Effect of AT13387 on C666-1 tumor sphere. (A) AT13387 was added on day 0 of C666-1 tumor sphere formation. Upper panel: image of tumor spheres formed from C666-1 single cell culture with and without AT13387 treatment. AT13387 significantly inhibited tumor sphere formation. Scale bar = 100 μm. Lower panel: Total number of tumor spheres per culture. Tumor spheres formed with diameter reaching 20 μm were counted. Results were expressed as the mean ± S.D. of three experiments; *p < 0.05. (B) Size profile of C666-1 tumor spheres. AT13387 was added to the tumor sphere culture after tumor spheres were formed for 7 days. The diameters of tumor spheres were measured after AT13387 treatment for 7 more days and presented as the size profile of tumor sphere. The mean diameters of tumor spheres with AT13387 treatment were significantly smaller than the untreated control (p < 0.05), showing the inhibitory effect of AT13387 on the growth of C666-1 tumor sphere. Scale bar = 50 μm. Results were expressed as the mean ± S.D. of three experiments. (C) Confocal image showing pseudocolor green for CD44 and red for SOX2. Loss of CD44 was observed in 1 μM AT13387-treated C666-1 tumor sphere and loss of both CD44 and SOX2 were observed in 10 μM AT13387-treated C666-1 tumor sphere. Scale bar = 20 μm. (D) FACS analysis showing the decrease of CD44hi and SOX2hi population in AT13387-treated C666-1 tumor sphere. Results were expressed as the mean ± S.D. of three experiments; *p < 0.05.