Poweruptm sybr green master mix assay

Total RNA was extracted from cultured H9C2 cardiomyoblasts with Trizol (Invitrogen, Carlsbad, CA, United States), then quantified on a spectrophotometer. cDNA was generated from total RNA (1 μg) using reverse transcriptase and oligo (dT) 15 primer. The PowerUp^TM SYBR Green Master Mix assay (Applied Biosystems, Life Technologies, Foster City, CA, United States) was used to detect and quantify the mRNA levels of each gene in an Applied Biosystems 7300 (Foster City, CA, United States).
All specific sequence primers included in the present work were listed as follows: atrial natriuretic peptide (ANP): 5′-GGG TAG GAT TGA CAA GGA TTG G-3′ and 5′-CTC CAG GAG GGT ATT CAC CAC-3′; brain natriuretic peptide (BNP):5′-CTC CAG AAC AAT CCA CGA TGC-3′ and 5′-CTT CCT AAA ACA ACC TCA GCC-3′; LPA3: 5′-TGT CAA CCG CTG GCT TCT-3′ and 5′-CAG TCA TCA CCG TCT CAT TAG-3′; Beclin1: 5′-TAC TGT TCT GGG GGT TTG CG5′- and 5′-GAA CTT GAG CGC CTT TGT CC-3′; GAPDH: 5′-CAA CGA CCC CTT CAT TGA CCT-3′ and 5′-CAG TAG ACT CCA CGA CAT ACT C-3′; beta-actin: 5′-GAA CCC TAA GGC CAA CCG TGA A-3′ and 5′-TAC GTA CAT GGC TGG GGT GT -3′. In this work, GAPDH or beta-actin were used as reference genes for data normalization. The relative expression of the target gene was normalized to the reference gene using the comparative 2^-ΔΔCt method.

Lysophosphatidic acid (LPA) (oleoyl C: 18:1) was obtained from Avanti Polar Lipids (Alabaster, AL, United States). 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonylamino)-3-methyl-5-isoxazolyl]benzylsulfanyl)propanoic acid (Kil6425) was purchased from Sigma (St. Louis, MO, United States). PI3K inhibitor LY294002, rapamycin, anti-phosphorylated mTOR (ser2448) antibody, anti-total mTOR antibody, anti-p70s6 kinase (Thr389) antibody, anti-phosphorylated p70s6 kinase, anti-4E-BP1 antibody, anti-phosphorylated 4E-BP1(Thr37/46) antibody, and anti-phosphorylated AMPKα (Thr172) antibody were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-p62/SQSTM1 (sequestosome1) polyclonal antibody and anti-beclin1 polyclonal antibody were from Proteintech Technology (Proteintech Group, Wuhan, China), and anti-LC3B polyclonal antibody and anti-GAPDH monoclonal antibody were obtained from Sigma (St. Louis, MO, United States). Lipofectamine^TM RNAiMAX, stealth siRNA, and siRNA negative control were purchased from life Technologies (Invitrogen, Carlsbad, CA, United States). The PowerUp^TM SYBR Green Master Mix assay (Applied Biosystems, Life Technologies, Foster City, CA, United States).