Penicillin

The transfection of cells and RNA extraction were mainly executed as previously described (21 (link)). In brief, human thymic 4D6 epithelial cells, a gift from Christophe Benoist (Harvard Medical School, Boston, Massachusetts, USA) (55 (link), 56 (link)) were cultured in RPMI 1640 (Lonza) supplemented with 10% FBS (Thermo Fisher Scientific), 10 mM HEPES buffer (Lonza, Basel, Switzerland), 1% nonessential amino acids (Lonza), 2 mM l-glutamine (Lonza), 100 U/mL penicillin (MilliporeSigma), and 100 μg/mL streptomycin (MilliporeSigma) at 37°C with 5% CO₂. Cells were then plated in a 6-well plate at a density of 5 × 10⁵ cells per well and incubated overnight. Samples (2.5 μg) of the pCMV6 plasmids were mixed with 10 μL FuGene HD transfection reagent (Promega) to a total volume of 160 μL supplemented with RPMI 1640 (without penicillin or streptomycin) and incubated for 5 minutes at room temperature before being added to the cells. Cells were incubated for another 24 hours before total RNA was extracted (RNeasy Mini Kit, QIAGEN). cDNA was prepared from 1 μg total RNA (High-Capacity RNA-to-cDNA Kit, Applied Biosystems).

Prior to bioluminescence analysis of Bmal1-dLuc fibroblast cultures on 35 mm dishes, growth medium containing control or experimental treatments (IL-6 or TNFα recombinant proteins, neutralizing antibodies, palmitate, or BSA) was removed. Cultures were rinsed, placed in DMEM recording medium containing 15uM forskolin, 25 mM HEPES, 292 µg/ml L-glutamine, 100units/ml penicillin, 100 μg/ml streptomycin and 10uM luciferin (Promega) and then sealed airtight with sterile glass coverslips and sterile silicon grease. The temporal patterns of Bmal1-dLuc bioluminescence were analyzed using an automated 32-channel luminometer (LumiCycle; Actimetrics) housed in a standard culture incubator at 35 °C. Bioluminescence from individual cultures was continuously recorded for ~70 sec at intervals of 10 min and analyzed using the LumiCycle Analysis program. As described previously^{5 (link),36 (link)}, rhythm parameters (period, amplitude) were determined from baseline-subtracted data using the damped sine fit and Levenberg–Marquardt algorithm (Y(t) = A*sin (2πft + ϕ)*e^−t/τ + C). The amplitude of phase shifts in response to treatment with IL-6 or TNFα recombinant proteins, or palmitate was determined by measuring the time difference between the peaks of the Bmal1-dLuc rhythms during the third cycle in PBS, BSA or BSA/vehicle (PBS) controls and experimental treatment groups.