Association of ASNS methylation with ASNS gene and protein expression levels in T-ALL cell lines. (A) Induction of ASNS gene expression by asparaginase treatment in T-ALL cell lines. Each cell line was cultured in the absence or presence of 1.0 U/mL of l-asparaginase for 12 hours. ASNS gene expression level in each cell line was determined by real time RT-PCR using ACTB gene expression level as an internal control. The vertical axes indicate relative ASNS gene expression level in each cell line, which was evaluated by using P12/ICHIKAWA as a control cell line. The values were compared between untreated cells and l-asparaginase–treated cells. P values in the Wilcoxon signed-rank test are shown. (B) Association between ASNS methylation status and ASNS gene expression in T-ALL cell lines. Horizontal axes indicate percent methylation of the ASNS gene, and vertical axes indicate basal (left) and asparaginase-induced (right) ASNS gene expression level. (C) Association of ASNS gene expression with asparaginase sensitivity in T-ALL cell lines. Vertical axes indicate log IC₅₀ value of l-asparaginase, and horizontal axes indicate basal (left) and asparaginase-induced (right) ASNS gene expression level. (D) Association between ASNS methylation status and basal ASNS protein expression in T-ALL cell lines. Horizontal axis indicates percent methylation of the ASNS gene, and vertical axis indicates ASNS protein expression level in each cell line as relative value to that in the control cell line (JURKAT). (E) Association of ASNS protein expression with asparaginase sensitivity in T-ALL cell lines. The vertical axis indicates the log IC₅₀ value of l-asparaginase, and the horizontal axis indicates ASNS protein expression level in each cell line as relative value to that in the control cell line (JURKAT). Panels B to E: red, yellow, and blue circles represent ASNS highly, intermediately, and weakly methylated cell lines, respectively. Correlation coefficients and P values in the Spearman correlation test are shown.