Figure 7. p53^3KR Inhibits Glucose Uptake, Glycolysis, Reactive Oxygen Species Level and Colony Formation.

(A) Glucose uptake measurement of p53^+/+, p53^3KR/3KR and p53^−/− MEF cells determined by the uptake of 2-[³H]-deoxyglucose. Results shown are the averages ± SD of three different experiments.

(B) Glycolysis rate analysis of p53^+/+, p53^3KR/3KR and p53^−/− MEF cells determined by monitoring the conversion of 5-[³H] glucose to ³H₂O as described in the Experimental Procedures. Values represent averages ± SD of three different experiments.

(C) Measurement of the reactive oxygen species (ROS) levels of p53^+/+, p53^3KR/3KR and p53^−/− MEF cells was performed by using mouse ROS ELISA kit as described in the Experimental Procedures. Data was reported as averages ± SD of three different experiments.

(D) Western blot analysis of p53-3KR, Mdm2 and Gls2 proteins in H1299 cells transfected with FLAG-tagged expression plasmids as indicated using anti-FLAG antibody. β-actin was used as a loading control.

(E) Colony formation assay of H1299 cells transfected with p53-3KR, Mdm2 and Gls2. H1299 cells were transfected with either empty vector, or FLAG-p53-3KR, FLAG-Mdm2, or FLAG-Gls2 expression plasmids for 48 hrs, and then split and subject to colony formation assay visualized by crystal violet staining.

(F) Quantification of colonies formed in H1299 cells transfected with empty vector, FLAG-p53-3KR, FLAG-Mdm2, and FLAG-Gls2 expression plasmids after 12 days culture in the presence of G418. Graphs are presented as percentage of colonies of empty vector transfected cells and values are shown as average percentage ± SD of three different experiments.