Fig. 6

MDM2 as a potential E3 ubiquitin ligase for FBW7 tumor suppressor. (A-B) The parental PC-9 cells were engineered to stably express the MDM2 ΔRING or MDM2 C464A mutants that lacked E3 ubiquitin ligase activity. Cells were seeded in 6 well plate and treated with DMSO or Osimertinib. Representative images of colony formation assay and TUNEL assay were shown. Scale bar = 200 μm. (C) PC-9 cells stably expressing MDM2 ΔRING or MDM2 C464A mutants were treated with increasing concentrations of Osimertinib (0, 0.1, 0.5, 1 µmol/L) for 48 h. Expression of indicated BCL-2 family members and fragmentation of PARP and caspase-3 were analyzed by immunoblotting. GAPDH was used as an equal loading control. (D) Sequence alignment of MDM2 recognition motif across FOXO3, HEXIM1, IRS1, HIPK1 and FBW7. The conservation of Q-enriched domain in the N-terminal proportion of FBW7 was assessed across different species. (E) The WCL of PC-9 cells was precipitated with anti-MDM2/anti-FBW7 antibody or equal amount of isotype IgG. The resultant immunoprecipitates were subjected to immunoblotting. (F) HEK293 cells were transfected with HA-FBW7 together with Flag-MDM2 construct. Forty-eight hours after transfection, WCL was prepared and precipitated with anti-HA/anti-Flag antibody or equal amount of isotype IgG. The cell lysates and immunoprecipitates were subjected to immunoblotting. β-actin was used as an equal loading control. (G) The effect of MDM2 on the ubiquitination status of FBW7. MDM2 was stably expressed in PC-9 cells. The ubiquitination level of FBW7 was determined by immunoprecipitation with an anti-FBW7 antibody and probed with an anti-Ub antibody. (H) HA-FBW7 and His-Ub, together with Flag MDM2 or its ΔRING and C464A mutant were transiently expressed in HEK293 cells. HA-FBW7 protein was pull down by an anti-HA antibody and probed with an anti-His antibody. (I) Myc-FBW7 and Flag-MDM2, together with His-Ub or its K48-only and K63-only construct were transfected into HEK293 cells. Forty-eight hours after transfection, Myc-FBW7 protein was precipitated with an anti-Myc antibody and probed with an anti-His antibody. (J) Flag-MDM2, His-Ub and HA-FBW7 or its ΔQ mutant were expressed in HEK293 cells. The ubiquitination status of HA-FBW7 was determined by immunoprecipitation. (K-L) The PC-9 OR cells stably expressing FBW7 or its ΔQ mutant were treated with increasing concentrations of Osimertinib and evaluated for cytotoxicity by MTT assay and apoptosis immunoblotting assay, respectively. *P < 0.05. **P < 0.01