Extended Data Fig. 9: Mechanism of STING inhibition by H-151.

a, THP-1 cells that had been pretreated with H-151 or DMSO were stimulated with cGAMP or triphosphate RNA, or left unstimulated. IP-10 production was quantified by enzyme-linked immunosorbent assay after overnight incubation (n = 3). b, Levels of TNF mRNA assessed by RT–qPCR in THP-1 cells that had been pretreated with H-151 and stimulated with cGAMP or triphosphate RNA (n = 4). c, [³H]-palmitate labelling of immunoprecipitated endogenous calnexin or transferrin receptor from HEK293T cells that had been treated with H-151 (1 μM). d, Structure of H-151-AL. e, Competition assay of H-151-AL with H-151 in HEK293T cells that express Flag–hsSTING. Flag–hsSTING labelled with H-151-AL was visualized by in-gel fluorescence. f, HEK293T cells or HEK293T cells that express Flag–hsSTING were treated with H-151-AL, lysed and clicked to a SiR azide. Whole-cell lysates were analysed by in-gel fluorescence and by immunoblot. g, HEK293T cells that express Flag–hsSTING, GSTO1–Flag, MAP27K–Flag, MGMT–Flag or GSTP1–Flag were exposed to H-151-AL, clicked to TAMRA azide and visualized by in-gel fluorescence or immunoblot. h, Deconvoluted electrospray ionization mass spectrum showing intact mass of Flag–hsSTING(C91S) with or without H-151. i, Flag–hsSTING was purified from HEK293T cells that had been pretreated with H-151 (1 μM) and analysed by top-down analysis using LC-HCD-MS/MS (with additional 8 residues due to N-terminal Flag). Data that were obtained with three different NCE values are combined to create fragmentation map with assigned b- and y-fragments. Achieved sequence coverage is 23% with 20 p.p.m. mass accuracy for fragment assignment. Data are shown as mean ± s.e.m. P values were calculated by two-tailed t-test. NS, not significant. One of n = 2 experiments is shown (c, e–h). For gel source data, see Supplementary Fig. 1. For electrospray ionization spectra, see Supplementary Information.