Figure 2. Inhibitory effects of dual JAK2 and mTOR inhibition on the PI3K/mTOR pathway.

(A) Phospho-flow cytometry analysis demonstrated increased phosphorylation of JAK-2(Tyr1008), STAT5(Ty694)), ERK(T202/Y204) and AKT/pS6 [AKT(Ser473)],4E-BP1(T37/46), and rS6(S240/244) after stimulation with TSLP(25ng/mL) for 30 min. MHH-CALL4 and MUTZ-5 cells were treated with indicated compounds for 1 h, then the phosphorylation signals of PI3K/mTOR pathway proteins were determined by phospho-flow cytometry. Heat map data depict the changes in phosphoprotein levels by median fluorescence intensity. Color scale represents normalization (Z-score) of each condition for each signal. (B) MHH-CALL4 cells were stimulated with TSLP for 30min followed by treatment with ruxolitinib (Ruxo), BBT594 (BBT), rapamycin (Rap), or AZD2014 (AZD) or one of their combinations at the indicated doses for 1 h, after which the expression of PI3K/mTOR pathway proteins were probed by Western blot. (C) MHH-CALL4 cells were infected with empty vector (EV), with a wild-type (WT) or with mutant (5A) 4E-BP1 constructs, then treated with indicated concentrations of doxycycline (Doxy) for 72 h to induce p-4E-BP1 expression. Levels of p-4E-BP1 were determined by Western blot. (D) MHH-CALL4 cells transfected with 4E-BP1 constructs were treated with doxycycline (1μg/mL) for 72 h to induce 4E-BP1 and then with ruxolitinib (Ruxo) or BBT594 (BBT) for 72 h. Cell viability was analyzed by CTG assay. ^* p<0.05, ^** p<0.005, ^*** p<0.0005, ^**** p<0.0001 as determined by unpaired Student t-test.