Figure 4. mTORC2 but not mTORC1 is required for ACL-catalyzed production of acetyl-CoA.

A. and B. 293T stable cell clones with doxycycline-inducible (pTRIPZ) lentiviral constructs for Sh-NT (control), Sh-Rap#2 or Sh-Ric#4 were induced with doxycycline for gene depletion. Cells were transfected with empty vector or ACL expression construct for 48 h, subjected to immunoblotting (A) and acetyl-CoA assay (B). ACL-dependent acetyl-CoA level (value of ACL-transfected normalized with the value of empty vector) obtained for each group are plotted. C. and D. MDA453 and MDA231 cells were infected with GIPZ-lentivirus encoding Sh-NT, Sh-Rap#2, Sh-Ric#4 or Sh-ACL#3. Puromycin-resistant cell pools were plated in 96-well plates for acetyl-CoA assay (C) or plated in 12-well plates, labeled with ¹⁴C-glucose for 2 h then subjected to measurement of total glucose-derived lipids as described in Methods. Statistical analysis: *, p < 0.05; **, p < 0.01; ***, p < 0.001.