Extended Data Fig. 4: ID increases H3K9 di-methylation independent of ATF4, the IRP system, and 2-HG.
From: Iron drives anabolic metabolism through active histone demethylation and mTORC1
(A) Immunoblot of H3K9me2 in hiPS-CM treated with 150 µM DFO. Representative image of one experiment. (B) Immunoblot of H3K9me2 in hiPS-Neurons treated with 150 µM DFO. Representative image of one experiment. (C) Immunoblot of H3K9me2 levels in MEFs with indicated treatments. Representative image of two independent experiments. (D, E) Summary of immunoblot in panel C (n = 3 replicates, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (F) Immunoblot of H3K9me2 in HepG2 cells expressing rtTA3/GFP-FPN with indicated treatments. Representative image of two independent experiments. (G) mRNA of indicated genes in WT and Arnt KO MEFs treated with 150 µM DFO. Internal control: 18S (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (H) Immunoblot of H3K9me2 levels in WT and Atf4 KO MEFs with indicated treatments. Representative image of one experiment. (I) Summary of immunoblot in panel H (n = 3 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (J) Immunoblot of H3K9me2 levels in WT and Irp1/2 KD/KO MEFs with indicated treatments. Representative image of two independent experiments. (K) Summary of immunoblot in panel J (n = 3 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (L) Immunoblot of IRP1 levels in Irp2 KO MEFs treated with indicated siRNA. Representative image of one experiment. (M) Summary of immunoblot in panel L (n = 3 replicates per condition, two-way unpaired t-test, mean ± s.e.m.). (N) mRNA of indicated genes in WT and Irp1/2 KD/KO MEFs treated with 150 µM DFO. Internal control: Snrk (n = 6 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (O) Ratio of 2-HG/succinate levels in HEK293T cells treated 150 µM DFO (n = 5 replicates control; n = 4 150 µM DFO, two-way unpaired t-test, median ± quartiles). (P) Immunoblot of H3K9me2 in HEK293T cells with indicated treatments. Representative image of one experiment. (Q) Immunoblot of H3K9me2 in HEK293T cells with indicated treatments. Representative image of one experiment. * indicates P value < 0.05 when noted for all panels.
