Extended Data Fig. 1: Long-term ID inactivates mTORC1 in multiple cell types.
From: Iron drives anabolic metabolism through active histone demethylation and mTORC1
(A) Non-heme iron levels in HEK293T cells treated with 150 µM DFO. (n = 5 replicates, two-way unpaired t-test, mean ± s.e.m.). (B) mRNA levels of TTP and TFRC at indicated time points in HEK293T cells treated with 150 µM DFO. Internal control: POLR2A (n = 6 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (C) Immunoblot of mTORC1 activity in HEK293T cells treated with indicated concentrations of DFO. Representative image of two independent experiments (D) Immunoblot of mTORC1 activity in HEK293T cells treated with 150 µM DFO or 50 µM BPD for 3 hours. Representative image of two independent experiments. (E) mRNA levels of indicated genes 3 hours after addition of 150 µM DFO or 50 µM BPD. Internal control: POLR2A (n = 8 TFRC Control; n = 7 TFRC 150 µM DFO and 50 µM BPD; n = 4 REDD1; n = 8 TTP replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (F) Immunoblot of mTORC1 activity in response to 250 nM Torin1 at indicated time points. Representative image of two independent experiments. (G) Immunoblot of mTORC1 activity in primary murine hepatocytes treated with increasing concentrations of DFO. Representative image of two independent experiments (H) mRNA of Ttp and Tfrc in primary murine hepatocytes treated with indicated concentrations of DFO. Internal control: Polr2a (n = 3 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (I) Immunoblot of mTORC1 activity in hPS-CM treated with 150 µM DFO. Representative image of one experiment (J) Immunoblot of mTORC1 activity in hiPS-neurons treated with 150 µM DFO. Representative image of one experiment. (K) Immunoblot of HEK293T cells chelated for 18 hours followed by addition of ferric ammonium citrate (FAC) for indicated times. Representative image of two independent experiments. (L) BrdU incorporation in HEK293T cells treated with 150 µM DFO. Representative image of six independent samples. (M) Quantification of cellular proliferation by Hoescht staining 48 hours after treatment with DFO (n = 6 replicates, two-way unpaired t-test, mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels. Source numerical data and unprocessed blots are available in source data files.
