Si tp53

Cellular oxygen consumption and extracellular acidification were measured with a Seahorse XF extracellular flux analyzer according to the manufacturer’s instructions (Agilent). PASMCs were cultured in a Seahorse XF 24-well assay plate in antibiotic-free medium at a density of 20,000 cells/well, and were treated with siRNA for p53 (si-TP53; Invitrogen) and negative control siRNA (Invitrogen) after attachment. The medium was replaced with antibiotic-containing medium after 24 hours. After incubation for a further 24 hours, the plates were washed and the medium was replaced with pre-warmed running medium (XF base medium supplemented with 10% D-glucose, 100 mM pyruvate, and 200 mM glutamine for the mitochondrial stress test, or with 200 mM glutamine alone for the glycolysis stress test). Then incubation was performed in a non-CO₂ incubator for 60 min at 37°C. The basal oxygen consumption rate and extracellular acidification rate were recorded for 24 min, followed by performance of the mitochondrial stress test (1μM oligomycin, 2 μM FCCP, and 0.5 μM rotenone/antimycin A) and glycolysis stress test (10 mM glucose, 1 μM oligomycin, and 50 mM 2-DG). All reagents were obtained from the Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience, #103015–100) and the Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience, #103020–100).

Small interfering RNA (si-RNA) targeting p53 (a mixture of si-TP53 #HSS186391, #HSS186390, and #HSS110905; 10 nM each) and the corresponding negative control (#46–2001) were purchased from Invitrogen. siRNAs were transfected by using Lipofectamine RNAi MAX (Invitrogen, #13778–150) and Opti-MEM (Gibco by Life Technologies, #31985–062). The medium was replaced after 24 hours, and the cells were incubated for a further 24 hours before experiments were performed, unless stated otherwise.

The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China). The synthetic siSETDB1, siTP53, or Scr NC at a final concentration of 20 nM were introduced into A549, PC14, or BEAS-2B cells using Lipofectamine 2000 kit (Invitrogen, Carlsbad, CA) as previously described by cell culture and transfection methods. Wild-type TP53 expression vector (pEGFP-TP53, plasmid 12091, GeneBank ID: AAD28628.1) was bought from Addgene. The SETDB1 ORF was amplified by PCR from human cDNA and inserted into the pcDNA3.1(+) to construct the pcDNA3.1-SETDB1 expression vector. The SETDB1 cloning primers were 5′-GGGGTACCACAAAAGCATGTCTT-3′ (forward) and 5′-AGGCTCTAGACTAAAGAAGACGTCC-3′ (reverse). The vector backbone pEGFP-N1 and pcDNA3.1(+), used as control vector, was purchased from Clontech Laboratories, Inc. (Mountain View, CA, U.S.A.). Transfected cells were harvested at 48 h.