Mtor inhibitor rapamycin

Gentamicin sulfate, dimethyl sulfoxide (DMSO), Triton X-100 (used at 1%), mTOR inhibitor rapamycin (used at 1 μM) and bafilomycin A1 (used at 100 nM) were all purchased from Sigma-Aldrich (Merck, Darmstadt, Germany). Sapitinib (AZD8931) and lapatinib were ordered from Selleck Chemicals (Houston, TX, USA) and SignalChem (Richmond, BC, Canada), respectively. Kinase inhibitors from the published kinase inhibitor sets (PKIS)1 and PKIS2 were kindly provided by GlaxoSmithKline Global Health Medicines R&D (GSK) and the Structural Genomics Consortium of the University of North Carolina at Chapel Hill (SGC-UNC) (Elkins et al., 2016 (link); Drewry et al., 2017 (link)). Larger quantities of GW296115X were obtained from AOBIOUS (Gloucester, MA, USA). All PKIS compounds were dissolved in DMSO at 10 mM. Adenosine monophosphate-activated protein kinase (AMPK) activator compound 991 (ex229) (Xiao et al., 2013 (link)) (Spirochem, Basel, Switzerland; used at 25 μM) was kindly shared by Eline Brombacher and Dr. Bart Everts (Leiden university Medical Center, Leiden, Netherlands). Chemical structures were generated from published simplified molecular-input line-entry specification (SMILES) using ChemDraw (PerkinElmer, Waltham, MA, USA).

Overnight bacterial cultures were diluted 20-fold in LB medium containing 0.3 M NaCl and subcultured in aerobic conditions for 3 h. Before infection, bacteria were quantified spectrophotometrically by determining the optical density at 600 nm along with viable plate counts. Mammalian cells were seeded in plates 16-24 h before infection. The next day cells were washed three times with PBS, incubated with HBSS for 0.5 h, and infected with Salmonella at a multiplicity of infection (MOI) of 100:1. Cells were washed with PBS, and fresh medium containing amikacin (100 μg ml⁻¹) was added to kill the extracellular bacteria at 1 h p.i. Baf (100 nM, Sigma), a microtubule-disrupting agent, was added to the complete medium 2 h before infection to inhibit autophagosome-lysosome fusion. To activate autophagic activity, cells were pretreated with mTOR inhibitor rapamycin (4 μM, Sigma) for 1 h before infection. At different time points following infection, cells were processed in the following ways.

The SA‐β‐Gal staining was performed as described previously.^[29, ³⁹, ^42] Co‐cultured tumor cells or the frozen tissue sections from in vivo experiments (4–8 µm) were washed in PBS, fixed in 4% formaldehyde, and incubated overnight at 37 °C with freshly prepared staining solution. The frozen sections were restored up to room temperature for 30 min before staining. Five sections were analyzed for each mouse. The blue‐stained senescent cells were counted manually under a microscope and calculated as a percentage of total cell numbers.
For some experiments, SA‐β‐Gal⁺ tumor cells were determined with the pretreatment of cells the following inhibitors: ATM inhibitor KU55933 (5 μm, #3544, Tocris Bioscience), MAPK inhibitors U0126 (4 μm, #19–147, Sigma‐Aldrich) and SB203580 (5 μm, #AG‐CR1‐0030, AdipoGen), mTOR inhibitor rapamycin (50 nm, #R0395, Sigma‐Aldrich), FASN inhibitor C75 (5 μm, #10 005 270, Cayman Chemical), HMGCR inhibitor simvastatin (1 μm, #10 010 344, Cayman Chemical), IDI1 inhibitor 25‐HC (0.25 µg mL⁻¹, #11 097, Cayman Chemical), and ACAT inhibitor avasimible (1 μm, #18 129, Cayman Chemical).

Cellular proliferation was tested by XTT Cell Proliferation Kit II (Roche, Penzberg, Germany). The assay is based on the cleavage of yellow tetrazolium salt XTT to form an orange formazan dye by metabolic active cells. The formation of the dye is quantified using a scanning multiwell spectrophotometer (ELISA reader). LCLs were synchronized by incubation for 24 hours in RPMI-1640 medium containing 0.5% FBS. After this period, cells were seeded at a density of 5 × 10⁴ cells/well in 96 multiwells plates in 100 μl/well RPMI-1640 medium containing 10% FBS in the presence or in the absence of 350 nM mTOR inhibitor Rapamycin (Sigma-Aldrich) or 10 μM STAT-3 inhibitor STATTIC (Sigma-Aldrich). Cell proliferation was stimulated with increasing doses of IL-6 (0.01–10 ng/ml) for 48 hours. Finally, 100 μl of yellow tetrazolium salt was added to each well and plates were incubated for 4 hours at 37 °C. The increase in number of living cells directly correlated to the amount of orange formazan formed, which has been monitored by absorbance at 450 nm.