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. Author manuscript; available in PMC: 2016 Dec 15.
Published in final edited form as: Cell Rep. 2015 Nov 25;13(10):2159–2173. doi: 10.1016/j.celrep.2015.10.073

Figure 4. Activation of JAK, AKT, and ERK Pathways Renders AML Cells Addicted to teHsp90.

Figure 4

(A and B) Immunoblot (A) and flow cytometry (B) of FL5.12 cells after 24 hr treatment with JAKi, AKTi, or PU-H71 (PU) in the presence or absence of IL-3.

(C) Percent dead cells in FL5.12 cells after 48 hr treatment with JAKi, AKTi, or PU.

(D and E) Immunoblots for pAKT (D) and percent apoptotic cells (E) of vector control- or mAKT-transfected cells treated with PU-H71, AKTi ± doxycycline (DOX) for 24 hr or 48 hr, respectively.

(F) Percentage of mCherry+ cells KG-1/N51 or KG-1/WT over 30-day cultures.

(G) Percentage of apoptotic cells after 48 hr treatment with PU-H71.

(H and I) Phosphorylated protein levels in cells evaluated by immunoblot (H) or flow cytometry (I) (fold change of MFI for KG-1/N51 relative to KG-1/WT).

(J) Immunoblots of Hsp90-interacting proteins as isolated by PU-beads.

(K) Immunoblots for Hsp90 and FLT3 immunoprecipitated with an anti-FLT3 antibody.

(L) Correlation of the apoptotic sensitivity to PU-H71 (x axis) and to AKTi, JAKi, MEK1/2i, or p38i (y axis) in cells treated for 48 hr.

(M) Percent cell death for FLT3-ITD− or FLT3-ITD+ primary AML cells after 48 hr exposure to PU-H71.

(N) Hsp90 expression of cells from (M) as measured by flow cytometry. Each symbol represents an individual sample.

Values denote mean ± SEM. *p < 0.05; **p < 0.01;***p < 0.001. See also Figure S4.