolated from quiescent (QUI) or senescent cells induced by IR, doxorubicin (DOXO) or mitochondrial dysfunction (MiDAS – antimycin A) and analyzed mass spectrometry for eNAMPT. Data are presented as means ± SEM for ≥ 3 experiments. * = p<0.05. ** = p<0.01 (t-test with Welch’s correction for A–E, I, J. One-way ANOVA for F, G). NS, non-senescent.