Extended Data Fig. 3: Leucine sensing is required for mTORC1 inactivation by ID.
From: Iron drives anabolic metabolism through active histone demethylation and mTORC1
(A) Ttp mRNA in WT and Tsc2 KO MEFs treated with 150 µM DFO. Internal control: Snrk (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (B) Immunoblot of mTORC1 activity in WT and Tsc2 KO MEFs with indicated treatments. Representative image of three independent experiments. (C) REDD1 mRNA in HEK293T cells treated with siREDD1. Internal control: POL2RA (n = 6 replicates per condition, two-way unpaired t-test, mean ± s.e.m.). (D) TFRC mRNA in HEK293T cells treated with siREDD1 and 150 µM DFO. Internal control: POL2RA (n = 6 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (E) Immunoblot of mTORC1 and AMPK activity with indicated treatments. Representative image of one experiment (F) Ttp mRNA in WT and Ampkα1/2 dKO cells treated with 150 µM DFO. Internal control: Polr2a (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (G) Acridine orange staining in HEK293T cells treated with 150 µM DFO. Representative image of six independent samples. (H) Summary of the results in Panel G (n = 7 replicates control; n = 6 150 µM DFO, two-way unpaired t-test, mean ± s.e.m.). (I) Leucine levels in HEK293T cells treated with 150 µM DFO (n = 5 replicates control; n = 4 150 µM DFO two-way unpaired t-test, median ± quartiles). (J) SAM levels in HEK293T cells treated with 150 µM DFO (n = 5 replicates control; n = 4 150 µM DFO, two-way unpaired t-test, median ± quartiles). (K) Immunoblot of mTORC1 activity in WT and NPRL2 KO HEK293T cells with indicated treatments. Representative image of one experiment. (L) mTORC1 localization to lysosomes in NPRL2 KO cells treated with 150 µM DFO. Representative image of six independent samples. (M) Quantification of images in panel L. (n = 5 replicates control; n = 6 150 µM DFO, two-way unpaired t-test, mean ± s.e.m.) (N) Cell death using Hoescht and propidium iodide (PI) in WT and NPRL2 KO HEK293T cells treated with 150 µM DFO for indicated times. Representative image of six independent samples. (O) Quantification of images in panel N. (n = 6 replicates, two-way unpaired t-test, mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels.
