Naringenin n5893

IC₅₀ determination was carried out using M. smegmatis strain mc²155 cells grown in 7H9 media (Difco, Middlebrook from Becton Dickinson and Company, Sparks, MD, USA) having 0.2% glycerol and 0.05% Tween 80 and supplemented with 10% OADC^{43 (link)}. Appropriate dilutions (0 to 500 µM) of both naringenin (N5893) and quercetin (Q4951); Sigma-Aldrich, St Louis, MO, USA, were added to the 7H9 media containing M. smegmatis cells and incubated for 48 hours and 72 hours in an assay volume of 200 μl and IC₅₀ values were calculated by using MTT assay (M6494; Invitrogen, Life Technologies). Ethambutol was used as a positive control. The values were calculated from at least three independent experiments and represented as Mean ± S.E.M. Obtained IC₅₀ concentration at 48 hours was used for all the subsequent experiments.

β-glucuronidase (G0751), ethyl acetate, hexane, isopropyl alcohol, methanol, Na₂-ethylenediaminetetraacetic acid (E5134) and pepsin (P7000) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). L-Ascorbic acid and NaOH were obtained from Avantor Performance Materials Poland S.A. (Gliwice, Poland). HCl and formic acid were purchased from Merck KGaA (Darmstadt, Germany). Naringenin (N5893) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Millipore Milli-Q system (Millipore Co., Millford, MA, USA) was used to produce ultrapure water.

Primary antibodies specific to mTOR were purchased from Abcam (Cambridge, MA, USA). Akt, phospho-mTOR (Ser2448), phospho-Akt (Ser473), phospho-AMPKα (Thr172), AMPKα, cleaved caspase 3, and cleaved caspase 9 (Cell Signaling; Danvers, MA, USA); Bcl-XL, Bcl-2, Bax, Bak, poly ADP-ribose polymerase (PARP), p62, microtubule-associated proteins 1A/1B light chain 3B (LC3B), and GAPDH (Genetex; Irvine, CA, USA); and cyclin-dependent kinase 1 (CDK1) and cyclin B1 (Merck Millipore; Burlington, MA, USA). Anti-rabbit polyclonal and anti-mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). ROS scavengers N-acetylcysteine (NAC) and H₂O₂-scavenging enzyme catalase were purchased from Merck Millipore (Burlington, MA, USA). Pan-caspase inhibitor z-VAD-FMK was purchased from R&D Systems (Minneapolis, MN, USA). Naringenin (N5893) and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).