Journal: PLOS Pathogens

Article Title: Interleukin-17A signaling promotes CD8 + T cell cytotoxicity against West Nile virus infection through enhancing PI3K-mTOR-mediated metabolism

doi: 10.1371/journal.ppat.1013218

Figure Lengend Snippet: (A) Eight weeks old Il17ra -/- , and WT (C57BL/6J) mice (n = 3) were infected with 100 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i., followed by purification of CD8 + T cells. The transcription profile was analyzed by RNA sequencing and the representative data were shown as a heatmap. (B-D) Eight to nine weeks old Il17a -/- (n = 5), Il17rc -/- (n = 4), and WT (n = 8) mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. CD8 + T cells were isolated from the splenocytes, cultured ex vivo with mouse rIL-17A (50 ng/ml) or RPMI (control) for 24 h and measured the gene expression of PIK3ca (B), mTOR (C), and S6K1 (D) by RT-qPCR, expressed as the relative fold changes (RFC). Data were analyzed by two-tailed student’s t- tests (B-D) and presented as mean ± s.e.m. with *, and ** denoting p <0.05, and p <0.01, respectively. (E-J) Eight to nine weeks-old WT mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. CD8 + T cells were isolated from the splenocytes, stimulated ex vivo with mouse rIL-17A (100 ng/ml) or RPMI (control), and co-treated with either rapamycin (20 nM), LY294002 (12.5 µM) for 15- or 60- minutes. Cell lysates were resolved with SDS-PAGE. The protein expressions of PI3K, p -PI3K Tyr458 , AKT, p -AKT Thr308 , mTOR, p -mTOR Ser2481 , pS6K, p -pS6K Thr389 , 4EBP1, and p-4EBP1 Thr37/46 were analyzed by immunoblot. GAPDH was used as the loading control. (E) The protein bands of PI3K, p -PI3K Tyr458 , AKT, p -AKT Thr308 , mTOR, and p -mTOR Ser2481 were from the 15-min treatment, and the remaining bands were from the 60-min treatment. (F-J) Densitometric quantification of the phosphorylated protein normalized to corresponding total protein and analyzed by Image Lab software.

Article Snippet: The proteins were then transferred into a nitrocellulose membrane and probed with primary antibodies against mouse PI3K (p85, 1:500, Cell Signaling Technology, 4257), phosphorated ( p )-PI3K (p85 [Tyr458]/p55 [Tyr199], 1:200, Cell Signaling Technology, 4228), AKT (1:500, Cell Signaling Technology, 9272), p -AKT (Thr308, 1:200, Cell Signaling Technology, 4056), mTOR (1:500, Cell Signaling Technology, 2972), p -mTOR (Ser2481, 1:200, Cell Signaling Technology, 2974), pS6K (1:500, Cell Signaling Technology, 9202), p -pS6K (Thr389, 1:200, Cell Signaling Technology, 9234), 4EBP1 (1:500, Cell Signaling Technology, 9644), p -4EBP1 (Thr37/46, 1:500, Cell Signaling Technology, 2855), and GAPDH as control (1:1000, Cell Signaling Technology, 5174) and incubated overnight at 4°C.

Techniques: Infection, Purification, RNA Sequencing, Isolation, Cell Culture, Ex Vivo, Control, Gene Expression, Quantitative RT-PCR, Two Tailed Test, SDS Page, Western Blot, Software