Fig 2: qRT-PCR analysis of G6pc, Glut1, Hk2, G6pdx, Mct4, and Gls in HCCs of mice treated with DEN and TCPOBOP. (A–E) qRT-PCR analysis of G6pc, Glut1, Hk2, G6pdx, and Mct4 in 15 HCCs of mice treated with a single dose of DEN, followed by repeated treatment with TCPOBOP and killed 28 weeks after DEN treatment. In the HCC group, each dot corresponds to 1 tumor analyzed. Gene expression is reported as fold change relative to livers from untreated mice. Results are expressed as means ± SD. *P < .05. (F) qRT-PCR analysis of Gls in HCCs from mice treated as in panel A. Gene expression is reported as fold change relative to livers from untreated mice. Results are expressed as means ± SD. (G) Immunohistochemical analysis of a mouse HCC. (H) Immunohistochemical analysis of a rat preneoplastic nodule stained with an antibody antiglutaminase (left, ×20; right, ×5). CTRL, control; DEN, Diethylnitrosamine; GLS, glutaminase; TCP, TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene).

Fig 3: Inhibition of Gls1 alters multiple signaling pathways in LSCs and synergizes with PMF in suppressing LSCs. (A–C) GSEA showed enrichment of gene sets with decreased expression including “HALLMARK OXIDATIVE PHOSPHORYLATION”, “NOTCH SIGNALING PATHWAY”, “EWSR1 FLI1 FUSION UP” and (D) increased expression for “HOXA9 DN.V1 UP” in GFP+ LSK cells post BPTES treatment. (E–F) The expression of gene in KEGG pathways were enriched and significantly altered (change fold=2, P < 0.05) post PMF and BPTES treatment. The Venn diagrams demonstrate that the expression of gene in the listed KEGG pathways were decreased post PMF treatment but were increased post BPTES treatment, as well as the expression of genes involved in the listed pathways were decreased after BPTES treatment, but increased after PMF treatment. (G) Left panel: the relative mRNA level of c-Myc, E2f1, Ezh2, Alox5 and Alox15 in RNA-seq data; Right panel: CML mice were induced by BCR-ABL(T315I), on day 14 post BMT, and recipients were treated with Placebo or BPTES (10 mg/kg) for 24 h, then LSCs were sorted with FACS (n = 3, each group). The mRNA levels of BCR-ABL, c-Myc, E2f1, Ezh2, Alox5 and Alox15 in LSCs were examined with RT-PCR. (H) LAMA84 cells were cultured in presence of the indicated concentration of free PM and BPTES for 24 h, then cells were harvested for western blotting analysis with anti BCR-ABL, c-MYC, EZH2, ALOX5, ALOX15, HOXA9, HSP90, P21 and P27 antibodies. GAPDH served as the total protein loading control. (I) A schematic diagram for the regulatory mechanisms of synergistic effects of PM and BPTES. Results were represented with the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered as significant difference.

Fig 4: PMF increases glutamine metabolism by increasing GLS1 expression. (A) GSEA demonstrated that PMF significantly enriched and increased the expression of genes involved in “GO REGULATION GLUTAMATE RECEPTOR SIGNALING PATHWAY” gene set. (B) LAMA84 and K562 cells were treated with free PM at indicated concentration for 24 h, then cells were harvested for western blotting analysis with anti GLS1 antibody. GAPDH served as the total protein loading control. (C) Primary leukemia cells of spleen from CML mice were treated with free PM at indicated concentrations for 24 h, then cells were harvested for mRNA examination with RT-PCR and western blotting analysis with anti Gls1 antibody, Gapdh served as the total protein loading control. (D) On day 14 post BMT, CML mice were treated with Placebo or PMF (10 mg/kg, n = 3, each group) for 24 h. LSCs were sorted by FACS and Gls1 expression was examined with cellular immunofluorescence, and nucleus was stained with DAPI. (E) A schematic diagram of glutaminolysis and TCA cycle. (F) K562 cells were treated with free PM at different concentration for 24 h, the contents of glutamate, a-ketoglutarate and Succinyl-CoA contents in cells were measured with LC-MS/MS. (G–I) CML mice treated with PMF (10 mg/kg) for 24 h, the contents of glutamate, a-ketoglutarate and Succinyl-CoA in the spleen were measured with LC-MS/MS. The relative content was normalized to that in Placebo treated mice. Results were represented with the mean ± SEM. *P < 0.05, and **P < 0.01 were considered as significant difference.