Figure 9
Effects of GDH inhibition on AMPK and mTORC1 activity and proliferation in HEK293 and T47D cells. HEK293 and T47D cells were plated in full media containing glutamine for 24 h. Cultures were washed with DPBS and fresh glutamine-depleted media with NH4Cl with or without hexachlorophene (Hex – 2.5 µM) was added. Cells cultured in glutamine-depleted media with or without NH4Cl or DMSO served as vehicle controls. Cultures were collected at 4 h and 3 days post-incubation for protein extraction or subjected to live imaging using IncuCyte FLR assay (n = 3) to measure cell proliferation rate over 7 days (A,D). Protein abundance of phosphorylated ACC (S79) (B,E) or S6K (T389) (C,F) relative to total ACC or S6K was quantified using western blot. Blots shown were representative from 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Full length blots are presented in Supplementary information Fig. 4.
