Ly294002

Nuclear and cytoplasmic protein extracts from RAW 264.7 cells were prepared after treatment with 50 μM of desoxyrhapontigenin for the indicated time periods. Total protein extracts from the specified time periods were also prepared for the detection of Keap1, an inhibitor of Nrf2. To elucidate the upstream signaling pathway involved in desoxyrhapontigenin-mediated Nrf2 activation and HO-1 induction, specific inhibitors of PI3K/Akt (LY294002, BioVision, Milpitas, CA, USA), p38 (SB202190), JNK (SP600125) and ERK (U0126) were used. Protein extracts were separated by 10% SDS-PAGE, electro-transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany), blotted with each primary antibody (1:1000) and the corresponding secondary antibody (1:5000), and detected with the WEST-ZOL^® (plus) Western Detection System (iNtRON, Korea). The target bands were quantified using the UN-SCAN-IT™ gel 6.1 software (Silk Scientific Corp, Orem, UT, USA).

Primary CRC cells CC-1/2 were obtained from the ascites of advanced colon cancer patients. HCT116 was obtained from ATCC. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; 4.5 g/L d-glucose) (Gibco, Grand island, NY) supplemented with 10% FBS and 1% antibiotic/antimycotic in tissue culture flasks in a humidified incubator at 37 °C in a humidified incubator with 5% CO₂. The medium was changed two times a week and cells were passaged using 0.05% trypsin/EDTA. PIK3CA-H1047R plasmid was purchased form Addgene (#14572, Cambridge, MA, USA)^{52 (link)}. HCT116 cell transfection was performed as previous report^{53 (link)}. In some experiments, the cells were exposed to FOLFOX (25 µM 5-FU and 0.625 µM oxaliplatin) for 72 h. Perifosine (KRX-0401, Selleck Chemicals, USA), LY 294002 (BioVision, USA), bpV (phen) (BioVision, USA) were administrated as described.

To evaluate the involvement of the PI3K-Akt-mTOR pathway during WSSV infection, shrimp were pretreated with inhibitors LY294002 (0.625–1.25 µg LY294002/g shrimp), MK2206 (0.625–1.25 µg MK2206/g shrimp) and BKM120 (buparlisib; 0.15625–25 µg/g shrimp) by intramuscular injection 2 h before being challenged. Stock solutions were prepared by dissolving LY294002 (Biovision), MK2206 (Biovision) and BKM120 (Selleckchem) in 10% DMSO, and these solutions were further diluted with PBS before use. Control shrimps were injected with 0.01% DMSO in PBS. At 24 h after the pretreated shrimps were injected with WSSV, hemocyte and pleopods samples were collected from 6–10 individual shrimp in each group. The hemocyte samples were subjected to real-time PCR to measure the mRNA levels of WSSV IE1, VP28 and ICP11 as described below. Real-time PCR was also used to measure the viral copy number in the pleopods samples.

The following reagents were added to in vitro culture from days 2.5 to 4: methyl pyruvate (10 mM; 371173; Sigma), methyl malate (5 mM; 355-17971; Wako), etomoxir (90 μM; E1905; Sigma), rotenone (120 nM; R8875; Sigma), metformin (1 mM; 150959; SIGMA), oligomycin (1 nM; O4876; Sigma), 2-deoxy-D-glucose (200 μM; 154-17-6; SIGMA), haemin (30 μM; H9039; Sigma), ascorbic acid (200 μM; A7506; Sigma), MitoTEMPO (64 μM; ALX-430-150; Enzo Life Science) and TPP (64 μM; 309567; SIGMA); from days 1 to 4: LY294002 (3 μM; 1667; Biovision), AZD5363 (5 μM; S8019; Selleckchem) and 5-ALA hydrochloride (450 μM; 3785; Sigma); and from days 3 to 4: cobalt (II) chloride (150 μM; 232696; Sigma).
For OCR and ECAR assay, all the reagents were added at 36 h of culturing and incubated for 12 h. Exposing duration of cells to reagents was therefore from 36 to 48 h of culturing.

Human bronchial epithelial cell line HBE cells were cultured in RPMI 1640 supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% heat inactivated fetal bovine serum (FBS). Lipopolysaccharide(LPS, Escherichia coli, 055:B5)was purchased from Sigma (Missouri, USA). Interferon-γ was purchased from R&D Systems (Minnesota, USA). PI3K/Akt/mTOR inhibitors (GDC0941, GSK690693, BEZ235, LY294002 and CAL101) were purchased from Biovision (California, USA). SHBM1009 (a new PI3K/mTOR inhibitor) was synthesized by Fudan University. CEACAM1 and GAPDH antibodies for western blot were purchased from Abcam (Hong Kong, China). SYBR Premix Ex Taq was from TaKaRa (Shiga, Japan). Lipofectamine™ 2000 Transfection Reagent was from Invitrogen (Grand Island, NY, USA).

The titers of the virus stocks were determined using TaqMan qRT-PCR for HIV-1 RNA (target in integrase) and normalized to a nominal 400 virion equivalents per cell for infection. Infections were performed by spinoculation in the presence of 5 µg/ml DEAE dextran (Sigma) for 2 h at 1,200×g and 37°C [22] (link), [132] (link). Cells were then washed and plated. For FKHR TransAM experiments, IL-7 treated CD4 T cells were infected with NLENHSA-ESI. At 7 dpi., cells were ficolled in order to remove dead cells, then stained with a phycoerythrin (PE) labeled anti-mCD24 and bound to anti-PE magnetic microbeads (Miltenyi Biotec). Positive and negative cells were separated using the MACS system (Miltenyi Biotec). Purity was routinely >95%.
Cell treatments included PMA (2 nM, Fisher Scientific), LY294002 (5 µM, BioVision), PI-103 (5 µM, EMD Millipore), TAPI-1 (50 µM, Santa Cruz Biotechnology). Non-nucleoside reverse-transcriptase inhibitor Efavirenz (2 µg/ml) and Protease inhibitor Indinavir (2 µM) were obtained from the NIH AIDS Research and Reference Reagent Program. The Foxo1 inhibitor 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856) [78] (link), was purchased from EMD Millipore and used at a final concentration of 50 nM. DMSO controls were performed with a corresponding dilution of 1 in 20,000.