Journal: Journal of Virology

Article Title: Seneca Valley virus 3C protease targets the Nrf2/HO-1 pathway to antagonize its antiviral activity

doi: 10.1128/jvi.01656-25

Figure Lengend Snippet: SVV infection retains Nrf2 in the cytoplasm. ( A and C ) BHK-21 cells infected with SVV (MOI = 0.5) or mock-infected with phosphate-buffered saline (PBS) with MG132 treatment (10 µM) ( A ) were monitored with confocal microscopy to examine subcellular localization of Nrf2 and HO-1 at 3, 6, and 12 hpi, respectively. Cells were stained with antibodies for detecting endogenous Nrf2 and HO-1 proteins (red), VP1 monoclonal antibody (green), and DAPI (blue), then examined by confocal microscopy. ( B ) BHK-21 cells mock-infected with PBS or infected with SVV (MOI = 0.5) with MG132 treatment (10 µM). Cells were stained with Nrf2 antibody (red), VP1 monoclonal antibody (green), and DAPI (blue), then examined by confocal microscopy. ( D ) BHK-21 cells mock-treated with dimethyl sulfoxide or CoPP or infected SVV (MOI = 0.5) with CoPP treatment (50 µM). Cells were stained with HO-1 antibody (red), VP1 monoclonal antibody (green), and DAPI (blue), then examined by confocal microscopy. ( E ) Nuclear and cytoplasmic fractions were collected at 0, 3, 6, and 9 hpi (MOI = 0.5) and analyzed by immunoblotting with antibodies against Nrf2, Keap1, histone H3, VP1, and β-actin as an internal control. ( F ) The relative gray intensity of cytoplasmic Nrf2 and nuclear Nrf2 was normalized against β-actin and histone H3, respectively, using ImageJ. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). ( G ) BHK-21 cells were transfected with HA-Nrf2 or co-transfected with HA-Nrf2 and either GFP, GFP-3C, GFP-3C-H48A, GFP-3C-C160A, or GFP-3C-DM (H48A-C160A). At 24 hpi, samples were processed for immunofluorescence staining using an HA antibody to detect exogenous HA-tagged Nrf2 proteins, followed by observation via confocal microscopy. ( H ) The graph shows the quantitative analysis of percentage of cytoplasmic Nrf2 from the results in panel G . Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). ( I ) Immunoblot was used to analyze the expression of Nrf2 and HO-1 after siRNA transfection-mediated knockdown with antibodies against Nrf2 and HO-1, respectively, with β-actin as an internal control. ( J and K ) Growth curves of SVV (MOI = 0.5) after transfection with siRNAs, HA-Nrf2, and HA-HO-1 plasmids in BHK-21 cells. At 3, 6, 9, and 12 hpi, the total viruses were titrated with the TCID 50 assay. Error bars indicate mean ± SD from three independent infection experiments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). ( L and M ) Immunoblot was used to analyze VP1 protein production in siRNAs, HA-Nrf2, and HA-HO-1 transfected BHK-21 cells at 6 and 12 hpi (MOI = 0.5).

Article Snippet: TBHQ (HY-100489), ML385 (HY-100523), Cobalt protoporphyrin IX (CoPP, HY-W250116), ZnPP (HY-101193), CORM-2 (HY-W033577), BV (HY-135005A), NAC (HY-B0215), bilirubin (HY-N0323, BR), ODQ (HY-101255, an sGC inhibitor), and KT5823 (HY-N6791, a PKG inhibitor) were obtained from MedChemExpress (Shanghai, China).

Techniques: Infection, Saline, Confocal Microscopy, Staining, Western Blot, Control, Transfection, Immunofluorescence, Expressing, Knockdown

Journal: Journal of Virology

Article Title: Seneca Valley virus 3C protease targets the Nrf2/HO-1 pathway to antagonize its antiviral activity

doi: 10.1128/jvi.01656-25

Figure Lengend Snippet: HO-1 agonist inhibits SVV infection. ( A ) BHK-21 cells were infected with SVV (MOI = 5) and treated with CoPP (50 µM). At 12 and 16 hpi, the cell lysates were collected and then analyzed by immunoblotting with indicated antibodies. ( B ) BHK-21 cells were infected with SVV (MOI = 5) and treated with CoPP at various concentrations (100, 50, 10, and 1 µM). At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. ( C ) BHK-21 cells were infected with SVV (MOI = 5) and treated with CoPP (50 µM). The transcriptional expression level of the indicated gene was analyzed using qRT-PCR and normalized to β-actin mRNA. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). ( D and E ) BHK-21 cells were treated with erastin or in the presence of HO-1 activator CoPP (50 µM) and inhibitor ZnPP (10 µM) or transfected with siHO-1. ROS was measured using DCFH-DA (10 µM) and examined by flow cytometry ( D ) and confocal microscopy ( E ), respectively. ( F ) Relative fluorescence intensity in BHK-21 cells from panel E . Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). ( G ) BHK-21 cells were infected with SVV (MOI = 5) and treated with HO-1 inducer hemin (50 µM). The cell lysates were collected at 12 and 16 hpi and analyzed by immunoblotting with indicated antibodies. ( H ) BHK-21 cells were infected with SVV (MOI = 5) and treated with varying concentrations of hemin (100, 50, 10, and 1 µM). At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. ( I ) BHK-21 cells were infected with SVV (MOI = 0.5), followed by treatment with CoPP (50 µM), ZnPP (10 µM), or hemin (50 µM). At 6 and 12 hpi, the viral titers were determined with TCID 50 assay. Error bars indicate mean ± SD from three independent infection experiments (*, P < 0.05; **, P < 0.01).

Article Snippet: TBHQ (HY-100489), ML385 (HY-100523), Cobalt protoporphyrin IX (CoPP, HY-W250116), ZnPP (HY-101193), CORM-2 (HY-W033577), BV (HY-135005A), NAC (HY-B0215), bilirubin (HY-N0323, BR), ODQ (HY-101255, an sGC inhibitor), and KT5823 (HY-N6791, a PKG inhibitor) were obtained from MedChemExpress (Shanghai, China).

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Transfection, Flow Cytometry, Confocal Microscopy, Fluorescence

Journal: Journal of Virology

Article Title: Seneca Valley virus 3C protease targets the Nrf2/HO-1 pathway to antagonize its antiviral activity

doi: 10.1128/jvi.01656-25

Figure Lengend Snippet: HO-1 inhibits SVV replication through the IFN-I pathway. ( A and G ) BHK-21 cells and PK-15 cells were transfected with HA-HO-1 and infected with SVV (MOI = 5), respectively. At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. ( B ) BHK-21 cells were transfected with HA-HO-1. The transcriptional expression level of the indicated gene was analyzed using qRT-PCR and normalized to β-actin mRNA. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). ( C and H ) BHK-21 cells and PK-15 cells were infected with SVV (MOI = 5) and treated with CoPP (50 µM), respectively. At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. ( D and F ) BHK-21 cells were infected with SVV (MOI = 5) and treated with CoPP (50 µM) and biliverdin (50 µM), respectively. The transcriptional expression level of the indicated gene was analyzed using qRT-PCR and normalized to β-actin mRNA. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). ( E ) BHK-21 cells were infected with SVV (MOI = 5) and treated with biliverdin (50 µM). At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. ( I ) PK-15 cells grown on six-well plates were co-transfected with GFP-3C and HA- HO-1 for 24 h, respectively, and GFP empty vector was used as a control. At 24 hpt, cells were collected and subjected to immunoblot with indicated antibodies. ( J and K ) HEK-293T cells plated in 24-well plates were transfected with 250 ng of IFN-β-Luc and ISRE-Luc separately, along with 25 ng of pRL-TK and either 250 ng of siHO-1 or siNC. After 24 h, HEK-293T cells were treated with poly(I:C) (2 ug/ml). Then the samples were prepared and analyzed by luciferase assays. ( L ) HEK-293T cells plated in 24-well plates were transfected with siHO-1 or siNC. After 24 h, the cells were treated with poly(I:C) (2 μg/mL). Then the samples were prepared and analyzed by qRT-PCR.Error bars indicate mean ± SD from three independent infection experiments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). ( M ) PK-15 cells grown on six-well plates were co-transfected with HA-Nrf2 and siRNA targeting HO-1; the GFP empty vector and siNC were used as controls. At 36 hpt, cells were collected and subjected to immunoblot with indicated antibodies.

Article Snippet: TBHQ (HY-100489), ML385 (HY-100523), Cobalt protoporphyrin IX (CoPP, HY-W250116), ZnPP (HY-101193), CORM-2 (HY-W033577), BV (HY-135005A), NAC (HY-B0215), bilirubin (HY-N0323, BR), ODQ (HY-101255, an sGC inhibitor), and KT5823 (HY-N6791, a PKG inhibitor) were obtained from MedChemExpress (Shanghai, China).

Techniques: Transfection, Infection, Western Blot, Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Luciferase

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: R11 modified tumor cell membrane nanovesicle-camouflaged nanoparticles with enhanced targeting and mucus-penetrating efficiency for intravesical chemotherapy for bladder cancer.

doi: 10.1016/j.jconrel.2022.09.055

Figure Lengend Snippet: Fig. 1. Hendeca-arginine peptide (R11) was polymerized with DNA to form nanocomplexes, which enabled one-step surface-functionalization of tumor cell membrane with large amounts of R11 at the extracellular side of cell membrane. The tumor cell membrane fragments with the R11 shell were used to encapsule gemcitabine-loaded PLGA nanoparticles by extrusion to construct a dual-targeting and mucus-penetrating drug delivery system for intravesical chemotherapy in bladder cancer.

Article Snippet: Gemcitabine elaidate was purchased from MedChemExpress.

Techniques: Membrane, Construct

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L positively regulated MDM2 expression and interacted with MDM2. (a) After GNL3L knockdown, qRT‐PCR, and western blot (WB) were performed to determine MDM2 expression in TE‐1 cells. (b) Differences in the expression of p53 and p21 in TE‐1 cells after the knockdown of GNL3L. (c) Co‐IP of GNL3L and MDM2 from ESCC cell lines. The input represents the total protein extract used in the IP. The GNL3L protein expression was normalized to that of GAPDH in ESCC cells. (IP, immunoprecipitation; IgG, negative control). (d) qRT‐PCR analysis of MDM2 and GNL3L expression in TE‐1 cells in different groups. (e) Protein expression of MDM2 and GNL3L of TE‐1 in different groups was determined by WB. (f) Cell proliferation was analyzed in different groups using the CCK8 assay for TE‐1 cell lines. (g) Proliferation was analyzed using an EdU assay. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, CCK-8 Assay, EdU Assay

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L knockdown reversed the increased malignant behaviors of ESCC cells induced by MDM2 overexpression. (a) Wound healing assay results in TE‐1 cells from different groups. (b) Invasion and migration of TE‐1 cells were assessed using Transwell assays. (c) Distribution of the cell phases of TE‐1 in the different groups. (d) In the different groups, apoptosis was measured in TE‐1 cells using flow cytometry. (e) Expression of P53 and P21 in TE‐1 cells in different groups. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Knockdown, Over Expression, Wound Healing Assay, Migration, Flow Cytometry, Expressing

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L overexpression reversed the decreased malignant behavior of ESCC cells induced by MDM2 knockdown. (a) Protein expression of MDM2 and GNL3L in TE‐1 cells in different groups was determined by western blot (WB). (b) Cell proliferation was analyzed in different groups using an EdU assay. (c) Migration and invasion of TE‐1 cells were assessed using Transwell assays. (d) In the different groups, apoptosis was measured in TE‐1 cells using flow cytometry. (e) Expression of P53 and P21 in TE‐1 cells in different groups. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Over Expression, Knockdown, Expressing, Western Blot, EdU Assay, Migration, Flow Cytometry

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: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 2: Bulk RNA-seq using iPSC-derived forebrain neurons treated with FGF21, PF-05231023 and vehicle. (A) Volcano plots indicate differentially expressed genes with FDR 0.05. (B) Venn diagram showing expression profiles for the 974 genes with expression that could be affected by both FGF21 and PF-05231023 as determined by RNA-seq (FDR < 0.05). The middle and bottom Venn diagrams illustrate overlapping up-regulated and down-regulated genes in response to FGF21 and the long-acting FGF21 molecule. Those results indicate that those overlapping genes displayed identical directionality. (C) Heatmap showing expression profiles for the 974 genes affected by FGF21 and PF-05231023.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: RNA Sequencing, Derivative Assay, Expressing

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 3: Plasma FGF21 concentrations were positively correlated with plasma GABA concentrations. (A) Plasma FGF21 concentrations were associated with plasma GABA levels in patients with alcohol use disorder. (B) both FGF21, and PF-05231023 could substantially increase the level of GABA in the culture medium of iPSC-derived forebrain neurons.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Clinical Proteomics, Derivative Assay

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 4: FGF21 and PF-05231023 induced GABA concentrations in iPSC-derived forebrain organoids. (A) A schematic outline of procedures used during the differentiation of iPSC-derived forebrain organoids. The middle panel displays images of iPSC-derived forebrain organoids during differentiation. The bottom panel of the schematic displays representative examples of staining for neural progenitors (SOX2) and neurons (TUJ1). (B) FGF21 and PF-05231023 substantially induced the concentrations of GABA in the culture medium of iPSC-derived forebrain organoids.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Derivative Assay, Staining

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 5: REST, EZH2 and H3K27me3 co-occupied the promoter regions of genes associated with GABA catabolism. (A) Heatmap showing expression profiles for the 15 genes involved in the GABAergic synapse pathway. (B) ChIP assays showing that REST, EZH2 and H3K27me3 co-occupied the promoter regions of the 13 selected genes as shown in Figure 5A that were associated with the GABAergic synapse pathway in response to FGF21 and PF-05231023 treatment of iPSC-derived forebrain neurons (n ¼ 4). Percentages of ChIP DNA/input were determined by qPCR. Data are represented as % input, (enrichment relative to IgG control) ¼ % input (antibody against REST, EZH2 and H3K27me3) e % input (IgG). ChIP-qPCR was performed in triplicate. Three independent experiments were performed. This figure represents results from one of the three experiments. (C) Co- Immunoprecipitation (IP) was used to determine whether REST protein could interact with EZH2 in iPSC-derived forebrain neurons. Whole cell lysates from iPSC-derived fore- brain neurons were immunoprecipitated with anti-REST antibodies or anti-IgG antibodies and protein samples were immunoblotted and probed with antibodies against REST or EZH2.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Expressing, Derivative Assay, Control, ChIP-qPCR, Immunoprecipitation

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 6: REST-EZH2-H3K37me3 complex-mediated gene repression in a drug dependent fashion. ChIP assays were performed in iPSC-derived forebrain neurons treated with FGF21, PF-05231023 and vehicle. We confirmed that changes in REST binding were correlated with changes in mRNA expression for all 13 genes. Specifically, in the presence of FGF21, all 13 binding sites except GNAI1 displayed significantly increased REST binding. In parallel, the recruitment of EZH2 and H3K27me3 also increased substantially, thus confirming that changes in REST-EZH2-H3K37me3 occupancy were highly correlated with transcription.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Derivative Assay, Binding Assay, Expressing

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 7: Schematic diagram illustrating the effects of FGF21 on GABA catabolism. Specifically, FGF21 and PF-05231023 significantly induced GABA levels in iPSC-derived forebrain neurons. We identified a drug-dependent occupancy of REST, EZH2, H3K27me3 in the promoter regions of genes associated with GABA catabolism i.e., ABAT (a key enzyme responsible for GABA catabolism), GRBBR1 (GABA B receptor 1), and SLC32A1 (GABA transporter), which resulted in transcriptional repression.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Derivative Assay