Figure 1. MTOR is required for autophagy-related PIK3C3 complex regulation. (A) ATG14 containing PIK3C3 is activated under conditions of MTOR inhibition. HEK293 cells were cultured in following conditions: nutrient-rich (NR), amino acid free (AA-, indicated time), Torin1 (100 nM, 1 h) or rapamycin (50 nM, 1 h) treatment. ATG14-containing PIK3C3 complex was immunoprecipitated with ATG14 antibody and used for lipid kinase assay. (B) Quantification of (A). (C) Induction of autophagy by MTOR inhibition. HEK293 cells were treated similarly as in (A) with or without bafilomycin A₁ (Baf A₁) (100 nM) treatment for 1 h before harvesting cells. LC3 and SQSTM1 level were detected using specific antibodies. (D) RHEB inhibits ATG14 containing PIK3C3 via activation of MTOR. HEK293 cells were transfected with Flag-ATG14, Myc-BECN1 and HA-PIK3C3 together with pcDNA3 or RHEB. ATG14-containing PIK3C3 complex was immunoprecipitated with Flag antibody and used for lipid kinase assay. (E) Quantification of (D). The error bars represent the standard error of the mean from independent experiments within same treatment group. Stars indicate a statistically significant difference.