Journal: NPJ Precision Oncology

Article Title: Synergistic effect of MDM2 inhibitors and radiotherapy in endometrial cancer

doi: 10.1038/s41698-025-01063-9

Figure Lengend Snippet: A Heat-map depicting 21 EC cell lines organized by decreasing radiation resistance (left to right) as quantified by area under the curve (AUC). The cell lines’ TP53 mutational status and variant allelic frequency (VAF) are depicted in the bars below. A significant association between AUC and TP53 mutation status ( p = 0.004) or VAF ( p = 0.01), respectively. A correlation between mutation and AUC (LR = 11.12 and p = 0.0038) as well as VAF and AUC (LR = 9.42 and p = 0.009) was observed. B The mean AUC for JHUEM1 and JHUEM2 cell lines are organized based on the CRISPR/Cas9 guide-RNA (gRNA) sequence for TP53 KO, as compared to the non-targeting control (NTC). Kruskal–Wallis test with Dunn’s Multiple Comparisons Test was performed with p < 0.001 for NTC vs KO 5.1, p = 0.0016 for NTC vs KO 6.1. C Western-blot analysis of two monoclonal isolates from each gRNA and NTC reveals loss of p21 signaling with knock-out of TP53 in JHUEM1 (left) and JHUEM2 (right). D Western blot analysis for downstream signaling after wild-type TP53 addback to 2 different KO clones of JHUEM2 under two different gRNAs (5.1 left / 6.1 - right). E Box-whisker plot depicting the Log2 fold-change in radiation response AUC with wild-type TP53 addback to JHUEM2 knock-out clones. Dunnett’s Multiple Comparisons test was performed with p = 0.0051 for KO 5.1 C vs NTC, p = 0.0199 for KO 6.1 C vs NTC.

Article Snippet: First, 5′ and 3′ fragments containing the mutated gene open reading frame (ORF) sequences were amplified by PCR from the pDONR223- TP53 WT plasmid (Addgene; Plasmid #82754).

Techniques: Variant Assay, Mutagenesis, CRISPR, Sequencing, Control, Western Blot, Knock-Out, Clone Assay, Whisker Assay

Journal: NPJ Precision Oncology

Article Title: Synergistic effect of MDM2 inhibitors and radiotherapy in endometrial cancer

doi: 10.1038/s41698-025-01063-9

Figure Lengend Snippet: A Western-blot analysis of JHUEM2 non-targeting control [NTC] isolates (1.1 and 3.1) with the addition of mCherry (complementation transfection control), wild-type TP53 (over-expression), and the addition of the R273C allele (co-expression with wild-type). B Waterfall plot depicting the overall effect on AUC (Log2-fold change) after introduction of a variant allele (or wild-type) across four different mono-clonal NTC isolates, NTC 5.1 and 6.1 for both JHUEM1 and JHUEM2. C Western-blot analysis of JHUEM2 NTC 3.1 (wild-type control) after complementation with each GOF/DN TP53 variant. D , E JHUEM2 isolate with R273C co-expression and the native NTC (wild-type alleles only) were irradiated at 2 Gy. RNA extracted 24 h after irradiation and gene expression assessed via RNAseq. On the volcano plot, the x-axis denotes a 1.5-fold change (FC) on a log2 scale and y -axis denotes a 0.05 false discovery rate (FDR) on a -log10 scale ( D ). Hallmark gene set analysis performed using MsigDB ( E ).

Article Snippet: First, 5′ and 3′ fragments containing the mutated gene open reading frame (ORF) sequences were amplified by PCR from the pDONR223- TP53 WT plasmid (Addgene; Plasmid #82754).

Techniques: Western Blot, Control, Transfection, Over Expression, Expressing, Variant Assay, Irradiation, Gene Expression

Journal: NPJ Precision Oncology

Article Title: Synergistic effect of MDM2 inhibitors and radiotherapy in endometrial cancer

doi: 10.1038/s41698-025-01063-9

Figure Lengend Snippet: A , C Drug response curve for treatment of EC cell lines representing three different allelic frequency populations – JHUEM2 (wild-type), Hec108 (heterogeneous-low VAF), and Hec1B (homogenous – high VAF) with increasing doses of Nutlin-3 ( A ) and AMG-232 ( C ). Error bars indicate standard error of the mean (SEM). Both demonstrate significant effects on cell viability in JHUEM2 and Hec108. Hec1B was resistant to treatment with either drug. B , D Western-blot analysis of all three cell lines after treatment with increasing doses of either Nutlin-3 ( B ) or AMG-232 ( D ), demonstrating an increase in p53/p21 with increasing concentrations of either drug.

Article Snippet: First, 5′ and 3′ fragments containing the mutated gene open reading frame (ORF) sequences were amplified by PCR from the pDONR223- TP53 WT plasmid (Addgene; Plasmid #82754).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a MYC-GFP-, DGCR8-, and Δ692-DGCR8-overexpressing LM2 cells with or without ATM inhibitor (ATMi) Ku55933 pretreatment (10 μM, 1 h) were treated with IR (8 Gy) and cultured for 30 min, followed by pulldown with anti-MYC beads and immunoblotting with antibodies against p-S/TQ and MYC. LE long exposure, SE short exposure. b LM2 and LM2-R cells were treated with IR (8 Gy) and cultured for 30 min, followed by immunoprecipitation with a DGCR8-specific antibody and immunoblotting with antibodies against p-S/TQ and DGCR8. c Consensus ATM phosphorylation site on human DGCR8 (S677) and alignment with the conserved site on Dgcr8 from other species. d Annotated tandem mass spectrometry (MS/MS) spectrum of the peptide encompassing phosphorylated serine 677 (QLASphosQKILQLLHPHVK) of DGCR8. MYC-DGCR8-overexpressing LM2 cells with or without IR (8 Gy followed by 1-h incubation) were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads, followed by MS analysis. PH indicates phosphorylation. NH 3 , H 2 O, and ++ indicate ammonia, water, and double charges, respectively. The graph shows the mass-to-charge ratios (m/z) of the doubly charged precursor peptide ions. The x and y axes represent m/z and relative ion intensity, respectively. e The extracted ion chromatograms for S677phos (peptide QLAS(phos)QKILQLLHPHVK) in non-irradiated and irradiated LM2 cells based on high-performance liquid chromatography (HPLC)-MS/MS analysis. The x and y axes represent the retention time of HPLC/MS analysis and the MS intensity, respectively. The area under the curve is used to indicate the relative abundance of S677phos with or without IR treatment. f HEK293T cells were transfected with DGCR8 (full-length or Δ692), S677A, or S677D mutant, treated with IR (8 Gy), and cultured for 30 min, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against p-S/TQ and MYC. g In vitro kinase assay. Purified WT DGCR8 and Δ692-DGCR8 or their S677A mutants were incubated with purified wild-type ATM or the kinase-dead mutant in kinase buffer. After the reaction, proteins were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. Purified MBP-p53 was used as a positive control for ATM kinase activity. h , i HEK293T ( h ) and LM2 ( i ) cells were transfected with MYC-tagged WT DGCR8 or the S677A mutant, treated with IR (8 Gy), and cultured for 30 min, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against p-DGCR8 (S677), DGCR8, and MYC.

Article Snippet: The DGCR8 (#10921), Drosha (#10921), Dicer (#10921), Exportin-5 (#10921), HA-ubiquitin (WT: #17608; K48R: #17604; K48: #17605; K63: #17606), RNF8 (#99396), FLAG-H2A (#63560), p53 (#81754), pLCN DSB Repair Reporter (DRR) (#98895), and pCAGGS DRR mCherry Donor EF1a BFP (#98896) constructs were from Addgene.

Techniques: Cell Culture, Western Blot, Immunoprecipitation, Phospho-proteomics, Mass Spectrometry, Tandem Mass Spectroscopy, Incubation, Irradiation, High Performance Liquid Chromatography, Transfection, Mutagenesis, In Vitro, Kinase Assay, Purification, SDS Page, Positive Control, Activity Assay

Journal: Cell Cycle

Article Title: Global tumor protein p53/p63 interactome

doi: 10.4161/cc.20863

Figure Lengend Snippet: Table 1. Overlapping cisplatin (CIS)-induced TP53- and ΔNp63- protein interactions in sensitive/resistant cells

Article Snippet: SCC cell line (expressing wt-Tp53 and TP63) was isolated from primary tissue at the Department of Otolaryngology/Head and Neck Surgery of the Johns Hopkins University School of Medicine.

Techniques:

Journal: Cell Cycle

Article Title: Global tumor protein p53/p63 interactome

doi: 10.4161/cc.20863

Figure Lengend Snippet: Figure 5. Complex formation between HDAC6, TP53 and splicing complex in SCC cells upon cisplatin exposure. Wt-ΔNp63α cells (left panels) and ΔNp63α -S385G cells (right panels) were exposed to control medium (CIS, -) or 10μg/ml cisplatin (CIS, +) for 16 h. (A) Nuclear lysates were immunoprecipitated (IP) with an anti-HDAC6 antibody and blotted with indicated antibodies. (B) Nuclear lysates were immunoprecipitated (IP) with an anti-TP53 antibody and blotted with indicated antibodies. Inputs (10%) were tested with anti-α-tubulin.

Article Snippet: SCC cell line (expressing wt-Tp53 and TP63) was isolated from primary tissue at the Department of Otolaryngology/Head and Neck Surgery of the Johns Hopkins University School of Medicine.

Techniques: Control, Immunoprecipitation

Journal: Cell Cycle

Article Title: Global tumor protein p53/p63 interactome

doi: 10.4161/cc.20863

Figure Lengend Snippet: Figure 8. Schematic representation of the TP53 and TP63 interactions/activities leading to regulation of RNA splicing/stability and cell death in SCC cells upon cisplatin exposure. Protein physical and functional interaction networks were based on data obtained from STRING 9.0 protein-protein interaction and Pubmed literature databases. Solid lines represent protein interactions shown in this work, dashed lines derived from the database and literature. Solid arrows represent activation of the target level or function, while gray arrows represent repression of target level or function. Dark gray ovals represent targets enhancing cisplatin sensitivity, while light gray ovals represent targets enhancing cisplatin resistance. White ovals represent targets, which play a neutral role in cellular response to cisplatin.

Article Snippet: SCC cell line (expressing wt-Tp53 and TP63) was isolated from primary tissue at the Department of Otolaryngology/Head and Neck Surgery of the Johns Hopkins University School of Medicine.

Techniques: Functional Assay, Derivative Assay, Activation Assay