Non targeting sirna 2

MIN-6 cells were maintained in Dulbecco’s modified Eagle's medium supplemented with 15% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. For overexpression of C/EBPβ, MIN-6 cells were transfected with expression plasmid carrying the full C/EBPβ by using Lipofectamine 3000 (Invitrogen) transfection reagent. For knockdown of AMPK, MIN-6 cells were re-plated in 12-well plates (60-mm dishes) at 24 h before transfection and transfected with siRNA for AMPKα1 and α2 (SMARTpool; Dharmacon, Lafayette, CO) or scramble controls (Non-Targeting siRNA#2; Dharmacon) with DharmaFECT2 transfection reagent (Dharmacon). After 48 h of further incubation for 48 h for protein, cells were harvested for evaluation of C/EBPβ expression.

siRNA targeting DCAF1 (siRNA bp3 from the siGENOME SMARTpool, M-021119-03 with the 5′-UCACAGAGUAUCUUAGAGA-3′ sequence targeting the DCAF1 mRNA ORF region 3148-3166) and non-targeting control siRNA (non-targeting siRNA #2) were obtained from Dharmacon. For transfection of HEK293T cells, 80 pmol of siRNA and plasmid DNA constructs encoding HA-tagged Vpr (50 ng) and DCAF1 (250 ng) were preincubated with 4.5 µl of lipofectamine 2000 and overlayed on cells at 50% confluency (final concentration of siRNA was 40 nM). To mark transfected cells, 1 µg of GFP-expressing plasmid (pQBI-25) was usually co-transfected in these experiments. All analyses were conducted at 48 h post-transfection.