Nucleofector 1

Plasmid transfection was performed by electroporation using an Amaxa Nucleofector-1.5 machine. 1 × 10⁶ VSMC was transfected with 3–5 μg of DNA or 100 pmol of siRNA using the standard Amaxa Nucleofector program A033. VSMCs were infected with adenovirus at 1 × 10⁸ plaque forming units/ml for 16 h.

MnSOD small interfering RNAs (siRNAs) and Silencer Negative Control siRNA were from Applied Biosystems (Darmstadt, Germany). MnSOD siRNAs (5′ GCU CUA AUC AGG ACC CAU Utt 3′ and 5′ AGG GAG AUG UUA CAA CUC Att 3′) were used and knocked down MnSOD with similar efficiencies. The 6 d predifferentiated cells were transfected using Endo-Porter (Gene Tools LLC, Philomath, Oregon, USA) according to the protocol supplied by the company. Differentiated 3T3-L1 cells were transfected using electroporation. Cells were suspended in 100 µl Amaxa Nucleofector solution (Amaxa Nucleofector Kit L, Lonza, Wuppertal, Germany) and 3 µg siRNA was added. Electroporation was performed using Nucleofector I (Lonza, Wuppertal, Germany) and the programme A33. Knockdown of SREBP2 was performed as described [27] (link).

Transfections with siRNAs against the GR were performed as described before (Fitzsimons et al., 2013 (link)). Briefly, a total of 100 pmol of a GR targeting (Hs_NR3C1_6_HP validated siRNA, Qiagen and siGENOME NR0B1 siRNA, Dharmacon) or a non-targeting control siRNA (AllStrars Neg. siRNA AF 546, Qiagen or siGENOME Non-Targeting Control siRNAs #1, Dharmacon) were transfected into A549 cells using the Nucleofector I (Lonza) and the Cell Line Nucleofector^® Kit T for the A549 cell line, according to the supplier’s instructions (Lonza) using the U-29 Nucleofector program. The control siRNA was tagged with a red fluorophore, to monitor transfection efficacy, which was always higher than 90%. The cell medium was refreshed 1 day after transfection and all ligand treatments were performed 3 days after transfection.

For knockdown experiments, ~2 million control LCLs were nucleoporated with 300 nM targeting siRNA or Non-Targeting siRNA#1 (Silencer Select, Invitrogen), using program X05 on Nucleofector I or X005 on Nucleofector II, and Cell Line Nucleofector Kit V (Lonza), and cells collected after 72–75 h. In experiments comparing single and double knockdowns, 200 nM of targeting siRNAs or Non-Targeting siRNA#1 was used in single knockdowns, whereas 150 nM of each siRNA was used for double knockdowns. For experiments with nuclear TRM editors, ~2 million LCLs were nucleofected with a mix of 95% crRNA encoding pU6-PspCas13b plasmid and pCMV-dCas13b-METTL3-NLS or dCas13b-METTL3^mut-NLS plasmid at 1:2 molarity, and 5% pmaxGFP plasmid (Lonza) at a final amount of 2 μg plasmid DNA per nucleofection, using program X05 on Nucleofector I and Nucleofector Kit V (Lonza), and cells harvested after 96 h. For experiments using the nuclear dCasRX-ALKBH5 eraser, ~2 million LCLs were nucleofected with a mix of 95% crRNA encoding pXR003 plasmid and pMSCV-dCasRx-ALKBH5-PURO plasmid at a 1:1 ratio of DNA and 5% pmaxGFP plasmid at a final amount of 2 μg plasmid DNA per nucleofection using kit and program as above, and cells harvested after 72 h.