Journal: Oncogenesis

Article Title: FLT4 activation promotes acute lymphoid leukemia survival through stabilization of MDM2/MDMX and inactivation of p53

doi: 10.1038/s41389-025-00552-7

Figure Lengend Snippet: A The frequency of p53 mutation in Diffuse Large B Cell lymphoma (DLBC) patients (DFCI, Nat Med, 2018 ), (Broad, PNAS, 2012), (BCGSC, Blood, 2013 ), (TCGA, PanCancer Atlas), (Duke, Cell, 2017) , ALL patients (St Jude, Nat Genet, 2016), (St Jude, Nat Genet, 2015) , and pediatric ALL patients (TARGET, 2018)—across public datasets in https://www.cbioportal.org . B U2OS cells were transfected with an empty or FLT4 expression plasmid along with a pg13 luciferase reporter vector or a luciferase reporter for p21 or MDM2 to determine p53 target genes activity. C U2OS cells were transfected with an empty or FLT4 expression vector for 24 h. The cells were then treated with 10 µg/mL of 5-FU for 6 h. The cell lysates were blotted for the indicated antibodies. D U2OS cells were transfected with different combinations of FLT4, MDM2, and MDMX plasmids along with pg13 luciferase reporter vector to determine endogenous p53 under genotoxic conditions with 5-FU. E HEK293T cells were transfected with various amounts of FLT4 expression plasmid for 24 h. The cell lysates were blotted for the indicated antibodies. F HEK293T cells were transfected with a combination of FLT4, MDMX and MDM2 expression plasmids for 24 h. The cell lysates were blotted for the indicated antibodies (LE low exposure, HE high exposure). G U2OS cells were transfected with a combination of FLT4, MDMX or MDM2 expression plasmids for 24 h. The cell lysates were blotted for the indicated antibodies. H U2OS cells were transfected with a combination of FLT4, MDMX, wild-type MDM2 or mutant MDM2 C464A expression plasmids for 24 h. The cell lysates were blotted for the indicated antibodies. I U2OS cells were transfected with a combination of FLT4, MDMX, and MDM2 expression plasmids and immunostained for MDMX (green) and MDM2 (red), while the nuclei were stained with Hoechst. Scale bar: 20 μm. * p < 0.05, **** p < 0.0001.

Article Snippet: Expression plasmids, including pCMV-Myc-MDM2, pCMV-MDM2 C464A mutant, pcDNA3-FLT4, and pcDNA3-Flag-MDMX, were obtained from Addgene (#16441, #12086, #119230, and previously described [ ], respectively).

Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Staining

Journal: Cancers

Article Title: NORE1A Regulates MDM2 Via β-TrCP

doi: 10.3390/cancers8040039

Figure Lengend Snippet: ( A ) HEK-293 cells were transfected with expression constructs expressing MDM2 paired with an empty vector or constructs expressing NORE1A, β-TrCP, or the dominant negative form of β-TrCP (ΔFBOX). NORE1A expression on its own has a substantial suppressive effect on MDM2 stability, and this effect is magnified when co-expressed with β-TrCP. However, the dominant negative β-TrCPΔFBOX reduces this effect, suggesting that NORE1A is cooperatively working with β-TrCP to regulate MDM2 protein levels; ( B ) MDM2 has the ability to self-ubiquitinate, and we sought to confirm whether NORE1A was degrading MDM2 via β-TrCP or inducing MDM2 auto-ubiquitination. We transfected HEK-293 cells with wild-type MDM2 and a mutant of MDM2 that is unable to auto-ubiquitinate (C464A) paired with an empty vector or NORE1A. We found that NORE1A was still able to suppress both the wild type and mutant MDM2, suggesting that indeed NORE1A is degrading MDM2 via β-TrCP, and not inducing MDM2 auto-degradation. Representative blots of 2 independent experiments are shown.

Article Snippet: Wild-type human MDM2 and mutant MDM2, unable to autoubiquitinate themselves (C464A), were obtained from Addgene (#20935 and #12086) [ ]. pcDNA-HA-NORE1A, pEGFP-NORE1A, pEGFP-β-TrCP, pEGFP-C1-β-TrCPΔFBOX, and pmKate-2-C-H-Ras12V have been previously described [ , , ].

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Dominant Negative Mutation, Ubiquitin Proteomics, Mutagenesis