Fig. 4.

Effect of 2HF on cell cycle progression in RCC. Inhibitory effect of 2HF on cell cycle distribution was determined by fluorescence activated cell sorting analysis. Experimental details are given in the Methods section. The stained cells were analyzed using the Beckman Coulter Cytomics FC500, Flow Cytometry Analyzer. The experiment was repeated three times and similar results were obtained (panel A). The cell morphology was observed and imaged at ×40 magnification in phase contrast microscope (Axio observer A1; Carl Zeiss microimaging, Thornwood, NY). Arrows in the panel point towards cells arrested in or completing cytokinesis (panel B). VHL-wild-type (Caki-2) and VHL-mutant (786-O) control and 50 μM 2HF-treated cells were processed for western blot analysis for cyclin B1 and CDK4 expression by using specific antibodies. Membranes were stripped and reprobed for GAPDH as a loading control (panel C).