Pantethine

M. tuberculosis H37RvMA and its coaBC Tet-OFF conditional knockdown derivative^{12 (link)} were grown in Difco Middlebrook 7H9 broth (BD) supplemented with Middlebrook albumin-dextrose-catalase (ADC) enrichment (BD), 0.2% glycerol (Sigma-Aldrich) and 0.05% Tween-80, unless otherwise indicated. Hygromycin and kanamycin were used at final concentrations of 50 μg/mL and 25 μg/mL, respectively, and pantethine (Sigma-Aldrich) supplementation was included at 2.5 mg/mL where required. The anhydrotetracycline (ATc) inducer was used at concentrations up to 200 ng/mL in order to transcriptionally silence coaBC in the Tet-OFF hypomorph.
The minimum inhibitory concentrations (MIC₉₉) of the compounds were determined by measuring fluorescence output using Alamar Blue, as previously described (Singh et al.^{56 (link)}). Briefly, 2-fold serial dilutions of compound were inoculated with a suspension of M. tuberculosis at a cell density of ~10⁵ CFU/mL in a 96-well microtiter plate and incubated at 37 °C for 10 days, following which 10 μL Alamar Blue solution was added and the plates were incubated for a further 24 h. Fluorescence as an indication of growth was measured using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices) in bottom-reading mode with excitation at 544 nm and emission at 590 nm.

Monoclonal anti-actin antibody, Prussian Blue, pantothenate, pantethine, vitamin E, L-carnitine, thiamine, Luperox^® and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mitochondrial acyl carrier protein (mtACP), anti-NF-Y, anti-FOXN4 and anti-hnRNPA/B were purchased from Invitrogen/Molecular Probes (Eugene, OR). Anti-phospho-PGC1α was purchased from RD systems. NFS1 antibody and Omega 3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PANK2, anti-PGC1α, complex 1 activity kit and PDH activity kit were purchased from Abcam (Cambridge, UK). Anti-TFAM was purchased from Cell Signaling. BODIPY^® 581/591 C11 was purchased from Thermo-Fisher (Waltham, MA). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).

Pantethine for cell cultures was purchased from Sigma-Aldrich. Purity of the preparation was ascertained using high performance liquid chromatography-coupled mass spectrometry (HPLC-LC-UV-MSD). Pantethine was added to astrocyte cultures at the concentration of 1 mM for 18 h. Previous experiments on primary human cells have shown that maximal cellular effects were obtained under these conditions without side effects [41 ]. Astrocytes were then washed in PBS and used in the assays.