Figure 2.

Differential effects of heat shock protein 90 (HSP90) inhibitors on cell survival and death between human uroepithelial cells and bladder cancer cells as determined by assessment of cell viability and the activity of caspases 3 and 7 using the Celigo image cytometer. 5637 and SV-HUC cells (1 × 10⁴ per well) were evenly distributed in 96-well plates overnight. Cells were incubated with AUY922 (10 nM), ganetespib (10 nM), SNX2112 (100 nM), or AT13387 (100 nM) for 48 h. (A) For analyzing cell viability, 5637 or SV-HUC cells were simultaneously stained with a mixture of calcein AM, propidium iodide, and Hoechst 33342 reagents for respective staining of live, dead, and all cells, and the percentage of viable cells was quantified with the Celigo imaging cytometer. Cell viability values are expressed relative to those cells without HSP90 inhibitor treatment (100% control value). *p < 0.01 versus the untreated control group; *p < 0.05 versus SV-HUC cells of the same group. (B) For the caspase 3/7 assay, the above mentioned HSP90 inhibitor-treated 5637 or SV-HUC cells were stained with Nexcelom ViaStain^TM Caspase 3/7 reagent and Hoechst 33342, as described in the “Methods”. Caspase 3/7 positive cells were identified using the Celigo imaging cytometer, and the percentage of apoptotic caspase 3/7 positive cells was calculated with the Celigo software. The data presented are representative of those obtained from three separate experiments. *p < 0.01 versus the untreated control group; *p < 0.05 versus SV-HUC cells of the same group. Control, untreated; STA9090, ganetespib.