Fig 2: Mitochondrial level of GLS1 isoforms: GAC (A) and KGA (B) and glutamate dehydrogenase (GDH) (C) in controls and gerbils subjected to 5 min ischemia and 1 and 96 h of reperfusion (IR 1 h, IR 96 h). 20 µg of pure mitochondrial fractions were separated by 10% SDS-PAGE and analyzed by Western blot with anti-GAC, anti-KGA, or anti-GDH and anti-pyruvate carboxylase (PC) to assess the gel loading. The immunoblots are representative of four independent experiments. Densities of GAC, KGA and GDH bands were evaluated, and data are expressed as a percentage of PC (mean ± SD, n = 4). * p < 0.05, ** p < 0.001.

Fig 3: Glutaminase activity (GLS1) is higher in CA2-4,DG than in CA1. Mitochondria enriched fractions obtained from hippocampal CA1 and CA2-4,DG of control gerbils and gerbils subjected to 5 min ischemia and 1 h of reperfusion (IR 1h) with or without PKCßII inhibitor ßIIV5-3 were used to measure glutaminase activity (A) and glutamine (B) and glutamate concentration (C). Results are mean ± SD (n = 4–7), * p < 0.05 ** p < 0.01 *** p < 0.001.

Fig 4: GLS1 regulates CSC properties via ROS/Wnt/ß-catenin signaling. Determination of cellular ROS in HCCLM3 cells. Cells were cultured with glutamine deprivation or 10 µM 968 for 48 h, and ROS was detected using DCFH-DA by flow cytometry (a) or confocal (b). H2O2 was used as positive control. Scale bars: 100 µm. (c) Colony formation assay was performed on HCCLM3 cell line treated with indicated concentration of H2O2. (d) H2O2 treatment suppressed the expression of KLF4, c-MYC, and ß-catenin. (e) Simplified schematic of the glutamine metabolism pathways. (f) HCCLM3 cells were assayed for colony formation in complete DMEM medium or without glutamine or in the presence of 10 µM 968 alone or together with 2 mM DM-a-KG or 4 mM NAC. (g) HCCLM3 cells were treated with 5 µM 968 for two weeks, then metabolomic analysis was performed to screen for the differentially expressed metabolites. (h) Genes associated with GLS1 were analyzed by gene set enrichment analysis (GSEA) based on TCGA dataset. Wnt/ß-catenin pathway was enriched. (i) HCCLM3 was treated with the indicated concentration of SKL2001 for 24 h, then GLS1 mRNA expression was examined. (j) HCCLM3 cells were treated with the indicated concentration of SKL2001 for 48 h (upper panel) or treated with 10 µM SKL2001 for 12, 24, and 48 h (lower panel), then GLS1 protein expression was measured. (k) HCCLM3 cells were treated with different concentration of 968 for 24 h. The mRNA levels of ß-catenin (CTNNB1) and its target genes (CCND1, BIRC5, BCL2, and AXIN2) were determined by q-PCR. (l) HCCLM3 cells were pretreated with 10 or 20 µM 968 for 24 h, then treated with SKL2001 for another 24 h. Cells were harvested and fractionated into cytoplasmic and nuclear fractions, thereafter the expression of GLS1, ß-catenin protein levels were determined by Western blot. GAPDH and lamin B1 were detected as fractionation and loading controls. (m) SMMC-7721 cells with stable knockout of GLS1 by CRISPR/Cas9 was treated with SKL2001 for 24 h, then cytoplasmic and nuclear fractions were prepared and subjected to Western blot with anti-GLS1 and anti-ß-catenin antibodies. Data are presented as mean ± SD. ****, p < .0001.

Fig 5: Expression of GAC and GLUD1 in TCGA and MLL AML datasets respectivelyA. Principal genes related to Gln metabolism. B. Boxplot comparing expression values of GAC and C. GLUD1 from the TCGA AML dataset. Expression represents batch-effect adjusted, normalized, log2-transformed RPKM (Reads per Kilobase of transcript per Million mapped reads) values. D. GAC and E. GLUD1 expression from the MLL AML dataset in different cytogenetic abnormality categories. The p-values were determined by ANOVA. Asterisk and red boxes denote categories with statistically significant higher GAC expression compared to healthy normal donors; median GAC in normal controls is shown by dotted red line. NK NPM, AML with normal karyotype, NPM-mutant; FLT3-LM NPM, AML with normal karyotype, FLT3-ITD length mutation and NPM-co-mutated; NK, normal karyotype AML.