Fig 2: GLS1 expression is associated with stemness in HCC. (a) HCC cells (HCCLM3, SMMC-7721, and Hep3B) were cultured in the presence or absence of glutamine for 72 h before cells were trypsinized and counted. (b) HCCLM3 were cultured in DMEM with glutamine withdraw for 24, 48, or 72 h. qRT-PCR was performed to detect the stemness markers. (c) HCC cells were cultured in the presence or absence of 10 µM 968 for 72 h before cells were trypsinized and counted. (d) HCCLM3 were treated with the indicated concentration of 968 for 48 h, then the expression levels of CSC markers were analyzed by qRT-PCR. (e) Cell lines with stable knockout of GLS1 were established using a CRISPR/Cas9 system. Knockout of GLS1 in SMMC-7721 (f), HCCLM3 (g) and Hep3B (h) downregulated the expression of stemness genes. Data are presented as mean ± SD. **, p < .01; ***, p < .001; ****, p < .0001.

Fig 3: GLS1 regulates in vivo tumorigenicity. 2 × 106 of HCCLM3 (a–c) and SMMC-7721 (d–f) stable cell lines were subcutaneously inoculated into both flanks of nude mice, with GLS1 knockout on the left flanks, and wide-type cells on the right flanks. Tumor volumes were monitored every three days. Five weeks after the injection, mice were photographed and tumors were collected. Representative tumor images and tumor growth curves are shown. The tumors volumes were analyzed by the repeated measures ANOVA. Data are presented as mean ± SD. **P < .01, ***p < .001. (g) The tumors isolated from nude mice were subjected to Western blot to validate the GLS1 knockout efficiency. (h) 5 × 105 of GLS1 KO or wide-type SMMC-7721 cells were subcutaneously inoculated into both flanks of nude mice. Mice were sacrificed after six weeks postinoculation and tumors samples were collected. (i) Schematic model illustrating the effect of GLS1 on Wnt/ß-catenin signaling and stemness in HCC. Targeting GLS1 triggers an increase of ROS, attenuates nuclear translocation of ß-catenin and subsequent inhibits the ß-catenin target genes, leading to the suppression of CSC properties in HCC. Abbreviations: Gln, Glutamine; GSH, Glutathione; Glu, Glutamate; Cys, Cysteine; Gly, Glycine; ROS, Reactive oxygen species; TCF/LEF, T-cell factor/lymphoid enhancer factor.

Fig 4: 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 5: Expression levels and localization of the GLS1 isoforms. (a) Pan-cancer analysis of GLS1 expression across cancers from TCGA. Cancer types with >10 paired normal samples were included in the analysis. The boxplot displays the first quartile, median, and the third quartile of the data. Abbreviations: BLCA: Bladder urothelial carcinoma; BRCA: Breast invasive carcinoma; CHOL: Cholangiocarcinoma; COAD: Colon adenocarcinoma; ESCA: Esophageal carcinoma; HNSC: Head and neck squamous cell carcinoma; KICH: Kidney chromophobe; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; PRAD: Prostate adenocarcinoma; READ: Rectum adenocarcinoma; STAD: Stomach adenocarcinoma; THCA: Thyroid carcinoma; UCEC: Uterine corpus endometrial carcinoma. (b) Western blot analysis of GLS1 expression in HCC and matched non-tumor tissues. N: normal liver tissue; C: cancer tissue. See also Fig. S1a. (c) Western blot analysis of GLS1 expression in HCC cell lines. (d) The expression levels of GLS1 mRNA in HCC cell lines. (e) The GAC/KGA mRNA ratio in HCC cell lines. (f) Relative mRNA levels of KGA and GAC isoforms in 67 pairs of HCC and matched non-tumorous tissues. (g) High expression of GAC predicts a poor prognosis based on TCGA dataset. (h) HCCLM3 cells stably expressing KGA (lvKGA) or GAC (lvGAC) or empty vector (lvCON) were established using lentiviral approach, then the cell clones derived from single cells were detected for gene expression with FLAG-tag antibody. (i) KGA and GAC location was confirmed by immunofluorescence of HCCLM3 cells stained with FLAG-tag antibody, MitoTracker and Hoechst 33342. Scale bars: 20 µm. (j) The mitochondrial location of KGA and GAC were confirmed by Western blot. (k) Cytoplasmic and nuclear fractions of HCCLM3 and SMMC-7721 cells were isolated to analyze the subcellular location of GLS1. Antibodies to Lamin B1 and vinculin were used as a marker for nuclear and cytoplasmic fractions. (l) Immunofluorescence using anti-Tom20 (OMM protein), anti-HSP60 (Matrix protein), and anti-GLS1 antibodies following permeabilization with 50 µg/mL digitonin or 1% Triton X-100 were shown of HCCLM3 cells. Cells were counterstained with DAPI to show the cell nucleus (blue). Scale bars: 20 µm. Data are presented as mean ± SD. *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001.