Journal: Cell Death & Disease

Article Title: p53-loss induced prostatic epithelial cell plasticity and invasion is driven by a crosstalk with the tumor microenvironment

doi: 10.1038/s41419-025-07361-1

Figure Lengend Snippet: A Prostate weight of control (Pten L2/L2 and Pten/Trp53 L2/L2 mice), Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 1 and 3 months AGI. n ≥ 6 per group. ns non-significant, p ≥ 0.05; *** p < 0.001. Two-way ANOVA followed by Tukey’s post-hoc test. B Representative hematoxylin-eosin (HE) staining of DLP sections from control, Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice, 3 months AGI. Scale bar: 100 µm. n ≥ 6 per group. C , D Representative Ki-67 staining (brown) of DLP sections from control, Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 3 months AGI ( C ) and quantification of Ki-67 positive PECs per gland at 1, 3 and 5 months AGI ( D ). n ≥ 3 mice per group. ns non-significant, p ≥ 0.05. Two-way ANOVA followed by Tukey’s post-hoc test. Scale bar: 100 µm. E Representative Senescence-associated ß-galactosidase (SA-ßGal) staining (blue) of DLP sections from Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 5 months AGI. Scale bar: 100 µm. n = 3 mice per group. F , G Representative immunohistochemical staining of p21 and p16 (brown) ( F ) and quantification of p16 positive (+) PEC per gland ( G ) in DLP of control, Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 5 months AGI. Scale bar: 100 µm. n ≥ 3 per group. ** p < 0.01; One-way ANOVA with post-hoc Tukey. H Representative SA-ßGal staining (blue) of DLP sections from Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 7 months AGI. Scale bar: 100 µm. n = 3 mice per group. I Representative immunohistochemical staining of p16 (brown) in DLP of Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 7 months AGI. Scale bar: 100 µm. n ≥ 3 per group.

Article Snippet: 5000 cells were seeded per Cultrex dome (R&D systems, 3533-010-02) and after 1 week, the culture media was replaced by conditioned medium from CAFs of Pten (i)pe−/− or Pten/Trp53 (i)pe−/− mice, with or without Ruxolitinib (10 μM) (MedChemExpress, HY-50856), with control IgG (R&D Systems; AB-108) or IL-6 neutralizing antibody (R&D Systems; AB-406-NA), or with “organoid medium” supplemented with IL-6 (at a final concentration of 2 or 4 ng/mL) (PeproTech, 216-16) for 48 h. To perform histological analyses of murine organoids, matrigel domes were digested for 1 h at 37 °C in dispase (2 mg/mL) (StemCell Technologies, 07913).

Techniques: Control, Staining, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: p53-loss induced prostatic epithelial cell plasticity and invasion is driven by a crosstalk with the tumor microenvironment

doi: 10.1038/s41419-025-07361-1

Figure Lengend Snippet: A Representative HE staining of DLP of Pten/Trp53 (i)pe−/− mice 7 months AGI. Top panel, intraductal carcinoma (IDC); bottom panel, invasive adenocarcinoma (pointed by an arrow). Scale bar: 100 µm. n = 4 mice per group. B Frequency of invasive and non-invasive tumors in DLPs of Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 5–7 months AGI. n = 4 mice per group, n = 100 glands per group. Paired t -tests. * p < 0.05. C Representative Ki-67 staining of DLP sections of Pten/Trp53 (i)pe−/− mice 7 months AGI with IDC and invasive adenocarcinoma. n = 4 mice per group. Scale bar: 100 µm. D Quantification of Ki-67-positive PECs per gland in DLP of control, Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 7 months AGI. n ≥ 4 mice per group. 2-way ANOVA followed by Tukey’s post-hoc test. ** p < 0.01; *** p < 0.001; ns non-significant, p ≥ 0.05; # p ≥ 0.05 compared to Pten (i)pe−/− 5 months AGI (Fig. ); $ p < 0.05 compared to Pten/Trp53 (i)pe−/− 5 months AGI (Fig. ). E Representative immunohistochemical detection of CD45 and pan-cytokeratin (panCK) in liver of Pten/Trp53 (i)pe−/− mice 6 months AGI. Scale: 100 µm. n = 15 mice. Arrows point to panCK + cells.

Article Snippet: 5000 cells were seeded per Cultrex dome (R&D systems, 3533-010-02) and after 1 week, the culture media was replaced by conditioned medium from CAFs of Pten (i)pe−/− or Pten/Trp53 (i)pe−/− mice, with or without Ruxolitinib (10 μM) (MedChemExpress, HY-50856), with control IgG (R&D Systems; AB-108) or IL-6 neutralizing antibody (R&D Systems; AB-406-NA), or with “organoid medium” supplemented with IL-6 (at a final concentration of 2 or 4 ng/mL) (PeproTech, 216-16) for 48 h. To perform histological analyses of murine organoids, matrigel domes were digested for 1 h at 37 °C in dispase (2 mg/mL) (StemCell Technologies, 07913).

Techniques: Staining, Control, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: p53-loss induced prostatic epithelial cell plasticity and invasion is driven by a crosstalk with the tumor microenvironment

doi: 10.1038/s41419-025-07361-1

Figure Lengend Snippet: A Uniform Manifold Approximation and Projection (UMAP) of single cell transcriptomic analysis of 5449 cells from dissociated prostates of Pten/Trp53 (i)pe−/− mice 5 months AGI. B Dotplot of the average expression level of the marker genes used to annotate the cell populations, and the proportion of cell expressing them. C Violin plots of the 9 genes of the refined cluster 15 signature in prostate tumors and healthy tissue of the TCGA PRAD cohort. D Violin plots of the ARPC, MSPC and NEPC signature score described in Han et al. in EMTc cells. E–G Violin plots of the score of the EMTc signature in patients with wild-type (WT) or mutated (mut) PTEN ( E ) or TP53 ( F ) and in patients with intermediate risk (Gleason score 6–7) or high risk (Gleason score 8–10) prostate cancer ( G ) in the TCGA PRAD cohort. Unpaired Student T -test. H Violin plots of the score of the EMTc signature in benign tissue, localized and metastatic primary prostate tumors of patients . Multiple unpaired Student T -tests.

Article Snippet: 5000 cells were seeded per Cultrex dome (R&D systems, 3533-010-02) and after 1 week, the culture media was replaced by conditioned medium from CAFs of Pten (i)pe−/− or Pten/Trp53 (i)pe−/− mice, with or without Ruxolitinib (10 μM) (MedChemExpress, HY-50856), with control IgG (R&D Systems; AB-108) or IL-6 neutralizing antibody (R&D Systems; AB-406-NA), or with “organoid medium” supplemented with IL-6 (at a final concentration of 2 or 4 ng/mL) (PeproTech, 216-16) for 48 h. To perform histological analyses of murine organoids, matrigel domes were digested for 1 h at 37 °C in dispase (2 mg/mL) (StemCell Technologies, 07913).

Techniques: Expressing, Marker

Journal: Cell Death & Disease

Article Title: p53-loss induced prostatic epithelial cell plasticity and invasion is driven by a crosstalk with the tumor microenvironment

doi: 10.1038/s41419-025-07361-1

Figure Lengend Snippet: A UMAP of Single-nuclei Assay for Transposase-Accessible Chromatin (ATAC) sequencing of 3973 cells from dissociated DLP of Pten/Trp53 (i)pe−/− mice 5 months AGI. B Heatmap representing the eRegulons specific to the cell populations identified by SCENIC + . Color scale represents the transcript levels of the activator transcription factors (TF), and the dot size the enrichment of their motifs in open chromatin regions. C Gene Set Enrichment Analysis (GSEA) of transcripts upregulated in EMTc compared to LumC cells. D Violin plots of the transcript levels of the indicated genes selected from EMT and IL6/Jak/Stat3 hallmarks identified in ( C ), in LumC cells and EMTc. E Representative immunofluorescent staining of pStat3 (magenta) and Trop2 (cyan) in DLP sections from Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 5 months AGI. Nuclei are stained with DAPI (white). Scale bar: 50 µm. n = 4 mice per genotype. Dashed line shows the limit of the gland. F Quantification of pStat3-positive cells located within less than 30 µm from the basal lamina of glands in DLP of Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice 5 months AGI. n ≥ 5 mice per group. Unpaired Student t -test. G Heatmap representing the number of interactions between the indicated cell populations in prostates of Pten/Trp53 (i)pe−/− prostates 5 months AGI, determined by CellPhoneDB analysis. H Schematic representation of the experimental procedure to evaluate the effect of culture media of CAFs isolated from mice 5 months AGI on organoids derived from DLPs of mutant mice 3 months AGI. I Representative immunofluorescent staining of E-cadherin (Ecad, cyan) and Vimentin (Vim, magenta) in Pten −/− and Pten/Trp53 −/− organoids, treated or not (NT) with supernatants from cultured CAFs isolated from prostates of Pten (i)pe−/− or Pten/Trp53 (i)pe−/− mice 5 months AGI. Nuclei are stained with DAPI (white). n = 3 independent experiments. Scale bar: 50 µm. J , K Representative immunofluorescent staining of Vimentin (Vim, magenta) ( J ) and quantification of Vim positive cells ( K ) in Pten/Trp53 −/− organoids, treated with supernatants from cultured CAFs isolated from prostates of Pten/Trp53 (i)pe−/− mice 5 months AGI, supplemented or not with Ruxolitinib (10 µM). Nuclei are stained with DAPI (white). n = 3 independent experiments. Scale bar: 50 µm. Unpaired Student t -test.

Article Snippet: 5000 cells were seeded per Cultrex dome (R&D systems, 3533-010-02) and after 1 week, the culture media was replaced by conditioned medium from CAFs of Pten (i)pe−/− or Pten/Trp53 (i)pe−/− mice, with or without Ruxolitinib (10 μM) (MedChemExpress, HY-50856), with control IgG (R&D Systems; AB-108) or IL-6 neutralizing antibody (R&D Systems; AB-406-NA), or with “organoid medium” supplemented with IL-6 (at a final concentration of 2 or 4 ng/mL) (PeproTech, 216-16) for 48 h. To perform histological analyses of murine organoids, matrigel domes were digested for 1 h at 37 °C in dispase (2 mg/mL) (StemCell Technologies, 07913).

Techniques: Sequencing, Staining, Isolation, Derivative Assay, Mutagenesis, Cell Culture

Journal: Cell Death & Disease

Article Title: p53-loss induced prostatic epithelial cell plasticity and invasion is driven by a crosstalk with the tumor microenvironment

doi: 10.1038/s41419-025-07361-1

Figure Lengend Snippet: A t-SNE of fibroblast subpopulations in prostates of Pten/Trp53 (i)pe−/− mice 5 months AGI. B Most significant unilateral interactions of the indicated secreted signaling between iCAFs and EMTc, or iCAFs and LumC cells. The first cell population in the interacting couple (interactant1_interactant2) expresses the ligand, and the second the receptor (ligand_receptor). C IL-6 levels in culture supernatants of Pten −/− and Pten/Trp53 −/− organoids and fibroblasts isolated from prostates of Pten (i)pe−/− and Pten/Trp53 (i)pe−/− mice, 5 months AGI. n = 3 mice per group. ns non-significant, p ≥ 0.05; *** p < 0.001. Two-way ANOVA followed by Tukey’s post-hoc. D Representative immunofluorescent staining of E-cadherin (Ecad, cyan) and Vimentin (Vim, magenta) in Pten −/− and Pten/Trp53 −/− organoids, treated or not (NT) with IL-6 at 2 and 4 ng/mL. Nuclei are stained with DAPI (white). Scale bar: 50 µm. n = 3 independent experiments. E , F Representative immunofluorescent staining of Vimentin (Vim, magenta) ( E ) and quantification of Vim positive cells ( F ) in Pten/Trp53 −/− organoids, treated with supernatants from cultured CAFs isolated from prostates of Pten/Trp53 (i)pe−/− mice 5 months AGI, supplemented with control IgG or IL-6 neutralizing antibodies. Nuclei are stained with DAPI (white). n = 3 independent experiments. Scale bar: 50 µm. Mann-Whitney test. G EMTc signature score in epithelial cells from IDC-containing prostate tissue from patients in the dataset of Wong et al. . H Violin plot of the score of the IL6/JAK/STAT3 Hallmark in epithelial cells with a low (below median) and a high (above median) EMTc signature score. I Dotplot of the average expression level of the indicated genes in cell populations reported by Wong et al. , and the proportion of cell expressing them. J , K Representative immunofluorescent staining of Vimentin (Vim, magenta) and E-cadherin (Ecad, cyan) ( J ) and quantification of Vim positive cells ( K ) in P20-11 patient-derived organoids, treated or not (NT) with IL-6 at 4 ng/mL. Nuclei are stained with DAPI (white). Scale bar: 50 µm. n = 3 independent experiments. Unpaired t -test. L Schematic representation of the crosstalk between PECs and stromal cells stimulating CAFs to produce IL-6 which in turn activates JAK/STAT3 signaling in PECs that promotes EMT.

Article Snippet: 5000 cells were seeded per Cultrex dome (R&D systems, 3533-010-02) and after 1 week, the culture media was replaced by conditioned medium from CAFs of Pten (i)pe−/− or Pten/Trp53 (i)pe−/− mice, with or without Ruxolitinib (10 μM) (MedChemExpress, HY-50856), with control IgG (R&D Systems; AB-108) or IL-6 neutralizing antibody (R&D Systems; AB-406-NA), or with “organoid medium” supplemented with IL-6 (at a final concentration of 2 or 4 ng/mL) (PeproTech, 216-16) for 48 h. To perform histological analyses of murine organoids, matrigel domes were digested for 1 h at 37 °C in dispase (2 mg/mL) (StemCell Technologies, 07913).

Techniques: Isolation, Staining, Cell Culture, Control, MANN-WHITNEY, Expressing, Derivative Assay

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 MBP-p53 construct was generated from pDONR-p53 (Addgene, #81754) through the LR reaction.

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