Fig. 7: JMJD1C is phosphorylated at T505 by mTOR in response to insulin/feeding.

a JMJD1C demethylase activity in HepG2 cells with or without insulin treatment, n = 4 wells of cells per group (left) and in livers from fasted and fed mice, n = 3 per group (right). b Immunoblotting with phospho-serine (p-Ser) and threonine (p-Thr) antibodies using lysates from HepG2 cells with or without Insulin treatment (left). Phosphorylated peptides detected by mass spec analysis (right). c Diagram for phosphorylation sites detected by mass spec analysis (top). FAS promoter activity in HEK293 that were transfected with JMJD1C serine or threonine mutants, n = 6 wells of cells per group (bottom). d IB with JMJD1C phospo-T505-specific antibody. JMJD1C T505 phosphorylation in HepG2 cells upon insulin treatment (top) in livers of mice upon feeding (bottom). e Purified GST-JMJD1C from overexpression in E.coli was incubated with mTOR complex with or without ATP. Immunoblotting with anti-phospho-threonine antibody after SDS-PAGE separation (left). IB with phospho-T505 antibody after co-transfection with JMJD1C WT or T505A mutant with mTOR complex, after IP with JMJD1C antibody (right). f Treatment of mice with vehicle or rapamycin (10 mg/kg), IB for phosphor-T505 and total Jmjd1c (left) and lipogenic gene mRNA levels in liver (right) n = 5. g Knockdown of Raptor in mouse liver, IB for phospho-T505, total Jmjd1c, Raptor, and Gapdh (left), and mRNA levels of lipogenic genes (right), n = 5. h IB following co-IP of JMJD1C mutants with USF-1. i IB of Usf-1 following IP of Jmjd1c (top) and IB of Jmjd1c following IP of Usf-1 (bottom) from mouse fasted and refed mouse liver. j ChIP–qPCR using FAS promoter luc construct to assess enrichment of JMJD1C mutants upon insulin treatment, n = 4 wells of cells per group. k IB for Jmjd1c from livers of WT and JMJD1C-LKO mice injected with designated virus (top). RT-qPCR for lipogenic gene mRNA levels in livers from WT and JMJD1C-LKO mice 10 days after tail-vein injection of JMJD1C or its mutant T505D adenovirus in refed (left) and fasted (right) conditions, n = 5 per group. l Representative schematic of insulin signaling pathways converging on USF-1 for recruitment of JMJD1C and other co-factors for the activation of transcription of lipogenic genes. Solid lines show established pathways, dashed lines show undefined pathways. a–k Data are expressed as means ± SEM. *denotes p < 0.05, determined by two-tailed t-test. Source data are provided as a source data file.