Figure 4. Ibrutinib suppressed GBM tumorigenic ability resembling Btk-gene silenced GBM cells.

A. The colony forming ability of both U87MG and DTBRG-05MG cells were significantly suppressed at low concentration of Ib (0.5 uM at IC10 for U87MG while 4 uM at IC₂₀ for DTBRG-05MG cells). B. Ibrutinib treatment significantly decreased the migratory ability of both GBM cell lines (at 0.25uM and 4uM for U87MG and DTBRG-05MG respectively, these concentrations represent the IC₂₀ values). C. Western blots of total protein lysates collected from both U87MG and DTBRG-05MG cells treated with ibrutinib (Ib), temozolomide (TMZ), and two drugs combined (Combo). The treatment conditions were as the follows: U87MG (Ib,10μM; TMZ300 μM); DBTRG-05MG (Ib, 30μM; TMZ 500 μM). The expression level of Btk, p-Akt and p-mTOR was significantly reduced by Ib treatment while TMZ only treatment moderately reduced Btk expression. The combination treatment led to the most potent suppressive effect in Btk, p-Akt, mTOR and Nestin expression. D. The GBM sphere-generating ability was significantly affected by different treatments. The most effective sphere-inhibitory effect was by the combined treatment followed by Ib alone and TMZ. (10x magnification, scale bar = 100μm). The lower panels depict the percentage survival of GBM spheres under different treatment conditions. E. Western blots analysis of total cell lysates collected from GBM spheres after different treatments. Similarly, Btk, p-Akt, p-mTOR, Nestin and Vimentin expression level was suppressed by Ib treatment, while TMZ slightly increased p-Akt expression. The combination of Ib and TMZ (combo) exerted the most inhibitory effect. Treatment conditions: U87MG (Ib, 10 μM; TMZ 300 μM), DBTRG-05MG (Ib,30 μM, TMZ 500 μM).