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: Coverslips were incubated with primary antibodies against MDM2 (86934S, Cell Signaling, 1:200) and MDMX (3807946, Millipore, 1:200) diluted in 1% BSA/PBS overnight.

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

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 HEK293T cells were transfected with a combination of MDMX, and Myc-MDM2 with or without FLT4 and harvested 24 h later for immunoprecipitation (IP) with Myc antibody-conjugated beads. Whole Cell Extract (WCE) and Myc IP samples were subjected to Western Blot analysis with the indicated antibodies. B HEK293T cells were transfected with a combination of Flag-MDMX, and MDM2 with or without FLT4 and harvested 24 h later for immunoprecipitation with Flag antibody-conjugated beads. Whole Cell Extract (WCE) and Flag IP samples were subjected to Western Blot analysis with the indicated antibodies. C HEK293T cells were transfected with various combinations of wild-type MDMX, mutant MDMX S314A, MDM2 and FLT4 plasmids. After 24 h of transfection, cells were harvested, and subjected to Western Blot analysis using the indicated antibodies. D HEK293T cells were transfected with either wild-type MDMX or mutant MDMX S314A in combination with Myc-MDM2 and FLT4 plasmids. After 24 h of transfection, cell lysates were harvested for IP with Myc-antibody-conjugated beads. WCE and Myc IP samples were subjected to Western Blot analysis with the indicated antibodies. E , F U2OS cells were transfected with various combinations of wild-type MDMX, mutant MDMX S314A, MDM2 and FLT4 plasmids. After 24 h of transfection, cells were harvested, and subjected to Western Blot analysis using the indicated antibodies. G HEK293T cells were transfected with a combination of MDM2, MDMX and FLT4 plasmids for 24 h, then treated with various concentrations of Palbociclib for 24 h. Cells were harvested and analyzed by Western Blot using the indicated antibodies. LE low exposure, HE high exposure.

Article Snippet: Coverslips were incubated with primary antibodies against MDM2 (86934S, Cell Signaling, 1:200) and MDMX (3807946, Millipore, 1:200) diluted in 1% BSA/PBS overnight.

Techniques: Transfection, Immunoprecipitation, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Regulation of the MDM2-p53 Nexus by a Nuclear Phosphoinositide and Small Heat Shock Protein Complex

doi: 10.1101/2025.04.11.648454

Figure Lengend Snippet:

Article Snippet: For double fluorescent IP-IB detecting MDM2-PIP 2 complex, anti-MDM2 rabbit monoclonal IgG antibody (clone D1V2Z, #86934, Cell Signaling) at 1:1,000 dilution and anti-PIP 2 mouse monoclonal IgM antibody (#Z-P045, Echelon Biosciences) at 1:1,000 dilution were incubated together in blocking buffer and incubated with the membrane at 4°C overnight.

Techniques: Binding Assay, Fluorescence, Microscale Thermophoresis, Concentration Assay, Incubation, Control, Software, Bacteria