Fig. 2: Flux through the HBP in cancer cells is up-regulated in response to drug stress.

a DOX induces increases in the UDP-GlcNAc levels. UDP-GlcNAc in the cell lysates were derivatizated with trimethylsilyldiazomethane. Chromatograms of polar metabolites in MCF-7/ADR and SMMC-7721 cell extracts from control (red line), and 1 μM DOX-treated cells for 6 h (blue line) and 24 h (black line) show regions corresponding to the UDP-GlcNAc derivative, UDP-GalNAc derivative and probenecid retention times. Quantitative analyses are shown as means ± SEM, deviation of three independent experiments. P-values were calculated using one-way ANOVA and the appropriate post test. *P < 0.05. b The GFAT transcriptional and protein levels are upregulated in parental and resistant cell lines after transient treatment with DOX. MCF-7, MCF-7/ADR and SMMC-7721 cells were treated with DOX (0.1 μM for MCF-7, 1 μM for MCF-7/ADR and SMMC-7721) for indicated time and the GFAT transcript level was analysed by quantitative PCR. DMSO was used as a control. Whole-cell lysates were analysed by immunoblotting. c MCF-7, MCF-7/ADR and SMMC-7721 cells were treated with DOX (0.1 μM for MCF-7, 1 μM for MCF-7/ADR and SMMC-7721) for the indicated time and cell lysates were used for the analysis of GFAT activity. d SMMC-7721 and MCF-7/ADR cells were treated with 1 μM DOX alone or together with 5 μM AZA for 6 and 24 h. The protein levels after treatment with DOX or AZA for 6 h were examined by immunoblotting. DMSO was used as a control. The cell viability after treatment with DOX or AZA for 24 h was assessed through an MTS assay. e The indicated cell lines were transfected with scrambled siRNA (siRNA-Scr) or GFAT siRNA (siGFAT) for 48 h and then treated with 1 μM DOX. The protein levels after treatment with DOX for 6 h were examined by immunoblotting. The cell viability after treatment with DOX for 24 h was assessed through an MTS assay. The data represent the means ± SEM, N = 3, *p < 0.05, **p < 0.01