Figure 2. HMGA1-mediated NAMPT expression drives the proinflammatory SASP.

a-c, The expression of indicated proteins in cells induced to senesce by oncogenic RAS at the indicated time points was analyzed by immunoblot (a). Expression of NAMPT (b) and the indicated proinflammatory SASP genes (c) were determined by qRT-PCR (n = 3 independent experiments). d-g, In established senescent cells, HMGA1 or NAMPT were knocked down using the indicated shRNAs. The NAMPT activity was also inhibited by FK866. The expression of the indicated proteins was determined by immunoblot (d). Expression of SASP genes was determined using quantitative RT-PCR (n = 3 independent experiments) (e,f). g, The secretion of soluble factors under the indicated conditions were detected by antibody arrays. Heat map indicates fold change in comparison to the control or RAS condition. Relative expression level per replicate and average fold change differences are shown (n = 4 independent experiments). h, V5-HMGA1 overexpressing cells had NAMPT knocked down and expression of NAMPT and the indicated SASP genes were determined using qRT-PCR (n = 3 independent experiments). i-j, In established senescent cells, HMGA1 was knocked down with or without ectopic expression of a FLAG-tagged wild type or catalytically-inactive NAMPT. The expression of the indicated proteins was determined by immunoblot (i). Expression of the indicated SASP genes was determined using qRT-PCR (n = 3 independent experiments) (j). 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.