Figure 6. HMGA1/NAMPT axis regulates the strengths of SASP.

a, Cells cultured at early passage (population doubling 30, PD30), late passage (population doubling 90, PD90), and oncogene-induced senescence (PD30 expressing oncogenic RAS) were compared. Expression of the indicated proteins was determined using immunoblot. b, Expression of NAMPT mRNA in the indicated cells was determined using qRT-PCR (n = 3 independent experiments). c, The indicated early passage and late passage cells were subjected to ChIP analysis for the NAMPT enhancer site using an anti-HMGA1 antibody. An isotype matched IgG was used as a control (n = 3 independent experiments). d, Cells from the conditions in (a) were assessed for their effects on the growth of co-cultured TOV21G cancer cells (n = 3 independent experiments). e-i, The indicated cells with or without NMN supplementation were compared. Cells were examined for expression of the indicated SASP genes using qRT-PCR (n = 3 independent experiments) (e), secretion of soluble factors using antibody arrays (f), NAD⁺/NADH ratio (g), NFκb reporter activity (h), and the effects on the growth of co-cultured TOV21G cancer cells (i). The heat map for the antibody array indicates fold change in comparison to the control or RAS + NMN condition. Relative expression level per replicate and average fold change differences are shown (n = 4 independent experiments). j, TOV21G cells overexpressing HMGA1 or NAMPT, or supplemented with NMN, were induced to senesce using etoposide (50 μM for 48 hours) and expression of the indicated SASP genes was determined by qRT-PCR (n = 3 independent experiments). All graphs represent mean ± s.d. P values were calculated using a two-tailed t-test. Statistical source data are provided in Supplementary Table 1. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 8.