Figure 3.

Inhibition of mTORC1/2 activates 4E-BP1, blocks protein synthesis, and reduces the level of the RRM2 protein. (A, B, C) Ewing sarcoma cell lines were treated with TAK-228 (1 μM) or temsirolimus (1 μM) for 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (D, E) Ewing sarcoma cell lines 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. (F) Sarcoma cell lines were treated with TAK-228 (1 μM) for 6 h. Cells were labeled with puromycin to quantify protein synthesis and then lysates were collected for immunoblotting. (G) EW8 and TC71 cells were treated with TAK-228 (100 nM), AZD1775 (100 nM), or the combination of the drugs for 6 h. (H, I) EW8 and A673 cells were treated for 72 h with a combination of TAK-228 and AZD1775, or TAK-228 in combination with prexasertib. Survival was assayed by Cell-Titer-Glo and each experiment was repeated 2 times. Loewe matrix plots for drug cooperativity are shown. Protein loading for all of the immunoblots was normalized using cell number.