Lipofectamin rnaimax transfection reagent

MNT1 cells were from Prof. Tiechi Lei (Renmin Hospital of Wuhan University) and grown in MEM medium (SH30024.01; HyClone) supplemented with 20% fetal bovine serum (10099-141; GIBCO), 10% AIM-V+AlbuMAX (31035025; GIBCO), 1% sodium pyruvate (11360070; GIBCO), and 1% Non-Essential Amino Acids Solution (100×) at 37°C and 5% CO₂. Melan-a cells were from the laboratory of Dorothy C. Bennett (St. George’s University) (Bennett et al, 1987 (link)) and grown in DMEM medium (11965092; GIBCO) supplemented with 10% fetal bovine serum (10099-141; GIBCO) at 37°C and 10% CO₂. siRNAs (Table S1) were transfected into cells to knock down VDAC1 using Lipofectamin RNAiMAX Transfection Reagent (13778100; Invitrogen) according to the manufacturer’s instructions.

Table S1 siRNA sequences used in knockdown experiments.

Small interfering RNA (siRNA) duplexes targeting human Arl1, arfaptin-1 and arfaptin-2 were synthesized and developed using the highly effective Stealth RNAi siRNA technology from Invitrogen. The siRNA sequences used in this study are shown in S3 Table. For gene knockdown, HeLa cells were transfected with siRNA using Lipofectamin RNAiMAX transfection reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. At 48 h after transfection, the cells were lysed for western blotting or stained for immunofluorescence assays.

HtrA1 siRNA (Silencer Select Mouse Htra1 s80179) and control scramble siRNA (Silencer Select Negative Control siRNA#1) were purchased from Ambion. RAW 264.7 cells (2 × 10⁴ cells per well) were transfected with 20 pmol of Lipofectamin RNAiMAX Transfection Reagent (Invitrogen).

A siRNA designed for the human Nrf2 gene (Merck KGaA) was incubated for at least 24 h to obtain the siNrf2 ARPE-19 cell line. A commercial negative siRNA (siNEG, Merck KGaA) having no known homology with any gene was used as a negative control in preliminary experiments to confirm the specificity of the transient Nrf2 silencing. The siRNAs were transfected into ARPE-19 cells using the lipofectamin RNAiMAX transfection reagent (Invitrogen, Thermo Scientific, Waltham, MA, USA) following the manufacturer’s instructions; siRNA treatment was maintained throughout the experiments (up to 72 h). To confirm that Nrf2 expression was silenced, 4 h before the end of the experiment, the proteasome inhibitor MG132 (5 µM) was added to the medium of selected plates to block the degradation of Nrf2 protein, that was evaluated by Western blotting.