Fig 2: SET7 has no obvious effect on EGLN1 protein level and vice versa.A, Western blot analysis of endogenous EGLN1 expression in HEK293T cells transfected with an increasing amount of Myc-SET7 expression plasmid. B, Western blot analysis of endogenous EGLN1 expression in SET7-deficient or WT HEK293T cells (SET7-/- or SET7+/+). C, Western blot analysis of endogenous EGLN1 expression in SET7-deficient HEK293T cells (SET7-/-) transfected with indicated plasmids. D, Western blot analysis of endogenous EGLN1 expression in H1299 cells transfected with an increasing amount of Myc-SET7 expression plasmid. E, Western blot analysis of endogenous SET7 expression in H1299 cells transfected with an increasing amount of HA-EGLN1 expression plasmid. F, Western blot analysis of endogenous SET7 expression in EGLN1-deficient or WT H1299 cells (EGLN1-/- or EGLN1+/+). EGLN, egg-laying defective nine; HA, hemagglutinin.

Fig 3: SET7 methylates EGLN1 on lysine 297.A, sequence alignment of partial EGLN1 proteins (285–307 amino acids) of human, mouse, rabbit, dog, and pig. The red box indicates a conserved consensus motif (R/K-S/T-K) methylated by SET7; the blue arrow indicates the conserved lysine (K297) methylated by SET7. The amino acid is numbered based on human EGLN1 amino acid sequence. B, Western blot analysis of EGLN1 or its mutant (K297A) methylated by WT SET7 or the enzymatic-deficiency mutant of SET7 (H297A) in HEK293T cells transfected with indicated plasmids. Anti-HA antibody–conjugated agarose beads were used for immunoprecipitation and anti-EGLN1-K297Me1 antibody was used for detecting monomethyl EGLN1. C, the methylated residue in EGLN1 identified by mass spectrometry analysis. HEK293T cells were cotransfected with HA-EGLN1 and Myc-SET7 plasmids. Cell lysate was immunoprecipitated with anti-HA antibody–conjugated agarose beads overnight. Immunoprecipitated EGLN1 proteins were subjected to 8% SDS-PAGE gel, and EGLN1 bands were excised from the gel and analyzed by mass spectrometry. D, Western blot analysis of endogenous methylated EGLN1 in SET7-deficient or WT HEK293T cells (SET7-/- or SET7+/+). Anti-EGLN1 antibody was used for immunoprecipitation, and normal rabbit IgG was used as a control. Anti-EGLN1-K297Me1 antibody was used to detect monomethyl EGLN1. E, Western blot analysis of endogenous methylated EGLN1 in SET7-deficient HEK293T cells (SET7-/-) transfected with indicated plasmids. Anti-EGLN1 antibody was used for immunoprecipitation, and anti-EGLN1-K297Me1 antibody was used to detect monomethyl EGLN1. EGLN, egg-laying defective nine; HA, hemagglutinin; IgG, immunoglobulin G.

Fig 4: The prolyl hydroxylase activity of EGLN1 is attenuated by SET7-mediated methylation on K297.A, Western blot analysis of endogenous EGLN1 and HIF1a expression in EGLN1-deficient or WT H1299 cells (EGLN1-/- or EGLN1+/+). B, Western blot analysis of exogenous Myc-HIF1a expression in EGLN1-deficient H1299 cells (EGLN1-/-) transfected with indicated plasmid. The cells were cotransfected with Myc-HIF1a and WT FLAG-EGLN1 or the enzymatically deficient mutant (H313A), the methylation-mimic mutant (K297F). FLAG empty was used as a control. The relative intensities of Myc-HIF1a were determined by normalizing the intensities of Myc-HIF1a to the intensities of ß-ACTIN. C, Western blot analysis of HIF1a hydroxylation with anti-hydroxy-HIF1a (Pro564) antibody in EGLN1-deficient H1299 cells (EGLN1-/-) transfected with indicated plasmid. The cells were cotransfected with Myc-HIF1a and WT FLAG-EGLN1 or the enzymatically deficient mutant (H313A), the methylation-mimic mutant (K297F), followed by MG-132 (20 µM) treatment for 8 h. FLAG empty was used as a control. The relative intensities of HIF1a-OH/Myc-HIF1a were determined by normalizing the intensities of HIF1a-OH to the intensities of Myc-HIF1a. D, Western blot analysis of HIF1a hydroxylation with anti-Hydroxy-HIF1a (Pro564) antibody in HEK293T transfected with indicated plasmid. The cells were cotransfected with Myc-HIF1a, FLAG-EGLN1, and HA-SET7 (HA empty was used as a control), followed by MG-132 (20 µM) treatment for 8 h. The relative intensities of HIF1a-OH/Myc-HIF1a were determined by normalizing the intensities of HIF1a-OH to the intensities of Myc-HIF1a. E, Western blot analysis of endogenous HIF1a expression in EGLN1-deficient H1299 cells (EGLN1-/-) transfected with indicated plasmid. The cells were transfected with WT FLAG-EGLN1 or the methylation-mimic mutant (K297F). FLAG empty was used as a control. The relative intensities of HIF1a were determined by normalizing the intensities of HIF1a to the intensities of GAPDH. F, Western blot analysis of HIF1a hydroxylation with anti-hydroxy-HIF1a (Pro564) antibody in EGLN1-deficient H1299 cells (EGLN1-/-) transfected with indicated plasmid. The cells were transfected with WT FLAG-EGLN1 or the methylation-mimic mutant (K297F) (HA empty was used as a control), followed by MG-132 (20 µM) treatment for 8 h. The relative intensities of HIF1a-OH/HIF1a were determined by normalizing the intensities of HIF1a-OH to the intensities of HIF1a. G, Western blot analysis of HIF1a hydroxylation with anti-hydroxy-HIF1a (Pro564) antibody in SET7-deficient or WT H1299 cells (SET7-/- or SET7+/+) treated with MG-132 (20 µM) for 8 h. The relative intensities of HIF1a-OH/HIF1a were determined by normalizing the intensities of HIF1a-OH to the intensities of HIF1a. H, Western blot analysis of exogenous Myc-HIF1a expression in HEK293T transfected with indicated plasmid. The cells were cotransfected with Myc-HIF1a, FLAG-EGLN1, and HA-SET7 (HA empty was used as a control), followed by CHX (50 µg/ml) treatment for indicated times. Myc empty was used as a control. The relative intensities of Myc-HIF1a were determined by normalizing the intensities of Myc-HIF1a to the intensities of ß-actin. CHX, cycloheximide; EGLN, egg-laying defective nine; HA, hemagglutinin; HIF, hypoxia-inducible factor.

Fig 5: SET7-mediated methylation of OTUB1 relieves its suppressive role on ferroptosis. A, quantitative real-time PCR (qPCR) analysis of PTGS2 mRNA in OTUB1-deficient H1299 cells (OTUB1-/-) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with or without TBH (100 µM) for 7 h. B, qPCR analysis of PTGS2 mRNA in OTUB1-deficient H1299 cells (OTUB1-/-) reconstituted with OTUB1 or its methylation-site mutant (OTUB1-K122A) by lentivirus and treated with or without TBH (100 µM) for 7 h. C, cell viability of OTUB1-deficient H1299 cells (OTUB1-/-) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with or without TBH (150 µM or 200 µM) for 7 h and examined by CCK8 assay. D, cell viability of OTUB1-deficient H1299 cells (OTUB1-/-) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with DMSO (as a control) or Erastin (20 µM) for 24 h and examined by CCK8 assay. E, cell viability of OTUB1-deficient H1299 cells (OTUB1-/-) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with or without cystine starvation for 24 h and examined by CCK8 assay. F and G, intracellular ROS in OTUB1-deficient H1299 cells (OTUB1-/-) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with or without Cystine starvation for 24 h and examined by flow cytometry. Quantification of the intracellular ROS levels in (F) and representative flow cytometry histogram in (G). H and I, intracellular ROS in OTUB1-deficient H1299 cells (OTUB1-/-) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with DMSO (as a control) or Erastin (20 µM) for 24 h and examined by flow cytometry. Quantification of the intracellular ROS levels in (H) and representative flow cytometry histogram in (I). Two-way ANOVA analysis; Data show mean ± SD; Tukey’s multiple comparisons test; *Adjusted p < 0.05, **Adjusted p < 0.01, ***Adjusted p < 0.001, ****Adjusted p < 0.0001; Data from three independent experiments. CCK8, Cell Counting Kit-8; ROS, reactive oxygen species; TBH, Tert-Butyl hydroperoxide.