Extended Data Fig. 2: Non-pharmacologic means of inducing ID inactivates mTORC1.

(A) Immunoblot of mTORC1 activity in HEK293T cells treated with high Tf-sat (66%) or low Tf-sat (6.6%) media for 18 hours. Representative image of two independent experiments. (B) Summary of immunoblot in panel A (n = 3 replicates, two-way unpaired t-test, mean ± s.e.m.). (C) Immunoblot of mTORC1 activity in HepG2 cells transfected with the FPN-GFP fusion protein and TET inducible rtTA3 plasmids in the presence and absence of 500 ng/ml doxycycline for 48 hours. Representative image of two independent experiments. (D) Summary of immunoblot in panel C (n = 3 replicates, two-way unpaired t-test, mean ± s.e.m.). (E) Fluorescent microscopy of cells transfected with FPN-GFP construct and treated with 500 ng/ml doxycycline for 24 hours demonstrating appropriate expression and localization of the FPN-GFP fusion protein. Representative image of three independent samples. (F) Immunoblot of puromycin incorporation in rtTA3/FPN-GFP stable HEK293T cells in the presence and absence of 500 ng/ml doxycycline for 48 hours. Representative image of one experiment. (G) mRNA expression of ER stress makers CHOP and BNIP3 in HepG2 cells transfected with rtTA3/FPN-GFP plasmids and treated with 500 ng/ml doxycycline for 48 hours. Internal control: 18S (n = 4 replicates, two-way unpaired t-test, mean ± s.e.m.). (H) Immunoblot of protein levels of the key components of the mTORC1 complex after 24 hours of 150 µM DFO in HEK293T cells. Representative image of two independent experiments. (I) Summary of results shown in panel A (n = 4 replicates, two-way unpaired t-test, mean ± s.e.m.). (J) Immunoblot of total and phosphorylated TSC2, AKT, ERK, GSK3β, and S6 proteins and total P53 at different time points after treatment with 150 µM of DFO in HEK293T cells. Representative image of one experiment. (K) Immunoblot of mTORC1 activity and mitochondrial function in HepG2 cells treated with DFO for 18 hours at the indicated doses. Representative image of one experiment. (L) Oxygen Consumption Rate (OCR) measured by the Seahorse Assay in HepG2 cells treated for 24 hours of DFO at the indicated doses (n = 10 replicates per group, mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels.