Exploratory experiment designed to discover new patterns targeting P2RY12, MTOR in cultured VSMCs. Primary outcome: MTOR activity and cholesterol efflux
This experiment examined the role of MTOR in P2RY12-mediated inhibition of autophagy and cholesterol efflux. The researchers investigated whether P2RY12 receptor activation leads to MTOR activation through the PI3K-AKT pathway in VSMCs. They used rapamycin as an MTOR inhibitor and employed genetic approaches to reduce MTOR expression. The study measured the effects of MTOR modulation on P2RY12-mediated inhibition of cholesterol efflux and autophagy.
Cells: Human aortic SMCs, passages 4-6. Cholesterol loading: 50 μg/mL AcLDL for 48h. Treatments: (1) 2-MeSADP (P2RY12 agonist, 10 μM). (2) Rapamycin (MTOR inhibitor, 100 nM, 1h pretreat). (3) Torin1 (ATP-competitive MTOR inhibitor, 250 nM). (4) PI3K inhibitor LY294002 (10 μM). (5) AKT inhibitor MK2206 (1 μM). Knockdown: siRNA for MTOR, RPTOR (mTORC1 component), RICTOR (mTORC2 component), 50 nM for 48h. Verify >70% knockdown. Signaling: Western blot for p-MTOR (Ser2448), p-S6K (Thr389, mTORC1 readout), p-AKT (Ser473, mTORC2 readout), total proteins. Time course: 0, 15, 30, 60, 120 min after agonist. Cholesterol efflux: NBD-cholesterol assay as in Exp 3, measure at 4h. Autophagy: LC3-II/I and p62 Western blot. Epistasis: Test if rapamycin rescues P2RY12-mediated effects.
...Quantitative predictions: (1) 2-MeSADP increases p-MTOR by 2-3 fold at 30 min, p-S6K by 3-4 fold (mTORC1 activation). Effect blocked by PI3K or AKT inhibition. (2) RPTOR (mTORC1) knockdown rescues cholesterol efflux (from 8% with agonist to 16-18%, ~baseline) and autophagy (LC3-II/I from 0.3 to 0.7). RICTOR KD has minimal effect (<20% rescue). (3) Rapamycin or Torin1 phenocopies RPTOR KD: restores efflux and autophagy despite P2RY12 activation. (4) Time course: p-MTOR peaks 30-60 min, returns toward baseline by 120 min. p-S6K follows similar kinetics but with longer duration.
...Primary: P2RY12 agonist increases p-S6K >2-fold (p<0.01, marker of mTORC1 activation), and this activation is blocked by PI3K or AKT inhibition (p>0.1 vs. vehicle). Secondary: (1) RPTOR knockdown or rapamycin treatment rescues efflux to >80% of vehicle level despite agonist (p>0.05 vs. vehicle, p<0.01 vs. agonist alone). (2) Same for autophagy: LC3-II/I restored to >70% of vehicle. (3) RICTOR knockdown does NOT rescue (<30% restoration, indicating mTORC1-specific effect). (4) Bafilomycin flux: ΔLC3-II restored >80% by rapamycin cotreatment.
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