1. mTORC1 signaling is required for STING-mediated type I IFN responses.
mTORC1 signaling is required for STING-mediated type I IFN responses. (A) Naive CD4+ T cells pretreated with (open column) or without (closed column) rapamycin were stimulated with anti-CD3/CD28 Abs in the presence or absence of graded doses of cGAMP for 48 h, and cell growth (upper) and IFN-β production (lower) were assessed by a WST-8 cell proliferation assay and ELISA, respectively. (B) qPCR analysis of Ifn-related genes in CD4+ T cells stimulated with anti-CD3/CD28 Abs in the presence (open column) or absence (closed column) of rapamycin or cGAMP. (C) Western blot analysis for mTORC1 activation in CD4+ T cells stimulated with anti-CD3/CD28 Abs in the presence of PF (PF), rapamycin (Rapa), or cGAMP. (D) Western blot analysis for phosphorylation of signaling molecules in CD4+ T cells stimulated with anti-CD3/CD28 Abs in the presence or absence of c-di-AMP or cGAMP. (E) qPCR analysis for the expression of IFN-related genes in CD4+ T cells stimulated with anti-CD3/CD28 with or without cGAMP for 48 h. (F) Western blot analysis of activation of mTORC1-related molecules in CD4+ T cells with or without Rapamycin pretreatment and upon stimulation with anti-CD3/CD28 Abs with or without cGAMP for 24 h. (G) Naive CD4+ T cells pretreated with or without rapamycin were stimulated with anti-CD3/CD28 Abs in the presence or absence of cGAMP (10 μg/ml) for 48 h, and cell growth (upper) and IFN-β production (lower) were assessed by a WST-8 cell proliferation assay and ELISA, respectively. Data are the mean from duplicate (A, G) or triplicate (B, E) ± SD. Data are representative of at least three independent experiments. (A—G) *P < 0.05, t test (compared with control cells treated with or without cGAMP).
Takayuki Imanishi et al. LSA 2019;2:e
© 2019 Imanishi et al.