Extended Data Fig. 7: The Jumonji-C KDM family inhibitor IOX1 mimics the actions of ID on mTORC1 activity.
From: Iron drives anabolic metabolism through active histone demethylation and mTORC1
(A) RT-PCR of AA transporters, RPTOR and TTP in HEK293T cells treated with various Jmj-C ___domain inhibitors for 12 hours. Internal control: POLR2A (n = 4 replicates per condition; except n = 3 TTP 1 mM DMOG, two-way unpaired t-test, mean ± s.e.m.). (B) Immunoblot of mTORC1 activity and H3K9me2 levels in A549 cells treated with 150 µM DFO or 250 µM IOX1 for 18hrs. Representative image of two independent experiments. (C) Immunoblot of indicated proteins in A549 cells treated with 250 µM IOX1 for 48 hours. Representative image of two independent experiments. (D) Immunoblot of cytosol and membrane fractions from HepG2 cells treated with 250 µM IOX1 for 12 hours. Representative image of one experiment. (E) Densitometry summary of data in Panel D (n = 3 replicates per condition, two-way unpaired t-test, mean ± s.e.m.). (F) 14C-leucine uptake into HEK293T cells treated with 150 µM DFO or 100 µM IOX1 for 18 hours (n = 5 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (G) 14C-leucine uptake into HeLa cells treated with 150 µM DFO or 100 µM IOX1 for 18 hours (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). CPM = counts per minute. (H) Immunoblot of mTORC1 activity WT and NPRL2 KO HEK293T cells treated with 250 µM IOX1 for 18 hours. Representative image of two independent experiments (I) Quantification of immunoblot in panel H (n = 3 replicates per group, one-way ANOVA with Tukey’s post-hoc test. mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels.
