Fig 2: Gene expression, protein abundance and effect of glutamine depletion in HB cell lines. A GLUL expression. GS mRNA levels in Huh-6 and HepT1 cells are comparable. GLUL expression was normalized to expression of TBP. Data show means ± SD (n = 3). B ASNS expression. Significantly higher ASNS mRNA level in HepT1 cells (**p < 0.01). ASNS expression was normalized to expression of TBP. Data show means ± SD (n = 3). C Western blot of GS and ASNS. A representative experiment is shown. d Densitometric quantification of GS band. Data show means ± SD (n = 5). e Densitometric quantification of ASNS band. Data show means ± SD (n = 5). GS and ASNS protein abundances in Huh-6 and in HepT1 cells is comparable. f Effect of glutamine depletion on cell number in Huh-6 cells. G Effect of glutamine depletion on cell number in HepT1 cells. H Effect of glutamine depletion on cell viability in Huh-6 cells. I Effect of glutamine depletion on cell viability in HepT1 cells. In each experiment, treatment with the combination showed a greater effect than the treatment with the single substances. Results are expressed as per cent [%] of untreated control cultures. Data show means ± SD (n = 3). The logarithmic plot of the X-axis is modified to show the values without ASNase. If MSO was used, the concentration was 1 mM

Fig 3: GLUL and ASNS expression in HB and overall survival. GLUL and ASNS expression levels of HB (n = 25) and normal liver tissues (NT) (n = 4) samples are shown. Each data point reflects the GLUL and ASNS level of one sample. Relative expression levels of genes are given as signal log ratio (SLR). A Scatter plot of the data. Expression of GLUL and ASNS is significant higher in HB than in NT (p = 0.0003 and p = 0.01, respectively). Pearson’s correlation coefficient for GLUL and ASNS expression is r = − 0.57. B Expression levels according to C1 and C2 tumour type. Expression levels by tumour type are different for GLUL (*p < 0.05) but not for ASNS (not significant). C GLUL expression values separated by a cut off value of SLR 12.0. Significant difference between high and low expression group (***p < 0.001). D ASNS expression values separated by a cut off value of SLR 6.75. Significant difference between high and low expression group (***p < 0.001). E Kaplan–Meier survival curves stratified according to high and low GLUL expression. Median overall survival time is 68 months for the low GLUL expression group, but not reached for the high GLUL expression group. Log rank test for group comparison p = 0.1855. F Kaplan–Meier survival curves stratified according to high and low ASNS. Median overall survival not defined for the groups. Log rank test for group comparison p = 0.7125.

Fig 4: Effect of ASNase (A) and ASNase + MSO (AM) on ASNS and GLUL expression in HB cell lines. A GLUL expression in Huh-6 cells and C in HepT1cells. GS mRNA is decreased in Huh-6 cells, but increased in HepT1 cells by ASNase + MSO. B ASNS expression in Huh-6 cells and D in HepT1 cells. ASNS mRNA in both cell lines is increased more by the combination than by ASNase alone. Relative GLUL expression (GLUL /TBP) and relative ASNS expression (ASNS /TBP) were normalized to expression in controls. Data show means ± SD (n = 3). *p < 0.05; ***p < 0.001; ****p < 0.0001

Fig 5: Overview of differentially expressed genes in tamoxifen-sensitive (TS) and tamoxifen-resistant (TR) estrogen receptor-positive (ER+) breast cancer patients visualized in a heatmap format. GLUL (glutamate-ammonia ligase) was one of the significantly upregulated genes in TR ER+ compared to TS ER+ breast cancer patients.