3T3-L1 fibroblasts (#CL-173, from American Type Culture Collection, Manassas, VA) were cultured and differentiated as described previously [20 (link)]. Briefly, cells were grown in the growth media [Dulbecco’s modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose, 10% fetal bovine serum, 1% glutamine, and 0.5% penicillin/streptomycin]. Differentiation was induced in post-confluent cells with growth media containing 500 μM isobutylmethylxanthine, 0.2 μM dexamethasone, and 2.5 μg/ml insulin for 3–4 days, and cells were replenished with growth media every 3–4 days. Experiments were performed in adipocytes 12–16 days post differentiation.
Electroporation of 3T3-L1 adipocytes was performed with cells on 10–12 days post differentiation. Differentiated 3T3-L1 adipocytes were electroporated at 200 V and 950 μF with 2 nmole siRNA using a Gene Pulser Xcell electroporator (Bio-Rad, Hercules, CA) and plated onto 12- or 24- well plates for experiments. The siRNA sequences were as follows: MEK1 and 2 (MEK1/2),5’-AGU CGG ACA UCU GGA GCA U-3’ , or 5’-CAG UCG GAC AUC UGG AGC A-3’ ; IKKβ, 5’-CGA CAG GAG CUC AGC CCA A-3’ , or 5’-GGA CAU CGU UGU UAG UGA A-3’ ; caveolin-1, 5’-AAC CAG AAG GGA CAC ACA G-3’ [21 (link)].
Electroporation of 3T3-L1 adipocytes was performed with cells on 10–12 days post differentiation. Differentiated 3T3-L1 adipocytes were electroporated at 200 V and 950 μF with 2 nmole siRNA using a Gene Pulser Xcell electroporator (Bio-Rad, Hercules, CA) and plated onto 12- or 24- well plates for experiments. The siRNA sequences were as follows: MEK1 and 2 (MEK1/2),
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