ps6k  (Cell Signaling Technology Inc)

Journal: Journal of Medical Virology

Article Title: Heterogeneous Activation of Signaling Pathways and Therapeutic Vulnerabilities in KSHV‐Associated Primary Effusion Lymphoma Cell Lines

doi: 10.1002/jmv.70534

Figure Lengend Snippet: Western‐blotting analysis of activation of signaling pathways involved in cell proliferation and survival. (A) KSHV‐infected cell lines have constitutively activated but reduced pS6K and S6K levels compared to BJAB cells. BCP1 and BCBL1 cells had elevated 4EBP1 and p4EBP1 levels with slightly higher molecular weights for p4EBP1. (B) AKT was consistently activated in all PEL cell lines. (C) All the KSHV‐infected cells had lower levels of FOXO1 compared to BJAB cells. However, all the KSHV‐infected cells except BCP1 cells had higher levels of pFOXO1 compared to BJAB cells. BJAB‐KSHV, BC3, and BCBL1 cells had higher levels of FOXO3a and pFOXO3a while BCP1 cells had lower levels of FOXO3a and pFOXO3a compared to BJAB cells. (D) All the KSHV‐infected cells had strong constitutive activation of p65, and higher levels of p50 and p52 compared to BJAB cells. Samples were prepared 2 days after seeding. GAPDH was measured as an internal control. Experiments were independently repeated at least three times and representative results were presented.

Article Snippet: Membranes were blocked in 5% skim milk for 1 h and incubated overnight at 4°C with rabbit primary antibodies against the following proteins: GAPDH (CST, Cat. No. 5174; 1:5000), AKT (CST, Cat. No. 4691S), phosphorylated AKT (pAKT‐T308) (CST, Cat. No. 2965L), 4EBP1 (CST, Cat. No. 96445), phosphorylated 4EBP1 (p4EBP1) (CST, Cat. No. 2855), S6K (CST, Cat. No. 9202S), phosphorylated S6K (pS6K/pp70) (CST, Cat. No. 9205L), FOXO1 (CST, Cat. No. 2880), FOXO3 (CST, Cat. No. 2497), phosphorylated FOXO1/FOXO3a (pFOXO1‐Thr24/FOXO3a‐Thr32) (CST, Cat. No. 9464), p65 (CST, Cat. No. 8242), phosphorylated p65 (pp65) (Ser536) (CST, Cat. No. 3033), p105/p50 (CST, Cat. No. 13586), and p100/p52 (CST, Cat. No. 4882).

Techniques: Western Blot, Activation Assay, Protein-Protein interactions, Infection, Control

Journal: Journal of Medical Virology

Article Title: Heterogeneous Activation of Signaling Pathways and Therapeutic Vulnerabilities in KSHV‐Associated Primary Effusion Lymphoma Cell Lines

doi: 10.1002/jmv.70534

Figure Lengend Snippet: Effects of mTORC1 inhibitors Torin 2 and Rapamycin (Rapa) on PEL cell lines. (A) PEL cell lines were treated with different concentrations of Torin 2 and live cells were counted for 5 consecutive days. (B, C) PEL cell lines were analyzed for apoptotic and dead cells (B) and cell cycle progression (C) following treatment with 30 nM Torin 2 for 2 days. (D) PEL cell lines were treated with different concentrations of Rapamycin and live cells were counted for 5 consecutive days. (E, F) PEL cell lines were analyzed for apoptotic and dead cells (E) and cell cycle progression (F) following treatment with 300 nM Rapamycin for 2 days. (G) Western‐blotting analysis of mTORC1 downstream targets S6K and 4EBP1 for their total and phosphorylated levels in all the KSHV‐infected cell lines following treatment with 30 nM Torin 2 or 300 nM Rapamycin for 2 days. GAPDH was measured as an internal control. Experiments were independently repeated at least three times and representative results were presented.

Article Snippet: Membranes were blocked in 5% skim milk for 1 h and incubated overnight at 4°C with rabbit primary antibodies against the following proteins: GAPDH (CST, Cat. No. 5174; 1:5000), AKT (CST, Cat. No. 4691S), phosphorylated AKT (pAKT‐T308) (CST, Cat. No. 2965L), 4EBP1 (CST, Cat. No. 96445), phosphorylated 4EBP1 (p4EBP1) (CST, Cat. No. 2855), S6K (CST, Cat. No. 9202S), phosphorylated S6K (pS6K/pp70) (CST, Cat. No. 9205L), FOXO1 (CST, Cat. No. 2880), FOXO3 (CST, Cat. No. 2497), phosphorylated FOXO1/FOXO3a (pFOXO1‐Thr24/FOXO3a‐Thr32) (CST, Cat. No. 9464), p65 (CST, Cat. No. 8242), phosphorylated p65 (pp65) (Ser536) (CST, Cat. No. 3033), p105/p50 (CST, Cat. No. 13586), and p100/p52 (CST, Cat. No. 4882).

Techniques: Western Blot, Infection, Control

Journal: Journal of Medical Virology

Article Title: Heterogeneous Activation of Signaling Pathways and Therapeutic Vulnerabilities in KSHV‐Associated Primary Effusion Lymphoma Cell Lines

doi: 10.1002/jmv.70534

Figure Lengend Snippet: Effects of PI3K/mTOR dual inhibitors BGT226 and Dactolisib on PEL cell lines. (A) PEL cell lines were treated with different concentrations of BGT226 and live cells were counted for 5 consecutive days. (B, C) PEL cell lines were analyzed for apoptotic and dead cells (B) and cell cycle progression (C) following treatment with 30 nM BGT226 for 2 days. (D) PEL cell lines were treated with different concentrations of Dactolisib and live cells were counted for 5 consecutive days. (E, F) PEL cell lines were analyzed for apoptotic and dead cells (E) and cell cycle progression (F) following treatment with 30 nM Dactolisib for 2 days. (G) Western‐blotting analysis of PI3K downstream target AKT, and mTORC1 downstream targets S6K and 4EBP1 for their total and phosphorylated levels in all the KSHV‐infected cell lines following treatment with 30 nM BGT226 or Dactolisib for 2 days. GAPDH was measured as an internal control. Experiments were independently repeated at least three times and representative results were presented.

Article Snippet: Membranes were blocked in 5% skim milk for 1 h and incubated overnight at 4°C with rabbit primary antibodies against the following proteins: GAPDH (CST, Cat. No. 5174; 1:5000), AKT (CST, Cat. No. 4691S), phosphorylated AKT (pAKT‐T308) (CST, Cat. No. 2965L), 4EBP1 (CST, Cat. No. 96445), phosphorylated 4EBP1 (p4EBP1) (CST, Cat. No. 2855), S6K (CST, Cat. No. 9202S), phosphorylated S6K (pS6K/pp70) (CST, Cat. No. 9205L), FOXO1 (CST, Cat. No. 2880), FOXO3 (CST, Cat. No. 2497), phosphorylated FOXO1/FOXO3a (pFOXO1‐Thr24/FOXO3a‐Thr32) (CST, Cat. No. 9464), p65 (CST, Cat. No. 8242), phosphorylated p65 (pp65) (Ser536) (CST, Cat. No. 3033), p105/p50 (CST, Cat. No. 13586), and p100/p52 (CST, Cat. No. 4882).

Techniques: Western Blot, Infection, Control

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