Dharmafect 4 transfection reagent

HepG2 cell signaling was targeted with specific siRNA silencing of AMPK (30 nM; Sigma-Aldrich) or TSC2 (100 nM; Sigma-Aldrich) using Dharmafect transfection reagent 4 (Cat #T-2004, Thermo Fisher Scientific). HepG2 cells were then treated with control scramble siRNA (Cat #SIC001, Sigma-Aldrich) or specific siRNA targeting AMPK or TSC2 in 10% FBS DMEM–F-12 media for 24 h. Cell media was subsequently exchanged for 10% FBS DMEM–F-12 media for 24 h and then cells were serum-starved with 0% FBS DMEM–F-12 media for 6 h. Cells were treated with control or glucose deprivation media as described earlier. Both cell media and lysate were collected for analyses, and the efficiency of target silencing was determined at the protein level using western blot analysis.

Sub-confluent HUVECs cultured in 12-well plates were treated with 150 pmol MCAM, PECAM siRNA (siGenome Smart pool human MCAM and PECAM, Thermo Fisher) or control non-target siRNA (siGenome control siRNA #1, Thermo Fisher) in serum-free medium for 5 min before addition of 2.5 μl DharmaFect Transfection Reagent-4 (Thermo Fisher) in 100 µl serum-free medium for 20 min at room temperature. After addition of 800 μl EGM, cells were incubated for 16 h at 37°C before the medium was replaced with fresh medium for a further 24 h. The cells were either lysed by SDS-sample buffer for analysis of MCAM and PECAM expression by immunoblotting, or introduced with 4 μg/ml PNA or BSA for 24 h at 37°C before IL-6 and MCP-1 concentrations in the culture media were determined by ELISA.