Extended Data Fig. 7: MD-224 results in rapid and reversible MDM2 destruction in hTERT-RPE1 cells.
From: MDM2 functions as a timer reporting the length of mitosis
a, A cartoon showing the mode of action for MD-224. b, hTERT-RPE1 p53WT cells depleted of CRBN with siRNA were treated with solvent (–) or 100 nM MD-224 (+) for 1 h, and Western blotted. MD-224 requires CRBN for MDM2 destruction. Non-targeting siRNA was used as a control. Relative depletion of MDM2 upon MD-224 treatment is plotted for each siRNA condition on the right (mean ± s.d.; n = 3 independent experiments; p = 1.5E−11 siControl –MD-224 vs. siControl +MD-224, p = 0.37 siCRBN –MD-224 vs. siCRBN +MD-224). c, hTERT-RPE1 p53WT or p53KO cells were treated with 100 nM MD-224 for 1 h (+) or a solvent control (–). MD-224 was then washed out with fresh growth medium for 0.5 to 2 h and Western blotted for MDM2, p53 and p21. Tubulin was used as a loading control. Relative recovery of MDM2 following MD-224 washout is plotted on the right for each cell line (mean ± s.e.m.; n = 3 independent experiments). d, The amount of MDM2 was measured by Western blotting in asynchronous (AS) hTERT-RPE1 p53WT and p53KO cells treated for 1.5 h with (+) or without (–) the proteasome inhibitor MG132 (20 µM) and for 1 h with (+) or without (–) 100 nM MD-224. Relative depletion of MDM2 in each condition is plotted on the right for each cell line (mean ± s.d.; n = 3 independent experiments; p53WT cells, p = 7.7E−7 –MG132 –MD-224 vs. –MG132 + MD-224, p = 0.46 + MG132 –MD-224 vs. +MG132 + MD-224; p53KO cells, p = 4.5E−5 –MG132 –MD-224 vs. –MG132 + MD-224, p = 0.32 + MG132 –MD-224 vs. +MG132 + MD-224). e, The amount of MDM2 was measured in asynchronous cells (AS) or mitotic hTERT-RPE1 p53WT and p53KO cells arrested for 1 h with 25 ng/ml nocodazole ( + Noc) in the presence (+) or absence (–) of 100 nM MD-224. MDM2 was detected by Western blot using two different antibodies. n = 5 independent experiments with similar results (see Fig. 4). f, The amount of MDM2 was measured using Western blotting in asynchronous cells (AS) or mitotic hTERT-RPE1 p53WT cells arrested for 45 min with 25 ng/ml nocodazole ( + Noc) in the presence (+) or absence (–) of 100 nM MD-224 and the proteasome inhibitor MG132 (20 µM). Relative MDM2 levels for each mitotic condition are plotted on the right (mean ± s.d.; n = 3 independent experiments; p = 2.8E−6 –MG132 –MD-224 vs. –MG132 + MD-224, p = 0.84 + MG132 –MD-224 vs. +MG132 + MD-224). Data were analysed using unpaired two-tailed t-test (b,d,f) (ns, not significant; ****, p < 0.0001). Source numerical data and unprocessed blots are available in source data.
