Fig 2: IPMK Enhances Transcription of Autophagy-Related Genes(A) qPCR analysis of LC3B, BNIP3, BNIP3L, p62, GABARAPL1, and ATG12 in IPMK WT and KO MEFs after GluStv.(B) Western blot of BNIP3L, ATG12, and GABARAPL1 in IPMK WT and KO MEFs after GluStv.(C) Western blot of BNIP3L, ATG12, and GABARAPL1 in F/F and Cre (IPMK KO) mouse livers after 24 h of food starvation.(D) IPMK KO MEFs were stably transfected with empty vector (myc), wIPMK myc, and kinase-dead myc (KSA) IPMK. Shown is a qPCR analysis of LC3B, BNIP3, BNIP3L, p62, GABARAPL1, and ATG12 in myc, wIPMK, and KSA MEFs.(E) Western blot analysis of histone 4 lysine 16 acetylation (H4k16ac).(F) Chromatin immunoprecipitation of H4k16 at the LC3B promoter from WT and KO MEFs. n = 3, ***p < 0.001.(G) Immunoprecipitation of Sirt-1 and western blot of DBC1 before and after GluStv in WT and KO MEFs.(H) HEK293 cells were transfected with FLAG Sirt-1 and myc AMPK or empty vector of myc. Immunoprecipitation of myc was followed by FLAG western blotting.(I) HEK293 cells were transfected with FLAG Sirt-1 and myc IPMK and empty vector of myc. Immunoprecipitation of myc was followed by FLAG western blotting.Data are means ± SD.

Fig 3: IPMK Regulates ULK Phosphorylation by Direct Binding Interactions(A) Empty myc, empty FLAG, myc IPMK, and FLAG ULK1 were co-transfected in HEK293 cells, and co-immunoprecipitation was performed (n = 4).(B) Glutathione S-transferase (GST), GST IPMK, and GST IPMK in combination with small interfering RNA (siRNA) of ULK1 and scrambled (sc) siRNA were co-transfected in HEK293 cells. Immunoprecipitation of GST was followed by western blotting of endogenous ULK1.(C) Schematic diagram of fragments of IPMK.(D) Different fragments of GST IPMK were cotransfected with myc ULK1. Immunoprecipitation of GST followed by western blot of myc was performed to map IPMK binding to ULK1 (n = 3).(E) Analysis of dominant negatives to assess binding of IPMK to ULK1 (n = 3).(F) Overexpression of an IPMK dominant-negative fragment (fragment 3) in HEK293 cells, followed by GluStv for 6 h. Functional evaluation of the dominant-negative action of fragment 3 was performed by immunoblotting LC3II and ULK serine 555 (n = 3).(G and H) Relative amounts of LC3II (G) and ULK serine 555 (H) are plotted (n = 3); ***p < 0.01, *p < 0.05. Data are means ± SD.(I) Schematic diagram of IPMK binding to ULK1, which facilitates ULK1 phosphorylation and activation of autophagy. Data are means ± SD.

Fig 4: IPMK Is Required for Lipophagy, Cytoprotection, and Cell Regeneration(A) Triglyceride levels in IPMK WT and KO MEFs treated in regular medium (RM) or oleate (OL), measured by staining with boron-dipyrromethene (BODIPY) 493/503. Relative amounts of lipid droplets (LDs) were plotted. ***p < 0.001. Data are means ± SD. Scale bar, 20 μM.(B) IPMK F/F and AlbCre mice were starved for 24 h, and livers were harvested for electron microscopy analysis of lipid droplets in untreated and 0-h- and 24-h-starved mice. Lipid droplets were counted and are represented as bars. ***p < 0.001. Data are means ± SD. Scale bar, 2 μM.(C) Histology of intact livers from IPMK F/F and AlbCre mice. Tissue sections were stained with H&E to check cellular texture and immunostained with F4/80 to monitor inflammatory cells and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for apoptosis of hepatocytes. Inflammatory cells and TUNEL-positive cells were counted and are represented as bars. n = 4, ***p < 0.001. Data are means ± SD. Scale bar, 200 μM.(D) Schematics of the experimental protocol. IPMK F/F and AlbCre mice were injected intraperitoneally with a single acute dose (2 μg/g) of Ccl4, followed by liver harvest after 48 h of treatment.(E) Staining of liver sections with H&E, F4/80, TUNEL, and Ki67. Inflammatory cells, TUNEL-positive cells, and Ki67-stained cells were counted and are represented as bars. n = 4, ***p < 0.001. Data are means ± SD. Scale bar, 200 μM.(F) EDU incorporation in vehicle-treated and Ccl4-treated mouse livers. EDU-positive cells were counted and are represented as bars. n = 4. ***p<0.001. Data are means ± SD. Scale bar, 20 μM.

Fig 5: IPMK Is Required for Autophagy(A) IPMK wild-type (WT) and (KO) MEFs were stably transfected with GFP-LC3. Cells were subjected to Baf A1 (100 nM), glucose starvation (GluStv), and GluStv + Baf A1 (100 nM). GFP-LC3 puncta were analyzed using confocal microscopy. Scale bar, 20 µM. The bar chart shows numbers of puncta per cell.(B) Transmission electron microscopy (TEM) of WT and KO MEFs subjected to different treatments. AV, autophagic vacuole. Scale bar, 2 µM. Autophagic vacuoles per cell are shown as bar diagrams.(C) The basal level of autophagy was evaluated by western blotting LC3 with Baf A1 (100 nM). The bar chart depicts the densitometric relative value of LC3-II and Actin. n = 3, ***p < 0.001.(D) LC3 western blot to check autophagic flux under GluStv and GluStv + Baf A1.(E) Western blot of the IPMK level in F/F and IPMK-deleted (Cre) livers.(F) LC3 western blot in F/F and Cre (IPMK KO) livers and after 24 h of food starvation.(G) IPMK KO MEFs were stably transfected with empty vector (myc), IPMK WT (wIPMK) myc, and kinase-dead myc (KSA) IPMK. Autophagy was evaluated by western blotting LC3 II levels with and without GluStv. Baf A1 (100 nM) was used to analyze autophagic flux.(H) NeoR-GFP was transiently transfected in WT and KO MEFs. Twenty-four hours after transfection, cells were analyzed using confocal microscopy. Scale bar, 20 µM. The bar chart depicts the mean fluorescence level of GFP.(I) The amount of NeoR-GFP was analyzed by western blotting NeoR-GFP in WT and KO MEF.Data are means ± SD.