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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Mol Cancer Res. 2019 Sep 24;17(12):2343–2355. doi: 10.1158/1541-7786.MCR-19-0245

Figure 4. ASNS mediates protein translation during asparagine deprivation.

Figure 4.

A-E) Translation rates as measured by incorporation of the methionine analog homopropargylglycine (HPG) over time and evaluated by flow cytometry. Each data point represents the slope of HPG incorporation for at least four time points using median fluorescence intensity (MFI) from an independent experiment. A) PC9 cells following 6 hr treatment as indicated. Thaps = 100 nM thapsigargin. CHX = 100 μg/mL cycloheximide, used as a control to completely block de novo protein translation. B) PC9 cells following 24 hr treatment as indicated. C) PC9 cells expressing the indicated shRNAs in +Asn or -Asn conditions for 24 hr. D) PC9 cells expressing control shRNA (Ctrl) or ectopic ASNS (ASNS) treated as indicated for 24 hr. E) PC9 cells expressing the indicated shRNAs with control or ectopic ASNS expression cultured in -Asn for 24 hr. n = 3 for all experiments, error bars show SD, and p-values by Unpaired t-test.