This study shows that MDMX is essential for the regulation of p53 protein levels in the context of an endogenous C-terminal tail mutant MDM2. Furthermore, MDMX activity in this context relies on MDM2-MDMX binding, is inhibited by DNA damage, and is localized to the cytoplasm. These results have implications for the function of MDM2-MDMX heterodimers and the viability of the MDM2Y487A/Y487A mutant mouse model.
Experimental procedures
Cell culture and reagents
Mouse embryonic fibroblasts (MEF) were generated from MDM2Y487A/Y487A and MDM2+/+ embryos and passage 0 MEF cells were frozen down in liquid nitrogen for future use. MEF cells were cultured in a humidified 37 °C, 5% CO2, 3% O2 incubator. Cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma) supplied with 10% fetal bovine serum (Sigma) and penicillin (100 IU/ml)/streptomycin (Sigma, 100 mg/ml). HEK293 cells were obtained through the ATCC and cultured in a humidified 37 °C, 5% CO2 incubator in Dulbecco’s modified Eagle’s medium (Sigma) supplied with 10% fetal bovine serum (Sigma) and penicillin (100 IU/ml)/streptomycin (Sigma, 100 mg/ml).
Antibodies
The following primary antibodies were used for western blot analysis: mouse anti-actin (MAB1501, Chemicon), mouse anti-p53 (pAb122), mouse anti-MDM2 (2A10), mouse anti-MDMX (Sigma MDMX Clone-82, M0445), mouse anti-GAPDH (ThermoFisher, MA5–15738), rabbit anti-Myc (gift from Yue Xiong), mouse anti-GFP (NeoMarkers, clone AB-2). The following antibody was used for immunofluorescent staining: mouse anti-MDMX (Clone 7A8, gift from Jiandong Chen).
siRNA treatment
Cells were treated with 20 μM of siRNA using the Lipofectamine RNAiMax reagent (ThermoFisher, catalog #13778150). Cells were analyzed 32–36 h after siRNA transfection. MDMX-targeting siRNA duplexes were purchased from Invitrogen (Stealth RNAi siRNA Duplex, siRNA 1 ID: MSS206594, siRNA 2 ID: MSS206595, siRNA 3 ID: MSS206596).
Western blotting
For western blot analysis, cells were lysed in 0.5% Nonidet P-40 buffer containing protease inhibitor mixture (leupeptin, catalog no. L2884; aprotinin, catalog no. A1155; benzamidine, catalog no. B6506; and trypsin inhibitor, catalog no. T9003; all from Sigma), 1 mM PMSF (Sigma, catalog no. P7626), 1 mM NaVO3 (Fisher Scientific, catalog no. S454–50), and 1 mM DTT (Roche, catalog no. 03117014001) for 30 min. 100-150 μg of protein was separated on a 12.5% SDS-PAGE gel prior to transfer to a nitrocellulose membrane (BioRad). Transfers were assessed by staining the membranes with Ponceau S (Sigma, catalog no. P3504) for 5 min, followed by several brief washes with double-distilled H2O. Membranes were blocked for at least 1 h in phosphate-buffered saline containing 5% nonfat milk and 0.1% Tween 20. Membranes were then incubated with the appropriate primary antibody diluted in blocking buffer for 2 h to overnight. Membranes were washed three times in phosphate-buffered saline containing 0.1% Tween 20 and then incubated with the appropriate HRP-conjugated secondary antibody diluted in blocking buffer for 1 h. Membranes were washed four times in PBS-T and then developed with Supersignal West Pico chemiluminescent substrate or Supersignal West Dura chemiluminescent substrate according to the instructions of the manufacturer (Pierce, catalog no. 34080 and 34,075).
Lentiviral transduction
Lentiviral overexpression constructs were generated using the gateway cloning system to insert MDMX constructs into the pLenti4 puro-DEST vector. Vector sequences were verified by DNA sequencing both before and after gateway insertion. For shRNA treatments, MDMX-targeting or nonspecific shRNAs were inserted into the pLKO.1 vector and verified by DNA sequencing. The following shRNA sequences were used to target MDMX: MDMX shRNA 1: GCAAGAAGTTTAATTCTCCAA, MDMX shRNA 2: GCAGAATTTCTTCGGAACAAA, MDMX shRNA 3: CCCGATTGTAGGAGAACCATT, MDMX shRNA 4: CTCAACTGATTTACAGACAAA, MDMX shRNA 5: GCGCGAGAGAACAAACAGATA. Lentiviral particles were generated by the UNC Lenti-shRNA Core Facility. For lentiviral transduction, cells were plated at 60% confluence in a 6-well plate and treated with 25-200 μL of lentiviral supernatant in 1 mL total media containing 8 μg/mL hexadimethrine bromide (polybrene, Sigma catalog #107689). Cells were incubated with lentiviral particles for 48 h prior to lysis for protein analysis.
Immunoprecipitation
HEK293 cells were plated at 70% confluence in a 60 mm dish and then transfected with 1 μg of plasmid mixture using the Effectene transfection reagent (Qiagen, catalog #301425). 24 h after transfection, cells were lysed in 0.1% nonidet P-40 buffer containing protease inhibitor, PMSF, NaVO3 and DTT. 100 μg of lysate was used as loading control, while 1 mg of lysate was used for immunoprecipitation (IP). Prior to IP, lysates were pre-cleared using 75-100 μL of CL4B beads. IP was performed using anti-Flag M2 affinity gel (Sigma, catalog #A2220) by rotating lysates with 5 μL Flag beads at 4 °C overnight. Beads were then washed three times with 0.1% nonidet P-40 buffer. Precipitated proteins were then isolated in 1X SDS loading buffer and analyzed via western blot.
Immunofluorescent staining
MEF cells were plated at low confluence and treated with a low volume (50 μL) of lentiviral particles for 48 h prior to fixation using 10% formalin solution (Sigma, catalog #HT501128). Cells were permeabilized using 0.2% Triton X-100 diluted in phosphate buffered saline for five minutes at 4 °C. Permeabilized cells were then incubated in 0.5% bovine serum albumin (BSA) solution for at least 30 min before overnight exposure to primary antibody solution. Cells were then treated with AlexaFluor 594-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories) for 30–45 min. Followed by incubating with 4,6-diamidino-2-phenylindole (DAPI) for 2 min for nuclear counterstaining. After staining, cells were analyzed using an Olympus IX-81 microscope fitted with a SPOT camera and software. Signal brightness and contrast was increased equally for all images using ImageJ software for clear signal visualization.