Fig. 2: IFNγ inducible expression of Nampt in macrophages through NRE1. | Nature Communications

Fig. 2: IFNγ inducible expression of Nampt in macrophages through NRE1.

From: A Stat1 bound enhancer promotes Nampt expression and function within tumor associated macrophages

Fig. 2

a Schematic of the first intron of Nampt, which contains the indicated predicted STAT1 binding sites, that we termed NRE1. CRISPR/Cas9-mediated deletion of this site was accomplished with the indicated constructs (CR1, CR2). b qPCR of Nampt expression in EV WT clones and NRE1 CR clones with and without LPS/IFNγ stimulation for 6 h. c Western blot of indicated cells treated with LPS/IFNγ for 24 h. Size markers are in kB. d Conservation of STAT1 binding site located within the first intron of Nampt. e Chromatin immunoprecipitation with anti-STAT1 antibody analyzed by qPCR of the indicated regions in RAW 264.7 Stat1 EV cells and Raw 264.7 Stat1 CR cells. f Schematic of the CRISPR mediated deletion of the STAT1 binding site (BS) located within NRE1 and the first intron of Nampt. g RT-qPCR expression measurement of RNA from Nampt mRNA or the STAT1 BS region of NRE1 from RAW 264.7 STAT1 BS CR cells and controls, with and without 6-h stimulation with IFNγ. h Oplophorus (NanoLuc) luciferase reporter constructs with no added sequence (EV), WT NRE fragment containing the STAT1 binding sites (SBS), or scrambled STAT1 binding sites (sbs-m) were transduced into RAW 264.7 cells treated with IFNγ or medium alone. Resulting luminescence was measured and normalized relative to a co-transduced Photinus luciferase positive control. For b, e, g, h, results presented are the average of N = 3 biological replicates, and are representative of at least three independent experiments. p values were determined by a two-tailed unpaired t-test: *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005. Error bars represent SEM. EV non-targeting CRISPR gRNA control. Uncropped images and raw scans provided in Source Data file in Supplementary information.

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