Fig 2: 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 3: Gene expression and GS protein abundance in HB tissue samples. A GLUL expression. GS mRNA is higher than in normal tissue (NT) in all but HB16 tissue samples. mRNA of HB sample 43 could not be extracted. GLUL expression was normalized to expression of TBP. Data show means ± SD (n = 3). B ASNS expression. ASNS mRNA in the HB tissue samples is increased in all samples but HB12 compared to normal liver tissue. mRNA of HB sample 43 could not be extracted. ASNS expression was normalized to expression of TBP. Data show means ± SD (n = 3). C Western blot of GS. A representative experiment is shown. D Densitometric quantification. Relative ratio of GS/GAPDH density was normalized to ratio obtained in NT. Data show means ± SD (n = 5)

Fig 4: Effect of ASNase (A) and ASNase + MSO (AM) on GS and ASNS protein abundance in HB cell lines. A Western blot of GS and ASNS in Huh-6 cells and B in HepT1 cells. Representative experiments are shown. C Densitometric quantification of GS band in Huh-6 cells and E in HepT1 cells. ASNase increases GS protein abundance more than ASNase + MSO. D Densitometric quantification of ASNS band in Huh-6 cells and f in HepT1 cells. ASNS protein abundance is increased by ASNase, but reduced by ASNase + MSO. Relative ratio of GS/GAPDH density and of ASNS/GAPDH density were normalized to ratio obtained in NT. Data show means ± SD (n = 5). **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig 5: 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.