Biomek 3000

Assays were realized in 96-well plates, prepared with a Biomek NKMC and a Biomek 3000 from Beckman Coulter, and read on a Wallac Victor fluorimeter from PerkineElmer [28 (link)]. Per well, 20 μL of farnesyl pyrophosphate (10 μM) was added to 180 μL of a solution containing 2 μL of varied concentrations of potential inhibitors (dissolved in DMSO) and 178 μL of a solution composed of 10 μL of partially purified recombinant human FTase (5 mg/mL) and 1.0 mL of Dansyl-GCVLS peptide (in the following buffer: 5.6 mM DTT, 5.6 mM MgCl₂, 12 μM ZnCl₂ and 0.2% (w/v) octyl-s-D-glucopyranoside, 52 mM Tris/HCl, pH 7.5). Fluorescence was recorded for 15 min (0.7 s per well, 20 repeats) at 30 °C with an excitation filter of 340 nm and an emission filter of 486 nm. Each measurement was reproduced twice (two independent experiments on different 96-well plates) in duplicate. The kinetic experiments were realized under the same conditions, either with FPP as varied substrate with a constant concentration of Dns-GCVLS of 2.5 μM or with Dns-GCVLS as varied substrate with a constant concentration of FPP of 10 μM. Nonlinear regressions were performed with Excel software.

Reverse transcription was completed using 1 μg of pooled RNA and a High capacity cDNA reverse transcription kit as specified by the manufacturer (Applied Biosystems, France).
PCR experiments were performed using a LightCycler480 System (Roche, France) in 384 well-plates using a liquid handler automated workstation (Biomek 3000 Beckman). The PCR was performed with 0.4 μM of each primer and EXPRESS SYBR GreenER qPCR Supermix (Invitrogen, France). Cycling conditions including a 2 min UDG activation at 50°C, 2 min activation at 95°C followed by 40 cycles of amplification (15 s denaturation at 95°C, 1 min primer annealing and fragment elongation at 60°C). A melt program was performed at the end of the PCR. The raw fluorescence data were analyzed using LightCycler480 software. Target gene mRNA expression was normalized to β-actin, and data were quantified using the 2־^∆∆Ct method. The primers used are listed in Table 1.

Compounds were purchased as powders from Sigma-Aldrich, except bedaquiline (Cellagen Technology), linezolid (Selleck Chemicals) and moxifloxacin (Fisher). Compounds were prepared in DMSO at 10 mM and stored in 70 μL aliquots at -20°C; stock solutions were thawed at RT overnight. Two-fold serial dilution series were prepared in 96-well polypropylene plates (Greiner) using a Biomek 3000 or 4000 (Beckman Coulter). Plates were sealed with DMSO-resistant thermal seals (Phenix) and stored at RT for up to 6 months.

Purified mRNA was converted to cDNA using the High-Capacity RNA-to-cDNA kit (4387406, ThermoFisher). RT-qPCR was performed with pre-mixed Taqman primer/probes (4331182, ThermoFisher; see S3 Table) and Taqman master mix (4369016, ThermoFisher) using 4 ng cDNA per well (10 μL reactions). 384-well plates were robotically loaded using the Biomek 3000 (Beckman Coulter, Indianapolis, IN) and analyzed using the 7900HT Fast Real-Time PCR System (Fisher Scientific). Each gene of interest was normalized to the geometric mean of three housekeeping genes (18s, rplp0, and actb); the Ct values of these housekeeping genes did not differ between dietary conditions. Statistical tests and linear modeling were conducted using ΔΔCt values to omit floor effects (normalized to RC NoDS) and data are presented as percent change from RC NoDS [30 (link)].

Plate uniformity was evaluated as previously described to investigate the signal variability across different plates^{36 (link)}. A library containing 960 small molecules (ChemBridge Co., US) with molecular weights ranging between 160 and 6600 Da was arrayed on three plates (320 compounds/plate) in triplicate to assess the assay for screening in high-throughput mode. A multidrop well liquid dispenser (MTX Lab System, Inc.) was used for the addition of all reagents to the assay plates. Twenty-five microlitres of complete medium without phenol red was added to 384-well polystyrene microtiter plates, followed by pin-arraying the compounds at 20 μg/mL,  pre-dissolved in DMSO (Hybri-Mix, Sigma, US), into the plates via an automation platform (Biomek^® 3000, Beckman Coulter). Porcine chondrocytes were dispensed at 3 × 10⁴ cells/cm² in 25 μL of complete DMEM. In each plate, the first and last two columns were reserved for nontreated controls (0.1% DMSO). The plates were incubated at 37 °C in 5% CO₂ for 3 days, followed by assessment of cell viability and GAG production using MTT and DMMB assays.

RNA from whole blood was extracted with PAXgene® blood RNA kits (Qiagen®, Valencia, CA) as per instructions. Quantity of RNA was determined using a Nanodrop 2000 (ThermoScientific™, Waltham MA). Maxima® first strand cDNA synthesis kit (ThermoScientific™) was used to synthesize cDNA with 2,000 ng of starting RNA. A Beckman BioMek 3000 liquid handling robot was used to set up qPCR reactions into 384 well plates using specific gene primers (27 (link)) (Supplementary Table 2) and 2X perfeCTA™ SYBR® Green SuperMix, ROX™ (Quantabio, Beverly, MA). qPCR reactions were run using an ABI ViiA7 with a holding stage of 3 min at 95°C, followed by 40 cycles of 10 s at 95°C, 30 s at annealing temperature and 30 s at 72°C. Gene expression was calculated using ΔΔCt method with TBP (TATA box binding protein) used as a house keeping gene (28 (link)).