MHY1485

MHY1485 suppresses the basal autophagic flux. MHY1485 causes the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner.^[1]

MHY1485 increases phospho-mTOR levels and phosphorylation of downstream S6K1 and rpS6 proteins without affecting total mTOR content, total S6K1 and rpS6 levels. Short-term treatment of ovaries with MHY1485 followed by allo-transplantation promoted secondary follicle growth. Treatment with MHY1485 and subsequent grafting allowed the derivation of mature oocytes and healthy offspring.^[2]

MHY1485 is a potent, and cell-permeable mTORC1 agonist.

• Activation of mTOR

  Western blot analysis is performed to detect the change of total protein level and levels of phosphorylated forms of mTOR and 4E-BP1 reflecting the activity of mTOR. Ac2F cells are treated with MHY1485 of different concentrations and rapamycin 5 mM as a positive control for 1 h. Cells are washed with cold PBS and harvested. Cell lysates are prepared using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM DTT, 0.1 mM NaF, 1 mM PMSF, 1 mg/mL pepstatin, 1 mg/mL leupeptin, and 1 mg/mL aprotinin). Protein concentration is determined by the bicinchoninic acid (BCA) method. Equal amounts of protein are separated on 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The gels are subsequently transferred onto a polyvinylidene difluoride membrane by electroblotting for 2 h at 60-75 V. The membranes are blocked in a 5% nonfat milk solution in Tris-buffered saline (TBS) with 0.5% Tween-20, and incubated with primary antibodies. Pre-stained protein markers are used for molecular-weight determination.

• Cell lines

  TU177 cells

• Concentrations

  10 µM

• Incubation Time

  24 h

• Method

  Cells were treated with indicated concentration of drug for 24 h.