Fig 2: MAPK13 phosphorylates PHGDH at S371 and promotes its protein degradation upon oxidative stress.a SFB-PHGDH was pulled down from SK-Hep1 cells stably expressing SFB-PHGDH treated with or without 0.5 mM H2O2 and 20 μM CQ for 3 h. The mass spectra of peptide harboring modified S371 residue was presented. Predicted b-type and y-type ions (not including all) were listed above and below the peptide sequence, respectively. b c57BL/6 J mice were intraperitoneally injected with 250 mg/kg APAP. Mouse livers were collected at 0 h, 4 h or 8 h after APAP treatment. Immunohistochemistry (IHC) staining of liver section using anti-PHGDH pS371 antibody was performed. Representative images of IHC staining were presented on the left panel, and statistical analysis were shown on the right panel (n = 5 in each time point). c SK-Hep1 cells were transiently transfected with Flag-PHGDH or Flag-PHGDH S371A. Cells were then treated with 0.5 mM H2O2 and 50 μg/mL CHX as indicated time. IB, immunoblotting. d SK-Hep1 cells were transiently transfected with Flag-PHGDH WT or 5 times Flag-PHGDH S371D plasmid and then treated with 50 μg/mL CHX as indicated time. e SK-Hep1 cells were transiently transfected with Flag-PHGDH WT or Flag-PHGDH S371D and then treated with or without 20 μM CQ for 16 h. f Flag-PHGDH was immunoprecipitated from SK-Hep1 cells transiently transfected with Flag-PHGDH WT or twice dose of Flag-PHGDH S371D plasmid. 36 h after transfection, the endogenous interacting proteins (LAMP2A and HSC70) were detected. g SK-Hep1 cells were transiently transfected with Flag-PHGDH WT or Flag-PHGDH S371A and HA-HSC70. 24 h after transfection, the cells were treated with 20 μM CQ and Flag-PHGDH was immunoprecipitated from the cells after treated with or without 0.5 mM H2O2 for 12 h. The interacting proteins (LAMP2A and HA-HSC70) were detected. h Flag-PHGDH was immunoprecipitated from SK-Hep1 cells co-transfected with EV or Flag-PHGDH and HA-MAPK13. i In vitro kinase assays were performed by mixing purified His-MAPK13 F324S and His-PHGDH WT or His-PHGDH S371A protein. j PHGDH was immunoprecipitated from hepatocytes isolated from WT and Mapk13 KO mice which were then treated with or without 0.5 mM H2O2 and 10 μM CQ for 4 h. k Hepatocytes isolated from WT and Mapk13 KO mice were treated with or without 0.5 mM H2O2 for 16 h. l SK-Hep1 cells stably expressing shNT or shMAPK13 were transiently transfected with Flag-PHGDH, Flag-PHGDH S371A or Flag-PHGDH S371D, and then treated with 0.5 mM H2O2 for 24 h. m Hepatocytes isolated from WT and Mapk13 KO mice transiently transfected with Flag-Phgdh WT or Flag-Phgdh S371D. Cells were treated with 0.5 mM H2O2 for 16 h. Immunoblots are representative of three independent experiments. Data are means ± SD. Each point represents an individual mouse. P values were determined by two-tailed Student’s t-test.

Fig 3: MAPK13 deficiency alleviates oxidative stress-associated acute or chronic liver injury.a Primary hepatocytes isolated from c57BL/6 J mice were transiently transfected with HA-Mapk13. Cells were then treated with 0.5 mM H2O2 for 0 h, 4 h or 8 h. HA-Mapk13 was immunoprecipitated and phosphorylation of Mapk13 was detected. b‒d WT and Mapk13 KO mice were intraperitoneally injected with normal saline (control) or 300 mg/kg APAP. After 24 h treatment, serum ALT and AST levels in these mice were detected by kit (b). Positive liver cells in TUNEL-stained sections were quantified (c). Liver necrosis area in H&E-stained sections were circled and quantified (d). Representative images of H&E staining or TUNEL staining were shown on the left panel and statistical analysis were shown on the right panel (n = 5 per group). e Survival during 72 h of WT and Mapk13 KO mice intraperitoneally injected with normal saline (control) or 800 mg/kg APAP (n = 13 per group). f‒h WT and Mapk13 KO mice were fed with irradiated diet with or without 0.1% DDC for 2 weeks. Serum ALT and AST levels were detected in these mice (f). Mouse livers were dissected and subjected to Sirius Red staining (g) and TUNEL staining (h) (n = 5 per group). i WT and Mapk13 KO mice were intraperitoneally injected with normal saline (control) or 300 mg/kg APAP. Livers were collected at 16 h after treatment. Immunoblotting analysis was performed (n = 2 per group). j WT and Mapk13 KO mice were intraperitoneally injected with normal saline or 300 mg/kg APAP. The GSH level in mouse livers was measured (n = 5 per group). Immunoblots are representative of three independent experiments. Survival data are based on Long-rank test (e). Other data are means ± SD. Each point or lane represents an individual mouse. P values were determined by two-tailed Student’s t-test.

Fig 4: MAPK13-dependent PHGDH pS371 sensitizes hepatocytes to oxidative stress-induced cell death.a, b Primary hepatocytes isolated from PhgdhLKO mice were transfected with Flag-Phgdh WT, Flag-Phgdh S371A or Flag-Phgdh S371D. Cells were treated with 10 μM CQ for 24 h (a). Transfected cells were treated with 0.5 mM H2O2 for 24 h. Cells were stained with 1 μg/mL PI and 10 μg/mL Hoechst, and the percentage of dead cells was quantified (b). c, d SK-Hep1 cells stably expressing shPHGDH were infected with lentivirus expressing shNT or shMAPK13 and then infected with lentivirus expressing Flag-rPHGDH WT or Flag-rPHGDH S371D respectively. Cells then were treated with 20 μM CQ for 24 h (c). Infected cells were treated with 0.5 mM H2O2 for 24 h. Cell viability was determined using trypan blue staining (d). e, f Primary hepatocytes isolated from PhgdhLKO mice were transfected with siNC or siMapk13 and then transfected with Flag-Phgdh WT or Flag-Phgdh S371D. Cells were treated with 10 μM CQ for 24 h (e). Transfected cells were treated with 0.5 mM H2O2 for 24 h. Cells were then stained with 1 μg/mL PI and 10 μg/mL Hoechst, and the percentage of dead cells was quantified (f). Immunoblots are representative of three independent experiments. Data represent the means ± SD of three independent experiments. P values were determined by two-tailed Student’s t-test.