Figure 4. DEPTOR is a cell cycle regulatory protein, controlled by the UBE2C/CDH1 axis.

(A) Knockdown of UBE2C, CDH1, SAG, and APC2 caused DEPTOR accumulation at the protein level, but not at the mRNA level. A427 cells were transfected with indicated siRNAs followed by IB with the indicated Abs (top), or by RT-qPCR analysis (bottom). (B) Knockdown of CDH1 and UBE2C caused the accumulation of DEPTOR and inactivation of mTORC1/2. A427 cells were transfected with indicated siRNAs followed by IB with the indicated Abs. (C) DEPTOR levels fluctuated during the cell cycle. HeLa cells were arrested at the G₁/S phase by double thymidine block (treatment with 2 mM thymidine for 14 hours and release for 9 hours, and then treatment with 2 mM thymidine for another 14 hours), and then released into the normal cell cycle. Cells were harvested at indicated times and analyzed by IB. (D and E) Knockdown of UBE2C and CDH1 abrogated the cell cycle fluctuation of DEPTOR. A427 cells were transfected with siRNA targeting UBE2C (D), CDH1 (E), or siCont for 24 hours, and then cells were synchronized at the G₁/S phase by thymidine block and released for the indicated time points. Cells were harvested for IB analysis with indicated Abs (top) or FACS analysis (bottom).