Siport amine

Small interfering ribonucleic acid (siRNA), a specific double-stranded 21-nucleotide RNA sequence homologous to the target gene, was used to silence PPARα expression. Respectively, siRNA for PPARα and Negative Control (NC) siRNA were purchased from Ambion (Austin, TX) and Santa Cruz Technology (Santa Cruz, CA). Inhibition of PPARα protein expression was assessed by immunoblot analysis after transfection HCM with PPARα–siRNA. Briefly, cells were grown in 100-mm dishes and transiently transfected with 20 nM siRNA using 8 ml of siPORT Amine (Ambion Inc., Austin, TX) in a total transfection volume of 0.5 ml of medium. After incubation at 37°C in 5% CO2 for 5 hours, 1.5 ml of normal growth medium was added and incubated with cells for 48 hours.

ARPE-19 cells were seeded at 2 × 10⁴ cells/well in 96-well plates. The cells were allowed to grow for 24 h and reached approximately 50–80% confluence. The siRNAs (all from Ambion) used were negative control siRNA (cat no. AM4611; final concentration 50 nM), siRNA against human Nrf2 (cat. no. s9491, final concentration 30 nM), and siRNA against human p62 (cat. no. s16962, final concentration 30 nM). Cells were transfected in medium without antibiotics and FBS for 24 h using the siPORT Amine (Ambion, cat. no. 4502) transfection agent according to the manufacturer’s instructions. After transfection, the cells were washed once with medium (antibiotic and FBS free) and further incubated with a 5 µM concentration of PS in medium without FBS for 6 h. RNA was isolated after PS incubation, and the expression levels of Nrf2 and HO-1 were determined as described in the previous section.
The effect of Nrf2 and p62 RNA interference together with PS and HQ exposure to cell viability was assessed using the MTT assay. Briefly, the cells were plated and transfected as described above. At 24 h post transfection, the cells were treated with a 5 µM concentration of PS and HQ as described. Finally, the cells were assessed with microcopy, and the MTT assay was performed.

HAECs were transfected with stealth RNAi targeting NOTCH1 (NM_017617.3_Stealth_775) or control duplexes (Invitrogen) using siPORT Amine (Ambion) as the transfection agent. For better efficiency, the cells were transfected twice with 24 h of recovery in between; cultures were then used for experiments 24 h after the second transfection.

Human pulmonary artery ECs obtained from Lonza (Basel, Switzerland) were cultured as previously described^{18 (link)} in EGM-2 complete medium (Lonza) with 10% fetal bovine serum at 37℃ in a humidified atmosphere of 5% CO₂ and 95% air, with passages 6–8 used for experimentation. Twenty-four hours before the PM challenge, EC media was changed to EGM-2 with 2% fetal bovine serum (Lonza). Human lung ECs were transfected with siRNA (Darmacon RNA Technologies, Inc., Lafayette, CO, USA) using siPORT Amine (Ambion, Austin, TX, USA) as the transfection reagent according to the manufacturer’s protocol. ECs (∼40% confluent) were serum-starved for 1 h, followed by incubation with 100 ng/mL total target siRNA (or control siRNA) for 6 h in serum-free medium. Serum-containing medium was then added (10% serum final concentration) for 42 h before further assays were conducted.