pcmv mdm2 plasmid  (Addgene inc)

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: bioRxiv

Article Title: ADAR1 Regulates Lipid Remodeling through MDM2 to Dictate Ferroptosis Sensitivity

doi: 10.1101/2025.01.16.633410

Figure Lengend Snippet: ADAR1 regulates MDM2 expression to dictate ferroptosis sensitivity A) Venn diagram of significantly downregulated genes in ADAR1-Deficient over -Intact TNBC cell lines (MDA-MB-231, MDA-MB-468, HCC1806, BT549). B) Venn diagram of significantly upregulated genes in ADAR1-Deficient over -Intact TNBC cell lines (MDA-MB-231, MDA- MB-468, HCC1806, BT549). C) The ten most significantly altered signaling pathways (BioPlanet_2019) in ADAR1-Deficient over -Intact TNBC cell lines. Genes significantly altered (both up- and down-regulated) in at least 3 out of 4 cell lines tested are included in the analysis. D) The ten most significantly altered disease-related biological functions (PheWeb_2019) in ADAR1-Deficient over -Intact TNBC cell lines. Genes significantly altered in at least 3 out of 4 cell lines tested are included in the analysis. E) GSEA enrichment plot of ferroptosis related genes in ADAR1-Deficient MDA-MB-231 cells. F) GSEA enrichment plot of fatty acid metabolism genes in ADAR1-Deficient MDA-MB-231 cells. G) Venn diagram of the most significantly downregulated genes (log2<-0.4) in ADAR1-Deficient over -Intact TNBC cell lines. Genes altered in all 4 cell lines are listed on the right. H) Venn diagram of the most significantly upregulated genes (log2>0.4) in ADAR1-Deficient over -Intact TNBC cell lines. Genes altered in all 4 cell lines are listed on the right. I) Immunoblot analysis showed significant induction of MDM2 protein levels in ADAR1-Deficient (shADAR1) MDA-MB-231 cells. Images are representative of three replicates. GAPDH, loading control. J) Reported ADAR1 editing site (bolded and underlined) in MDM2 3’UTR. Numbers mark the location within MDM2 open reading frame. K) Reduced editing of MDM2 in ADAR1-Deficient TNBC cells. T/C were read with reverse primers in Sanger sequencing to indicate WT (T) and edited (C) RNA. Ratio of C/T peak heights indicates level of editing. Sequence chromatograms showed are representative of 3 independent experiments. L) Immunoblot analysis showed overexpression of MDM2 in MDA-MB-231 cells. Anti-MDM2 antibody detected both endogenous and overexpressed MDM2, and anti-Myc-tag antibody detected overexpressed MDM2 containing Myc-tag. GAPDH, loading control. ADAR1 level was not affected by MDM2 overexpression. M) Cell viability analysis was performed to determine IC 50 values of ferroptosis inducers RSL3 for MDA-MB-231 cells with (MDM2) or without (Ctrl) MDM2 overexpression. Cell viability graph is representative of three independent experiments. FC, fold-change. N) Relative abundance of PUFA PC species, listed in the order of lipid unsaturation, in MDA-MB-231 cells with (red dots) or without (blue dots) MDM2 overexpression for 96 h. Values were log10 transformed, mean centered, and shown as mean over standard deviation ratio for each PC species. n=5. O) Relative abundance of PC42:7 between control (Ctrl) and MDM2- overexpressing MDA-MB-231 cells. Detected peak intensity was log10 transformed and mean normalized. n=5. P) Relative cell viability within each of ADAR1-Intact (shNT) and -Deficient (shADAR1) MDA-MB-231 cells treated with RSL3 (100nM), as well as ADAR1-Deficient MDA-MB-231 cells co-treated with RSL3 and MDM2 inhibitors MX69 (10µM), MEL23 (10µM), and SP-141 (10µM), or PPAR-α agonist pirinixic acid (PA; 10µM) for 72h. n=3. Error bars mark standard error. See also Figure S3 and Table S1-4 .

Article Snippet: Tet-inducible MDM2- overexpressing construct was generated by subcloning MDM2 open-reading frame (ORF) from pCMV-myc3-MDM2 (Addgene 20935) into pSBtet-GP sleeping beauty vector (Addgene 60495) using the Sfil restriction enzyme site. pSBtet-GP-MDM2 and pCMV(CAT)T7-SB100 transposase construct (Addgene 34879) were co-transfected into MDA-MB-231 cells to generate a puromycin-resistant stable cell line.

Techniques: Expressing, Western Blot, Control, Sequencing, Over Expression, Transformation Assay, Standard Deviation

Journal: bioRxiv

Article Title: ADAR1 Regulates Lipid Remodeling through MDM2 to Dictate Ferroptosis Sensitivity

doi: 10.1101/2025.01.16.633410

Figure Lengend Snippet: ADAR1 loss synergizes with cobimetinib to suppress tumorigenesis A) Drug screen designed to identify ferroptosis modulators synergizing with ADAR1 loss. B) Anticipated results in identifying ADAR1-sensitized ferroptosis modulators. Small molecules (Exampled by blue dots) that display ADAR1-sensitized activity against cancer cells will reduce viability of ADAR1-Deficient (shADAR1) cells more strongly compared to ADAR1-Intact (shNT) cells. C) Screening result with a ferroptosis-focused library (654 small molecules) in MDA-MB-231 cells. X and Y axis represent cell viability for ADAR1-Intact (shNT) and - Deficient (shADAR1) cells after treatment, respectively. Data were normalized to cells treated with DMSO. RSL3 treatment was included as positive control for ADAR1-sensitized hits (red dots). Dashed line denotes expected locations for molecules without discriminating activity based on ADAR1 expression. D) Cell viability ratio of 0.51 (horizontal red line) between shADAR1 and shNT cells was used as the cutoff to identify ADAR1-sensitized hits (red dots). RSL3 treatment was included as positive control. E) List of hits identified from C) and D), ranked by cell viability ratio between treated shADAR1 and shNT MDA-MB-231 cells. F) Progression of overall tumor volumes throughout the treatment regimen, n=5-10 each group. Error bars mark standard deviation. G) Quantification of areas under curve (AUC) for overall tumor volumes from F). Error bars mark standard deviation. H) Two-way ANOVA analysis to compare weekly measured overall tumor volumes. Statistically significant p-values (<0.05) were highlighted in red. I) ADAR1 protects TNBC cells from ferroptosis through regulating MDM2 and lipid compositions between MUFA and PUFA. MUFA, monounsaturated fatty acid. PUFA, polyunsaturated fatty acid. FIN, ferroptosis inducer. See also Figure S4 .

Article Snippet: Tet-inducible MDM2- overexpressing construct was generated by subcloning MDM2 open-reading frame (ORF) from pCMV-myc3-MDM2 (Addgene 20935) into pSBtet-GP sleeping beauty vector (Addgene 60495) using the Sfil restriction enzyme site. pSBtet-GP-MDM2 and pCMV(CAT)T7-SB100 transposase construct (Addgene 34879) were co-transfected into MDA-MB-231 cells to generate a puromycin-resistant stable cell line.

Techniques: Activity Assay, Positive Control, Expressing, Standard Deviation

Journal: PLOS ONE

Article Title: Alcohol exposure suppresses ribosome biogenesis and causes nucleolar stress in cranial neural crest cells

doi: 10.1371/journal.pone.0304557

Figure Lengend Snippet: Alcohol-responsive genes related to ribosome biogenesis in early neural folds * .

Article Snippet: Expression vector plasmids for dominant-negative p53 (XE150DN P53-pCS2P+, #17033; AddGene, Watertown MA) and MDM2 (pCMV MDM2, #16441, AddGene) were grown in E . coli strain DH5α and purified using cesium chloride ultracentrifugation.

Techniques:

Journal: PLOS ONE

Article Title: Alcohol exposure suppresses ribosome biogenesis and causes nucleolar stress in cranial neural crest cells

doi: 10.1371/journal.pone.0304557

Figure Lengend Snippet: (A) Immunostain reveals that p53 protein (red) is detected predominantly in the cytosol of untreated O9-1 cells, and this protein becomes stabilized within the nuclei (blue) 6hr after alcohol exposure. Right panel, alcohol increases the percentage of p53 + nuclei. (B) Alcohol exposure causes a six-fold rise in p53 protein content in O9-1 cells at 6hr following exposure. (C) Transfection of dominant-negative p53 (p53-DN) into O9-1 cells prevents the alcohol-induced apoptosis as detected using TUNEL (red signal) and does not affect the survival of unexposed cells, whereas transfection with the parent pCS2 vector does not affect cell survival. Right panel, alcohol increases the percentage of TUNEL + cells and this is prevented by overexpression of dominant-negative p53 (+p53DN) but not by transfection with the empty vector (-p53DN). (D) Overexpression of MDM2 prevents the alcohol-induced apoptosis as detected by TUNEL (red) but does not affect the survival of control cells, compared with cells transfected with the empty vector pCS2. Right panel, alcohol increases the percentage of TUNEL+ O9-1 cells and this is normalized by MDM2. All values are mean ± SEM of 2 independent experiments as detailed in Methods . Data were analyzed using equal variance t-test (A, B) or one-way analysis of variance with Holm-Sidak multiple comparisons (C, D).

Article Snippet: Expression vector plasmids for dominant-negative p53 (XE150DN P53-pCS2P+, #17033; AddGene, Watertown MA) and MDM2 (pCMV MDM2, #16441, AddGene) were grown in E . coli strain DH5α and purified using cesium chloride ultracentrifugation.

Techniques: Transfection, Dominant Negative Mutation, TUNEL Assay, Plasmid Preparation, Over Expression, Control

Journal: PLOS ONE

Article Title: Alcohol exposure suppresses ribosome biogenesis and causes nucleolar stress in cranial neural crest cells

doi: 10.1371/journal.pone.0304557

Figure Lengend Snippet: (A) Zebrafish embryos injected with nonsense MO, or MOs against RPL5A , RPL11 , RPS3A , NOLC1 , p53 , or MDM2 had no or modest deficits in the size or shape of cranial cartilage elements as compared with no-MO controls. Low-dose alcohol-only also did not worsen cranial development. The combination of low-dose alcohol and morpholinos directed against genes that promote ribosome biogenesis ( rpl5a , rpl11 , rps3a , nolc1 ) and p53/MDM2 signaling ( mdm2 ) synergized to worsen cranial development and, in some instances, ablated cranial cartilage. Similarly, low-doses of the RNA polymerase I inhibitor CX5461 modestly affected cranial cartilage and synergized with low-dose alcohol to eliminate facial cartilage. Conversely, high-dose alcohol (boxed panels) caused craniofacial reductions that were prevented by morpholinos directed against p53. All views are ventral at 4 dpf with equivalent magnification. (B) Depiction of morphometry measurements, and quantification of cranial length, Meckel area, and ceratohyal area. Units are arbitrary and normalized to the mean of the uninjected controls. Open bars, no-alcohol; shaded bars, alcohol at the low dose or high dose as indicated. All values are mean ± SD with 4–14 embryos per treatment as detailed in Methods. Data analyzed using two-way analysis of variance followed by post hoc analysis using Holm-Sidak multiple comparisons (morpholino, compound) or within-group comparisons (alcohol vs. no-alcohol). * Different at p≤0.001 from its exposure-matched control for either nonsense-MO vs. gene-morpholino or no-compound vs. compound-treated. # Different at p≤0.001 from its treatment-matched control, Alcohol vs. no-Alcohol.

Article Snippet: Expression vector plasmids for dominant-negative p53 (XE150DN P53-pCS2P+, #17033; AddGene, Watertown MA) and MDM2 (pCMV MDM2, #16441, AddGene) were grown in E . coli strain DH5α and purified using cesium chloride ultracentrifugation.

Techniques: Injection, Control