Figure 10.

The growth inhibitory effects of CBB3001 is not reversible in PA-1 cells.

A. Actively growing PA-1 cells were treated with various concentrations of CBB3001 for 24 hours and the chemicals were removed. The cells were extensively washed and re-cultured in fresh cell culture medium without CBB3001 for 24 additional hours. Cells were examined and cells images were acquired as I Figure 3A. Triplicated cells (technical repeats) were used for examination and one set of representative treated cells was shown.

B. Triplicated treated cells (technical repeats) from Figure 10A were harvested by trypsin digestion, diluted, and blindly spotted onto a hemacytometer. Cells in four corners of the hemacytometer were counted to obtain average cells per dish. The differences between control and CBB3001 cells in triplicated samples were plotted. Experiments were repeated for three independent times with the same conclusion and one example is shown.

C. PA-1 cells were transfected with 50 nM control (luciferase, Luc) or SOX2 siRNAs for 48 hours. Left and middle panels: Cells were examined and cells images were acquired as in Figure 4A. Right panel: The levels of SOX2 and CUL1 (loading control) were examined in transfected cells by specific antibodies.