Figure 2

AMBRA1 deregulation dynamically altered the cell cycle arrest in hTERT-RPE1 cells. (A) Flow cytometry analysis of AMBRA1-altered hTERT-RPE1 cells. Cells were stained with Ki67, a cell proliferation marker rarely detected in the non-dividing G0 phase and highly expressed in proliferating cells (maximum in the G2 and early M phases), and PI, a DNA marker, staining proportionally to the DNA content of the cell. Staining cells with both Ki67 and PI successfully differentiated each cell cycle phase, represented in boxes of different colors (G0—black; G1—blue; S—pink; G2/M—orange). Analysis was performed in triplicate. A representative graph for each genotype is shown. (B) Bar graph representing the percentage of AMBRA1-depleted and Q30R mutant hTERT-RPE1 cells distributed in the S+G2/M phase under nutrient-sufficient conditions, calculated from the flow cytometry result (A, top). Statistical significance was determined by ANOVA. (C) Bar graph representing the percentage of starved AMBRA1-depleted and Q30R mutant hTERT-RPE1 cells distributed in the S+G2/M phase, calculated from the flow cytometry result ((A), bottom). Statistical significance was determined by ANOVA. (D) Stacked bar graph representing the percentage of AMBRA1-depleted and Q30R mutant hTERT-RPE1 cells distributed in different cell cycle phases under nutrient-sufficient conditions. (E) Stacked bar graph representing the percentage of starved AMBRA1-depleted and Q30R mutant hTERT-RPE1 cells distributed in different cell cycle phases.