Figure 6.

Impact of ectopic expression of ASNS variants in ASNS-null JRS cells. Stable cell lines were generated from ASNS-null JRS cells transduced to express WT, G373V, or R519H variant ASNS protein. A, GFP was coexpressed with the ASNS and shows the relative expression of the transduced plasmids. The top panels show the bright field image (BF). B, flow cytometry was used to quantitate the percent of cells in the population that expressed GFP for nontransduced JRS cells (“JRS only”) or cells transduced with vector encoding WT, G373V, or R519H ASNS. C, immunoblotting of total cell extracts from the stable cell lines described in panel (B) was used to detect relative variant protein abundance, and (D) qRT-PCR analysis was used to measure ASNS mRNA levels. E, cell proliferation of the stable cell lines described in panel (B) was analyzed after incubation in DMEM ± Asn for 24, 48, and 72 h. The results are presented as viable cell numbers only measured with an automated cell counter using a trypan blue exclusion method. For panels (D) and (E), the data are shown as the averages ± SDs for assays in triplicate. Each experiment was repeated at least once with different batches of cells to establish reproducibility. For panel (D), an asterisk indicates a p value of ≤ 0.05 compared to JRS only. For panel (E), where not shown, the SD bars are within the symbol and an asterisk indicates a p value of ≤ 0.05 compared to the DMEM + Asn condition. ASNS, asparagine synthetase; JRS, Jensen rat sarcoma.