Ampk sirna

AMPK siRNA was synthesized by Gene Pharma (Shanghai, China). Following the manufacturer’s instructions, cells were instantaneously transfected with Lipofectamine 2000 reagent (Invitrogen, ThermoFisher, Waltham, MA, USA). NIH 3T3 cells were cultured in six-well plates in DMEM (2 mL per well) without antibiotics or FBS, then transfected with 5 μL of 20 μM siRNA per well using 5 μL Lipofectamine 2000 reagent. The steps were as follows: each siRNA (5 μL) was diluted in 100 μL of OPTIMEM I reduced serum medium; 5 μL Lipofectamine was diluted in 100 μL of OPTIMEM I reduced serum medium; the transfection complexes (200 μL) were placed in 6-well culture plates and incubated at 37 °C in 5% CO₂ for 24 h. AMPK activity, assessed by monitoring the phosphorylation of AMPK at Thr172, was significantly downregulated when NIH 3T3 cells were transfected with AMPK siRNA for 24 h (Figure 1C,D). All siRNA sequences used in this study are shown in Table 2. We chose Prakaa1-mus-1337 for the next stage of the experiment.

Transfection with siRNA was performed according to our previous report [26 (link)]. Briefly, FLS were transfected with 100 nM AMPK siRNA (Genepharma, Shanghai, China), 100 nM CPT1 siRNA, or scrambled siRNA (sc-156134 and sc-37007; Santa Cruz, CA, USA) using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) with reduced serum medium Opti-MEM. The mRNA and protein levels of AMPK were measured by immunoblot and real-time PCR analyses. AMPK sequence: siRNA1: 5′-GCACGAGUUGACUGGACAUTT, AUGUCCAGUCAACUCGUGCTT-3′, siRNA2: 5′-CCUUUCUGGUGUGGACUAUTT, AUAGUCCACACCAGAAAGGTT-3′, siRNA3: 5′-CCAUUCUUGGUUGCCGAAATT, UUUCGGCAACCAAGAAUGGTT-3′. After 6 h of transfection, the solution was exhausted and incubated in a cell culture medium for 24 h to validate the transfection efficiency.