Slhv004sl

Lentiviral production and transduction were conducted according to the manufacturer’s instructions (Gene Copoeia). Briefly, lentiviral vectors and LonP1 or control shRNA were packed with the Lenti-Pac HIV Expression Packaging Kit using HEK293T cells and incubated overnight, followed by replacement of the old culture medium with fresh DMEM supplemented with 5% heat-inactivated FBS and penicillin-streptomycin. Titer Boost reagent (1/500 volume) was added to the culture medium and incubated at 37 °C in a humidified incubator with 5% CO₂. At 48 h after transfection, the supernatants containing lentivirus particles were collected, filtered through 0.45-μm syringe filters (SLHV004SL, Millipore), and used immediately to infect H9c2 cells. To select stably transfected cells, the old medium was replaced by fresh complete medium containing puromycin (2.0 μg/ml) every 3 days until drug-resistant colonies become visible. The knockdown of LonP1 was validated by qRT-PCR and Western blot analysis. Positive clones with stable knockdown of LonP1 were expanded and maintained in medium supplemented with puromycin (2.0 μg/ml).

Palmitate was first dissolved in DMSO (Millipore, Cat # 41639), and subsequently this solution was dissolved at 45 °C in DMEM media (no glucose) containing 6.7% fatty acid-free BSA (EMD Millipore, Cat # 126609) to make a 4 mM (10×) stock. For control BSA conditions, a 10× stock of DMEM media containing 5% BSA and 1% DMSO was used. For the treatment conditions, the 10× stocks were added to DMEM media containing 5% FBS, 50 U/mL penicillin, and 50 g/mL streptomycin. The pH of the treatment media was then adjusted to 7.4, and the adjusted media was sterile filtered using a 0.45 µm syringe filter (Millipore, Cat # SLHV004SL) before treating the HepG2 cells for 16 h.