Torin2

Reagents were from Sigma (St. Louis, MO) unless noted. Iodoacetate, lactate, pyruvate, and cycloheximide stocks were prepared in water. Rotenone, oligomycin A, and AICAR were dissolved in DMSO. BEZ235 (Axon Medchem, Reston, VA), Torin-1 (Tocris Bioscience, Bristol, UK), Torin-2 (Selleck, Houston, TX), BKM120 (Axon Medchem), GDC0941 (Axon Medchem), Gefitinib (Axon Medchem), MK2206 (Selleck), PD 0325901 (Sigma and Selleck), and Rad001 (SU2C PI3K Dream Team Mouse Pharmacy, which obtains compounds from Shanghai Haoyuan Chemexpress; [Elkabets et al., 2013 (link)]) were dissolved in DMSO. For GF titration, EGF (Peprotech, Rocky Hill, NJ) and insulin (Sigma) were diluted in PBS and added at indicated concentrations.

Rapamycin (catalog #S1039) and Torin2 (catalog #S2817) were purchased from Selleckchem and resuspended in the minimum amount of DMSO to dissolve the compounds and prepare the initial 20 mM stocks. Confluent endothelial cells were treated with Rapamycin (5 nM) and Torin2 (12.5 nM) for 2 h to inhibit mTOR activity. The medium was then removed, and the cells were infected with R. rickettsii for 48 h. Endothelial cells treated with DMSO were used as a mock control. In some experiments, mTORC1-specific Raptor siRNA (25 or 50 nM, catalog #sc-44069), mTORC2-specific Rictor siRNA (25 or 50 nM, catalog #sc-61478), or control siRNA (50 nM, catalog #sc-44230) (Santa Cruz Biotechnology, Dallas, TX, USA) were transfected into endothelial cells using Lipofectamine RNAiMAX (ThermoFisher, catalog #13778075) according to the manufacturer’s instructions for 48 h prior to infection with R. rickettsii, and total cell lysates were prepared as described above.

Anti-mouse S6K, S6, 4EBP, PDCD4, beta-Actin, phospho-S6K, phosphor-S6, phosphor-4EBP, and phospho-4EBP-Alexa647 antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-mouse phoshoS6-PE, anti-mouse CD45.1 Pacific Blue, and anti-mouse Ki67 Brilliant Violet 605 antibodies were purchased from Biolegend (San Diego, CA, USA). Anti-GFP antibody purchased from Abcam (Cambridge, UK). Cytarabine arabinoside (Ara-C) and doxorubicin were purchased form Sigma-Aldrich (St. Louis, MO, USA). Rapamycin, Rad001, Torin2, AZD0855, and MLN(0128) were purchased from Selleckchem (Houston, TX, USA).

For intracellular staining of Ki67 (SolA15, 1:100; eBioscience), murine NK cells were first surface stained, fixed, and permeabilized using the FoxP3 transcription factor staining buffer set of eBioscience (00-5523-00) according to the manufacturer’s recommendations. For assessment of Myc levels, intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s protocol. Cells were stained for 1 h in a permeabilization buffer with an Myc antibody (Myc/n-Myc D3N8F rabbit mAb PE antibody, #35876, 1:50; Cell Signaling). Myc expression (expressed as geometric MFI) was measured by flow cytometry and the background of Myc-knockout NK cells was subtracted. For Bcl2 staining, BM cells were fixed, permeabilized as above, and stained for 1 h in the permeabilization buffer with Bcl2 antibody (Bcl2 10C4, 1:100; Invitrogen).
To assess the Myc stability, enriched mouse splenic NK cells or human DERL7 NK cell line were stimulated 2 or 3 h, respectively, with recombinant mouse or human IL-15. The cells were then treated with CHX (20 μg/ml) alone or in combination with Torin2 (250 nM from Selleckchem), PD98059 (10 μM) or both for 70 min. The Myc content was then assessed by intracellular staining as mentioned above.

Succinimidyl ester conjugated to Alexa Fluor 647 and DAPI were purchased from Life Technologies (Burlington, ON). The following small molecule inhibitors were purchased from Selleckchem (Houston, TX): Rapamycin, Torin2, SB203580, VX-702, SB202190 (FHPI), BIRB 796 (Doramapimod), FR 180204, PD184352 (CI-1040), PD98059, JNK Inhibitor II, JNK Inhibitor IX. Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155). ON-TARGETplus SMARTpool siRNAs for the genes of interest as well non-targeting negative control siRNAs were obtained from Dharmacon (Lafayette, CO). Phospho-p38 MAPK (Thr180/Tyr182) antibody (RRID:AB_2139682) for immunofluorescence was purchased from Cell Signaling (Beverly, MA).

The AZD8055 (Chresta et al., 2010 (link)), Torin2 (Liu et al., 2011 (link)), KU-63794 (García-Martínez et al., 2009 (link)), and WYE-132 (Yu et al., 2010 (link)) were purchased from Selleckchem. PF-4708671 (Pearce et al., 2010 (link)) and allyl/sinigrin were purchased from Sigma-Aldrich. 4MSB and 3MSP GSLs were purchased at C2 Bioengineering. But-3-enyl GSL was purified from Brassica rapa seeds while 3OHPGSL was purified from the aerial parts of 4–5 weeks old greenhouse-grown plants of the Arabidopsis accession Landsberg erecta (Kliebenstein et al., 2001c (link); Crocoll et al., 2016 ). The concentration of 3OHP and but-3-enyl GSL was determined by LC-MS/TQ as desulfo-GSLs. All inhibitors were dissolved in DMSO and stored as 10 mM stocks at –20°C. For allyl, 3MSP, and 4MSB ~ 100 mM GSL stocks were made with H2O and the concentration of GSLs within these stocks was determined by LC-MS/TQ (see above).