Nt sirna

Small interfering RNA (siRNA) targeted for OGR1 (OGR1-siRNA) and non-targeting siRNA (NT-siRNA) as a control were purchased from Thermo Fisher Scientific. The ID number was L-005591-00-0005 for OGR1-siRNA and D-001810-10-05 for NT-siRNA. OGR1-siRNA or NT-siRNA was transfected at a final concentration of 3 nM into cells using RNAiMAX reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. After ASMCs suspended in the complete culture medium without gentamicin and amphotericin B were mixed with siRNA and the RNAiMAX reagent, they were then cultured in 6 or 12-well plates for 48 h before using them in further experiments.

For silencing experiments, the human fetal CB660 cell line (1 × 10⁶ cells) was transiently nucleofected with 100 pmol small interference RNA (siRNA) against the 5′ end of the SEL1L coding sequence and non‐targeting siRNA (NT siRNA) (Ambion, Life Technologies, Monza, Italy) using the Nucleofector® and Amaxa nucleofector kit V (Lonza, Basel, Switzerland). Scrambled siRNAs were used in order to guarantee minimal or no off‐target activity and the reliability of the silenced phenotype.

To prepare siRNA transfection solution for each well of cells in a 24-well plate, 20 pmol of non-targeting control siRNA (NT siRNA) (Ambion, CT, USA, AM4636), siRNA specific for mouse Tet3 (Tet3 siRNA; Ambion, 4390815/s101483), mouse Pgc1a (Pgc1a siRNA; Ambion, 4390771/n253420), human TET3 (TET3 siRNA; Ambion, 4392420/s47239) or human PGC1A (PGC1A siRNA; Ambion, 4392420/s21394) was mixed with 100 μl of OPTI-MEM (Gibco, 31985-070) by gentle pipetting. In parallel, 6 μl of Lipofectamine RNAiMAX (Invitrogen, MA, USA, 13778-150) was mixed with 100 μl of OPTI-MEM by gentle pipetting, then the two were combined. Following 5 min of incubation at room temperature, the resulting 200 μl of transfection solution was added to each well of cells. For treatment with NT siRNA alone, 20 pmol of NT siRNA was used for each well of cells. For treatment with Tet3 siRNA alone, 10 pmol of NT siRNA and 10 pmol of TET3 siRNA (or Tet3 siRNA) were used for each well of cells. For TET3/PGC-1α double knockdown, 10 pmol of TET3 siRNA (or Tet3 siRNA) and 10 pmol of PGC1A siRNA (or Pgc1a siRNA) were used for each well of cells. Therefore, the total amount of siRNAs for each well of cells was 20 pmol. After 12 h of incubation at 37°C in a 5% humidified CO₂ tissue culture incubator, 300 μl of medium was added and incubation was continued for an additional 24 or 48 h until further analyses.