Extended Data Fig. 10: Additional characterisation of hTERT1-RPE1 GFP-MDM2 overexpressing cells.
From: MDM2 functions as a timer reporting the length of mitosis
a, Western blot of undiluted parental hTERT-RPE1 p53WT and a dilution series of GFP-MDM2OE cell lysates. GFP-MDM2 level relative to endogenous MDM2 shown in Fig. 6a was measured from this data; n = 3 independent experiments with similar results. b, MDM2 and GFP-MDM2 half-lives were measured using cycloheximide treatment of parental hTERT-RPE1 p53WT and GFP-MDM2OE cells for up to 4 h. Samples were Western blotted and MDM2 levels measured and plotted in the graphs. Half-lives were estimated using a 1-phase curve fit; n = 3 independent experiment. c, Titration of MD-224 to reveal the threshold MDM2 concentration (orange line, circles) required for p21 induction. Total p21 levels were measured by Western blot in hTERT-RPE1 p53WT (grey line, circles) and GFP-MDM2OE cells (green line, circles) (mean ± s.e.m.; n = 3 independent experiments with similar results). GFP-MDM2 levels in GFP-MDM2OE cells were also measured (orange line, circles) (mean ± s.e.m.; n = 3 independent experiments with similar results). Source numerical data and unprocessed blots are available in source data.
