Figure 4.

The CRISPR/Cas9-mediated knockout of 4E-BP1 in Ewing sarcoma cell lines rescues the effects of mTORC1/2 inhibitors on protein synthesis. (A) Immunoblot showing 4E-BP1 expression level after CRISPR/Cas9-mediated gene knockout. This immunoblot reflects 4E-BP1 levels in the bulk cell population prior to single cell cloning. (B, C) The 4E-BP1-KO and parental cells were treated with TAK-228 (1 μM) for 3 h or 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (D) The 4E-BP1-KO and parental cells were treated with AZD2014 (1 μM) for 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (E, F) Dose response curves for the 4E-BP1-KO and parental (4E-BP1-WT) cells. Cell viability was assessed 72 h after drug addition using the AlamarBlue assay. Error bars represent the mean ± SD of three technical replicates. The results are representative of two independent experiments. (G) RT-qPCR for RRM2 mRNA in EW8 and TC71 cells treated with TAK-228 (100 nM) for 24 h. Protein loading for all of the immunoblots was normalized using cell number.