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. 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.