Allstars cell death sirna

Commercial miR-941 mimic, MIMAT0004984:5′CACCCGGCUGUGUGCACAUGUGC-3′, AllStars Cell Death siRNA (Cat.no: 1027298) and AllStars negative control siRNA (Cat.no: 1027280) (Qiagen, USA) were used for transfection. The transfection was accomplished using HiPerFect transfection reagent (Qiagen, USA) according to the manufacturer’s protocol. Transfected cells were further incubated under normal conditions for 48 h, and downstream assays were applied.

Human U2OS cells stably expressing the pBmal-dLuc construct were reverse transfected with a total of 9.6 nM pooled siRNAs (n = 4/gene) using Lipofectamine/RNAiMAX (Invitrogen) transfection reagent and Opti-MEM media (Gibco), totaling 6 pmol/siRNA for each gene. All siRNAs were purchased from Qiagen. Allstars Negative siRNA (Qiagen) and AllStars Cell Death siRNA (Qiagen) were used as experimental controls. Transfected cells were seeded in triplicate at a density of 200,000 cells/mL in DMEM containing 10% FBS (Gibco), 0.1 mM nonessential amino acids (NEAA, Invitrogen), 1% L-Glutamine (Invitrogen) without antibiotics into 96-well plates and incubated for 24 h. siRNA/transfection medium was removed 24 h post-transfection and replaced with 250 µl of luminescence recording medium: phenol red-free DMEM (Sigma D-2902), sodium bicarbonate (Invitrogen), D-(+)-glucose (Sigma), 10 mM HEPES (Invitrogen), 1% pen/strep/L-Glutamine (Invitrogen), and 0.1 mM luciferin (Promega). The medium is supplemented with 0.1 µM dexamethasone (Sigma-Aldrich) to synchronize the cells to the same circadian phase. The Synergy 2 plate reader (BioTek) was used to measure luminescence every 1 h for 6 days. Period length was determined using the WaveClock algorithm implemented in R.