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. Author manuscript; available in PMC: 2015 Jun 7.
Published in final edited form as: Leukemia. 2012 Jan 6;26(6):1277–1285. doi: 10.1038/leu.2012.2

Figure 3.

Figure 3

GSK3 inhibition significantly enhances ATRA's activity. (a, b) GSK3 inhibitors enhance ATRA-mediated differentiation. Cells were treated with SB (15 μm), AT (50 nm for HL-60 and OCI-AML3 and 10 nm for NB4) or a combination for 4 days and differentiation was assessed by NBT reduction and CD11b staining. *P < 0.05, **P < 0.001 when comparing single treatments (AT or SB) to the combination treatment. In all cases, SB and AT demonstrated synergistic effects in inducing NBT reduction and CD11b expression as measured by the excess over Bliss independence method. (c) GSK3 inhibition and ATRA dramatically inhibit colony formation in AML cell lines. Cells were treated with vehicle, ATRA (10 or 50 nm), SB (15 μm), Lithium (10 mm) or GSK3 inhibitor and ATRA for 72 h and a colony assay was performed as described in Figure 2. (d) The combination of GSK3 inhibition and ATRA can differentiate HL-60 cells into neutrophils. HL-60 cells were treated as indicated and stained as described in Figure 1c to assess morphology. (e) GSK3 inhibition and ATRA can inhibit colony growth in an AML patient sample. An AML patient sample (AML-M2) was treated as described in Figure 3c and tested for colony growth. (f) GSK3 inhibition does not significantly impair colony growth from normal bone marrow. Normal human bone marrow cells were treated as described in Figure 3c and plated in methylcellulose with cytokines and the resulting erythroid and myeloid colony types were counted after 14 days. (g) The combination of GSK3 inhibition and ATRA lead to enhanced changes in the expression of growth and differentiation related genes. OCI-AML3 cells were treated with ATRA (0.1 μm), SB (15 μm) or a combination for 72 h and the expression of p21 and cyclin A was measured by western and c-myc and CEBP/ε by real-time PCR. (h) GSK3 inhibition can enhance differentiation mediated by retinoids besides ATRA. HL-60 cells were treated for 4 days with AM80 (10 μm), Adapalene (1 μm), ATRA (0.1 μm), SB (15 μm) or a combination and the NBT reduction assay was performed.