Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Schematic workflow for identifying DSS1 as a metastasis-associated driver gene in ccRCC. We leveraged Catalogue of Somatic Mutations in Cancer (COSMIC) cancer gene census due to its expert-curated cancer hallmark annotations (e.g., invasion, metastasis) from experimental evidence. b Scatter plot showing DSS1 / SEC31B mRNA levels (log 2 transformed count per million [log 2 CPM]) vs. wound healing scores in TCGA-KIRC cohort ( n = 578 distinct samples, two-sided Spearman’s rank correlation test). Dashed lines: linear regression fit; Shaded area: the 95% confidence interval. c DSS1 mRNA expression in ccRCC versus adjacent normal tissues across datasets ( n = 560 distinct samples, two-tailed Welch’s t-test). Boxplot: Center line = median; box = 25th to 75th percentiles; whiskers = minima to maxima. d Kaplan-Meier survival curves for DSS1 expression (DESeq2 log 2 CPM) in TCGA-KIRC cohort ( n = 522 distinct patients, Log-rank test). e Immunohistochemistry (IHC) of DSS1 protein in ccRCC tumor and paired normal tissues ( n = 74 distinct patients; boxes: zoom-in regions; scale bar: 200×, 100 μm, 400×, 50 μm; error bar: mean (centre) ± Standard Deviation [SD]; two-tailed Welch’s t-test). ISUP, the International Society of Urologic Pathologists. f Survival analysis stratified by DSS1 IHC levels in the in-house cohort ( n = 74 distinct patients; Log-rank test). g , i Representative images of metastatic lung lesions from xenograft mouse models (BALB/c-nu, male, 7 weeks before harvest, tail vein injection) using Caki-1 cells ( g ) or of 786-O cells ( i ). Scale bar: lung, 5 mm; 5×, 2 mm; 200×, 50 μm. Boxes: zoom-in regions. h , j Quantification of metastatic foci number and total lesion area for Caki-1 based models ( h ; Lentivirus [Lv]-sh NC as control) or 786-O based models ( j ; Lv- Vector as control). k Subcutaneous tumors (axillary fossa injection) from Caki-1 cells-based mouse models (BALB/c-nu, male, Lv-sh NC as control, 4 weeks before harvest). l , m Tumor growth curves ( l ) and final weights ( m ) of subcutaneous tumors. Lv-sh NC as control. g –m n = 6 mice per group, Error bar: mean ± SD, two-tailed Welch’s t-test. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: Transformation Assay, Expressing, Two Tailed Test, Immunohistochemistry, Standard Deviation, Injection, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Schematic diagram illustrating KEGG autophagy pathway negatively associated with DSS1 expression (DESeq2 log 2 CPM). b Transmission electron microscopy (TEM, left) images and quantification (right) of autophagic vacuoles (arrows) in ACHN and Caki-1 cells transfected with si DSS1 or si NC ( n = 3 independent experiments; scale bars: 8000×, 2 μm, 20000×, 0.5 μm; boxes: zoom-in regions; error bar: mean ± SD; two-tailed Welch’s t-test, Benjamini-Hochberg [BH] adjustment). c Confocal microscopy images of LC3 puncta (magenta) and LAMP1 (turquoise) co-localization in cells treated with si NC , si DSS1 , with or without chloroquine (CQ: 10 μM, control: 0.02% DMSO, 24 h before harvest; n = 3 independent experiments; scale bar: 10 μm; error bar: mean ± SD; two-tailed Welch’s t-test, BH adjustment). COSMIC, Catalogue of Somatic Mutations in Cancer. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: Expressing, Transmission Assay, Electron Microscopy, Transfection, Two Tailed Test, Confocal Microscopy, Control

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a , b Transwell migration and Matrigel invasion assays in ACHN and Caki-1 cells treated with sh NC , sh DSS1 , pcDNA3.1, DSS1 plasmids, with or without CQ (Chloroquine, representative images; scale bar: 100 μm). Quantification of migrated/invaded cells (error bar: mean ± SD; two-tailed Welch’s t-test; n = 3 independent experiments). c Morphology images of ccRCC cells between DSS1 knockdown (pebble-shaped) and negative control (spindle-shaped; scale bar: 100 μm; error bar: mean ± SD; two-tailed Welch’s t-test). The average cell aspect ratio was determined from 100 randomly selected cells per group per experiment ( n = 3 independent experiments). d , e Immunoblotting (IB) of autophagy markers (LC3-II, p62) and EMT proteins (E-cadherin, N-cadherin, Vimentin) in si NC - and si DSS1 -treated (with or without CQ, 0.02% DMSO as control) cells. Endogenous control: β-actin. Densitometry quantification ( e , n = 3 independent experiments, error bar: mean ± SD, two-tailed Welch’s t-test). The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. f RT-qPCR analysis of MAP1LC3B and SQSTM1 mRNA levels in si DSS1 vs. si NC cells (normalized to GAPDH ; n = 3 independent experiments; error bar: mean ± SD; two-tailed Welch’s t-test). Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: Migration, Two Tailed Test, Knockdown, Negative Control, Western Blot, Control, Derivative Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Identification of TWIST1 as a potential downstream effector of DSS1-autophagy axis. Gene signatures (Molecular Signature DataBase v2022.1) were analyzed by single-sample gene set enrichment analysis (ssGSEA) in TCGA-KIRC ( n = 522 distinct tumors). b Fold changes (DESeq2) of transcription factor (TF) mRNA expression in metastatic (Met.) vs. primary ccRCC and primary ccRCC vs. normal kidney (TCGA-KIRC). c Immunoblotting of EMT-TFs (ZEB1, ZEB2, SNAI1, SLUG/SNAI2, FOXC2, TCF3) in sh DSS1 vs. sh NC cells (two-tailed Welch’s t-test, Benjamini-Hochberg [BH] adjustment). β-actin: endogenous control. d Immunoblotting showing TWIST1 protein levels in cells treated with si NC /si DSS1 , autophagy inhibitor (CQ, 10 μM; Control: 0.02% DMSO; 24 h before harvest), or both (two-tailed Welch’s t-test). e Immunoblotting showing TWIST1 levels in cells treated with si NC , TWIST1 knockdown, pcDNA3.1 or TWIST1 plasmids (two-tailed Welch’s t-test). f Rescue experiments showing protein levels of EMT markers in ccRCC cells transfected with different combinations of control, DSS1 , and TWIST1 siRNAs, and DSS1 and TWIST1 plasmids (two-tailed Welch’s t-test). g Transwell assays following the same treatments as in panel ( f ) (scale bar: 100 μm, two-tailed Welch’s t-test). h Immunohistochemistry using lung metastatic lesions of xenograft mouse models generated by tail vein injection of Caki-1 cells treated with Lv-sh DSS1 or control Lv-sh NC ( n = 5 independent samples per group; scale bar: 100 μm; two-tailed Welch’s t-test). IOD, Integrated Optical Density. i Caki-1 cells were analyzed by immunoprecipitation with antibody to the LC3 or TWIST1 epitope, followed by SDS-PAGE and immunoblotting (IgG: negative control, input: 5% lysate). j Immunofluorescence microscopy showing the co-localization of Bcl-2 (magenta), p62 (orange), and TWIST1 (turquoise) in ACHN cells treated with DSS1 knockdown or control (scale bar: 5 μm). DAPI (blue): nucleus. c –f The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. c – g , i , j n = 3 independent experiments. c – h Error bars: mean ± SD. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: Expressing, Western Blot, Two Tailed Test, Control, Knockdown, Transfection, Immunohistochemistry, Generated, Injection, Immunoprecipitation, SDS Page, Negative Control, Immunofluorescence, Microscopy, Derivative Assay

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Co-Immunoprecipitation (Co-IP) with antibody to the FLAG epitope, followed by SDS-PAGE and immunoblotting with antibody to LC3 (endogenous, IgG: isotype control; Input: 5% lysate). Short/long: exposure time of the same membrane. b Co-IP with antibody to the LC3 epitope, followed by SDS-PAGE and immunoblotting showing endogenous DSS1. a , b IgG light chain-specific secondary antibody was used. c Glutathione-S-Transferase (GST)-pulldown analysis for DSS1-LC3B interaction using 50 ng recombinantly expressed human full-length LC3B and 1 mg GST/GST-DSS1 (GST: negative control; Purified LC3B: positive control; Glutathione agarose beads: 50 µL). d Cells were lysed with EDTA (10 mM) or not, followed by metal bath at 95 °C for 5 min and immunoblotting with antibody to DSS1. e GST-pulldown analysis showing the interaction of LC3 and DSS1 mutant using 50 ng recombinantly expressed human LC3 and 1 mg GST-DSS1 W27GW39GW43GF52A (Glutathione agarose beads: 50 µL). f Caki-1 cells were transfected with plasmids encoding HA-DSS1 W27GW39GW43GF52A . Cell lysates and cell immunoprecipitants were immunoprecipitated with antibody to the LC3 epitope and HA epitope, respectively. g Knockdown of DSS1 or treatment with MG-132 (20 μM, 6 h before harvest) elevates LC3 protein levels in HEK293T cells (control: siNC and/or 0.02% DMSO, error bar: mean ± SD, two-tailed Welch’s t-test). h Immunoblotting showing the indicated protein levels in HEK293T cells transfected with control or DSS1 siRNA, and/or treated with 0.02% DMSO or MG-132 (20 μM, 6 h before harvest). i Immunoblotting of cycloheximide (CHX, 50 μg/mL) chase assay in sh DSS1 cells compared to sh NC cells (error bar: mean ± SD, two-tailed Welch’s t-test). c –e , g–i The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. a – i n = 3 independent experiments. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, FLAG-tag, SDS Page, Western Blot, Control, Membrane, Negative Control, Purification, Positive Control, Mutagenesis, Transfection, Knockdown, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Representative images of immunofluorescence showing the subcellular localization of DSS1 (magenta), LC3 (yellow), and PSMD3 (azure) in ACHN cells ( n = 3 independent experiments). Nuclei: DAPI (blue). Scale bar: 5 μm. b LC-MS/MS analysis of LC3 interactomes identifies E3 ligases potentially associated with LC3 ( n = 3 independent experiments; Proteome Discoverer 2.4, Thermo Scientific; False discovery rate <0.01). c The mRNA expression of TRIM25 in ccRCC patients from the TCGA-KIRC dataset ( n = 595 distinct samples, tumor vs. normal, two-tailed Welch’s test). Boxplot: Center line = median; box = 25th to 75th percentiles; whiskers = minima to maxima. d Representative images of immunohistochemistry analysis showing TRIM25 protein levels upregulated in ccRCC tissues vs. normal ( n = 66 distinct pairs; scale bar: 50 μm; error bar: mean (centre) ± SD; two-tailed paired t-test). e , f Immunoblotting showing TRIM25 knockdown increases LC3 levels, while TRIM25 overexpression reduces LC3 levels ( n = 3 independent experiments, error bar: mean ± SD, two-tailed Welch’s t-test). g Co-IP analysis in Caki-1 cells showing the interaction between endogenous TRIM25 and LC3 ( n = 3 independent experiments). h GST-pulldown analysis for TRIM25-LC3B interaction using 50 ng recombinantly expressed human full-length LC3B and 1 mg GST-TRIM25 ( n = 3 independent experiments; Glutathione agarose beads: 50 µL). Asterisk: GST-TRIM25. i Immunoblotting of HEK293T cells transfected with HA-Ub , pcDNA3.1, and Myc-LC3B (wildtype) or Myc-LC3B mutant plasmids ( n = 3 independent experiments). j Immunoblotting of HEK293T cells transfected with Myc-LC3B and HA-Ub -wildtype or HA-Ub -K48R/K63R mutant plasmids ( n = 3 independent experiments). k Recombinantly expressed human GST-TRIM25 was incubated with recombinant Ub, LC3B, UbcH5a/UbcH5c, and His-UBE1 in a reaction system containing MgATP at 37 °C for 4 h. Samples were analyzed by SDS-PAGE and immunoblotting with antibody to Ub epitope ( n = 3 independent experiments). As shown in the result of GST-TRIM25, the polyubiquitination of LC3B (LC3B-Ub) is shown only when all the components are present in the mix. e , f , h –j The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: Immunofluorescence, Liquid Chromatography with Mass Spectroscopy, Expressing, Two Tailed Test, Immunohistochemistry, Western Blot, Knockdown, Over Expression, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Incubation, Recombinant, SDS Page, Derivative Assay

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Quality control for single-cell RNA-seq data from ccRCC tissues ( n = 49 distinct samples). b , c Uniform Manifold Approximation and Projection (UMAP) of cells of ccRCC tissues before ( b ) and after batch effect correction ( c ). Right panel: annotation of major cell types. d , e Identification of malignant cells using inference of copy number variation (inferCNV, CNV score threshold: 0.04) of epithelial cells. Del, deletion; Amp amplification. f Comparison of DSS1 pseudo-bulk expression between malignant cells (tumor samples) and normal epithelial cells (normal samples, error bar: mean ± SD, n = 47 distinct samples with detected DSS1 RNA; two-tailed Welch’s t-test). g DSS1 amplification correlates with higher DSS1 expression (DESeq2 log 2 CPM) in tumors ( n = 513). h Functional module analysis for malignant cells. Upper graph: hierarchical clustering dendrogram of genes; lower graph: gene module (x-axis, different genes). i UMAP showing DSS1-driven ccRCC cells and other malignant cells. j Comparison of the epithelial-mesenchymal transition (EMT)/autophagy module scores between DSS1-driven cells and other malignant cells. KEGG v113.0, Kyoto Encyclopedia of Genes and Genomes. k Proportion of DSS1-driven cells across different stages in treatment-naive patients (I-IV: n = 1, 6, 6, 3 patients, respectively). l DSS1-driven cell signature scores in patients with different grades and pathologic stages ( n = 521). m Comparison of DSS1-driven cell signature scores across molecular subtypes defined by Clinical Proteomic Tumor Analysis Consortium (CPTAC) in TCGA-KIRC dataset ( n = 495). n High DSS1-driven cell signature predicts poorer survival (Log-rank test, n = 506 and 509, respectively). HR, Hazard Ratio. o – q Differences in signature scores of DSS1-driven cells between treatment-naive/response and treatment/resistance groups (VEGFR inhibitors) in ccRCC patients or patient-derived xenograft (PDX) models (left to right, TCGA-KIRC, n = 430; GSE65615 , n = 122; GSE64052 , n = 28; GSE76068 , n = 16). MUT, mutant; WT, wildtype. g , j , l , m , o –q Two-tailed Mann-Whitney U test. Box plots (Turkey style): Center line = median; box = 25th to 75th percentiles; whiskers = ±1.5×interquartile range; Violin plots: data distribution (minima to maxima) via kernel density estimation. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: Control, RNA Sequencing, Amplification, Comparison, Expressing, Two Tailed Test, Functional Assay, Derivative Assay, Mutagenesis, MANN-WHITNEY

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Cell type deconvolution of DSS1-driven cells using spatial transcriptome data from ccRCC tissues. b Spearman correlations of proportions between DSS1-driven cells and tumor microenvironment (TME) cells in spatial spots. c Correlations between the proportion of DSS1-driven cells and signature scores (AddModuleScore) of endothelial subsets in spots. Scores were rescaled (to a 0–1 range), then adjusted by dividing by the summed weights of TME cell types within a spot. d Re-clustering (single-cell RNA-seq) and annotation of endothelial cell subsets. e Cell-cell communication analysis between DSS1-driven cells ( n = 1403 cells) and microvascular cells ( n = 7916 cells, single-cell RNA-seq). f Correlations between DSS1-driven cell weights and coexpression strength of predicted ligand-receptor pairs in spots (pT1/pT2: n = 8 distinct tumors, pT3: n = 27 [21 distinct tumors with 1 slide, and 3 distinct tumors with 2 slides each], pT4/IV: n = 2 distinct tumors available, error bar: mean ± standard error of mean). Ligand-receptor pair coexpression (log-normalized counts) strength: rescaled (to 0-1) product of ligand L and receptor R expression (R1 + R2, if two receptors). g SPP1-integrin colocalizations in late-stage sample slides. h Multiplex immunohistochemistry showing expression and colocalization of ligand SPP1 from DSS1-driven cells (DSS1 high and LC3 low , and pan-CK + ) and receptor ITGB1 of microvascular cells (CD105 + ) ( n = 4 distinct patients; boxes: zoom-in regions; scale bar: 20 μm). i Quantification of DSS1-driven cells and colocalized puncta in ccRCC patient samples ( n = 4 distinct patients). j Proportions of SPP1-ITGB1 interaction within 25, 50, or 75 μm radius of CD105 puncta. b , c Boxplot: Center line: median; box: 25th to 75th percentiles; whiskers: ±1.5×interquartile range. a – c , f , g . n = 38 (32 distinct tumors with 1 section each, 3 distinct tumors with 2 sections each). Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: RNA Sequencing, Expressing, Multiplex Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Multiplex immunohistochemistry showing spatial distribution of DSS1-driven cells with a high epithelial-mesenchymal transition (EMT) feature (E-cad low , Vimentin high ) around vascular cells (CD31 + , arrows, n = 4 distinct patients). Scale bar: 40 μm. b SPP1 RNA and SPP1 protein levels in tissues of diverse cancer types (kidney cancer: RNA, n = 35 distinct cell lines, protein, n = 110 distinct samples) from the Human Protein Atlas (HPA, www.proteinatlas.org ). c SPP1 Protein levels in normal tissues from the HPA database (kidney, high expression in proximal tubules, n = 3 slides). d Pseudo-bulk RNA levels of SPP1 in epithelial and immune cell types. AvgExp, Average expression. e Protein abundance of SPP1 between tumor and normal tissues in Clinical Proteomic Tumor Analysis Consortium (CPTAC)-ccRCC dataset ( n = 194 distinct samples, two-tailed Mann-Whitney U test). Boxplot: Center line = median; box = 25th to 75th percentiles; whiskers = minima to maxima. f Spearman correlation between SPP1 and DSS1 mRNA expression (log 2 Count Per Million, two-tailed spearman’s rank correlation test). g Immunoblotting showing SPP1 protein levels in ccRCC cells (sh DSS1 vs. sh NC , n = 3 independent experiments, error bar: mean ± standard deviation [SD], two-tailed Welch’s t-test). The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. h Spearman correlation between CD68 expression and coexpression of SPP1 - ITGB1 in spots of sample sections from either tumor core or tumor-stromal interface (Li2022 spatial dataset, n = 13 sections from 10 distinct patients; error bar: mean ± SD). i Schematic diagram (by Figdraw.com) illustrates the proposed mechanistic model of DSS1-driven cells in ccRCC metastasis: DSS1 (oligomer) interacts with pro-LC3B or LC3B-I, promoting LC3B degradation via E3 ubiquitin ligase TRIM25-mediated Lys-63 (K63)-linked polyubiquitination at LC3B-K51, leading to impaired macroautophagic flux and p62 accumulation, TWIST1 stabilization and increased TWIST1 nuclear transport, promoting EMT activation. DSS1 highly expressed (DSS1 hi ) tumor cells were increased in late-stage tumors and linked to microvascular invasion within a vascularized invasive niche at the tumor-stromal interface, mediated by SPP1-ITGB1 interactions. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: For multiplex immunofluorescence, primary antibody against DSS1 (Proteintech, 13639-1-AP, 1:200), LC3 (ServiceBio, GB11124, 1:500), PSMD3 (Proteintech, 12054-1-AP, 1:200), Bcl-2 (ServiceBio, GB154830 , 1:500), TWIST1 (Proteintech, 25465-1-AP, 1:200), and p62 (Proteintech, 18420-1-AP, 1:200) were applied.

Techniques: Multiplex Assay, Immunohistochemistry, Expressing, Quantitative Proteomics, Two Tailed Test, MANN-WHITNEY, Western Blot, Standard Deviation, Derivative Assay, Ubiquitin Proteomics, Activation Assay

Journal: Frontiers in Physiology

Article Title: Sedentary Conditions Promote Subregionally Specific Changes in Brain-Derived Neurotrophic Factor in the Rostral Ventrolateral Medulla

doi: 10.3389/fphys.2021.756542

Figure Lengend Snippet: Primary antibodies.

Article Snippet: p75NTR , Peptide CEEIPGRWITRSTPPE, corresponding to amino acids 188–203 of human p75NTR (extracellular domain) , Alomone Labs, ANT-007, rabbit polyclonal , AB_2039968 , 1:200.

Techniques: Recombinant

Journal: Frontiers in Physiology

Article Title: Sedentary Conditions Promote Subregionally Specific Changes in Brain-Derived Neurotrophic Factor in the Rostral Ventrolateral Medulla

doi: 10.3389/fphys.2021.756542

Figure Lengend Snippet: Expression of Glycosylated (Glyco-p75) and non-Glycosylated (nonGlyco-p75) forms of the p75NTR receptor in the RVLM/RVLM RE following 12weeks of sedentary vs. physically active conditions. (A) Representative Western blot of Glyco-p75 (arrow ~75kDa), nonGlyco-p75 (arrow ~50kDa), and GAPDH expression at different rostrocaudal levels of the RVLM and the RVLM RE . (B) Group data from sedentary (black bars) vs. physically active (white bars) conditions ( n =6 ea) demonstrate no significant overall difference in the expression of Glyco-p75 in sedentary compared to active animals [ F (1, 30)=1.150, p =0.309, main effect of group] and there was also no overall significant effect of rostrocaudal distribution [ F (3,30)=1.877, p =0.155; main effect]. The interaction between experimental groups and rostrocaudal levels did not reach a significance [ F (3,30)=2.029, p =0.131], which precluded further post hoc testing. (C) Group data from sedentary vs. physically active conditions demonstrate a significant interaction term [ F (3,30)=3.384, p =0.031] and revealed a significantly lower expression of nonGlyco-p75 in both RVLM subregions ( ** p =0.004 for FN-480 and p =0.006 for FN-240) and in the FN+240 subregion of the RVLM RE ( ** , p =0.001) of sedentary rats. Sedentary rats showed significantly higher expression of nonGlyco-p75 in the FN+480 of the RVLM RE compared to the FN+240 (##, p =0.012) and both RVLM subregions (##, p =0.004 for FN-240 and p <0.001 for FN-480). Physically active rats exhibited significantly higher expression of nonGlyco-p75 in the FN+240 subregion of RVLM RE compared with the FN-480 of RVLM (##, p =0.006). See for results of all rostrocaudal comparisons within groups. (D) Group data showing that the Glyco-p75/nonGlyco-p75 ratio was overall significantly higher in sedentary rats vs. physically active [*, F (1,30)=20.829, p =0.001, main effect] and that there was an overall significant main effect of rostrocaudal distribution [#, F (3,30)=7.205, p <0.001, main effect]. Simple main effect testing revealed a significantly higher Glyco-p75/nonGlyco-p75 ratio in the most caudal FN-480 subregion of the RVLM compared with two subregions (FN+240 and FN+480) of the RVLM RE ( p =0.009 and p <0.001, respectively, see for all simple main effect comparisons). The interaction between main effects did not reach a significance [ F (3,30)=2.895, p =0.051] which precluded further post hoc testing. Data in (C,D) were log10 transformed in order to achieve normal distribution prior to running two-way mixed ANOVAs.

Article Snippet: p75NTR , Peptide CEEIPGRWITRSTPPE, corresponding to amino acids 188–203 of human p75NTR (extracellular domain) , Alomone Labs, ANT-007, rabbit polyclonal , AB_2039968 , 1:200.

Techniques: Expressing, Western Blot, Transformation Assay

Journal: World Journal of Gastroenterology

Article Title: Mucosal healing effect of nilotinib in indomethacin-induced enterocolitis: A rat model

doi: 10.3748/wjg.v21.i44.12576

Figure Lengend Snippet: Small intestine platelet-derived growth factor receptor α and β scores. PDGFR: Platelet-derived growth factor receptor.

Article Snippet: A primary antibody was then applied: PDGFR α, in a 1:100 dilution (NOVUS Biologicals, NBP1-19 423, United States) and PDGFR β, in a 1:50 dilution (NOVUS Biologicals, NBP1-19 473, United States).

Techniques: Derivative Assay

Journal: World Journal of Gastroenterology

Article Title: Mucosal healing effect of nilotinib in indomethacin-induced enterocolitis: A rat model

doi: 10.3748/wjg.v21.i44.12576

Figure Lengend Snippet: Colon platelet-derived growth factor receptor α and β scores. PDGFR: Platelet-derived growth factor receptor.

Article Snippet: A primary antibody was then applied: PDGFR α, in a 1:100 dilution (NOVUS Biologicals, NBP1-19 423, United States) and PDGFR β, in a 1:50 dilution (NOVUS Biologicals, NBP1-19 473, United States).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: LM11a-31 Inhibits p75 Neurotrophin Receptor (p75 NTR ) Cleavage and is Neuroprotective in a Cell Culture Model of Parkinson’s Disease

doi: 10.1101/2024.09.10.612299

Figure Lengend Snippet: (A) Confocal micrographs of cell surface p75 NTR (green) and plasma membrane (red) staining in LUHMES cells that were differentiated for five days and then treated for 18H with 7.5 µM 6-OHDA or vehicle solution following a one-hour pretreatment with 10 µM SP600125 or vehicle solution. Cell surface p75 NTR was labeled by immunostaining fixed, unpermeabilized cells with an antibody specific for the p75 NTR -ECD. Plasma membrane labeling was performed through staining with Wheat germ agglutinin-CF®568 conjugate, and nuclei (blue) were labeled with 4’,6-diamidino-2-phenylindole (DAPI). (B) Quantification of cell surface-localized p75 NTR in LUHMES cells that were treated, stained, and imaged as described in 1A ( n = 4, ANOVA with Tukey’s HSD). Abbreviations: Veh , vehicle; 6OH , 6-hydroxydopamine; SP6 , SP600125; A.U ., arbitrary units; *, p<0.05; n , number of experiments, each featuring an independent cell culture preparation.

Article Snippet: Fixed slides were blocked with 10% normal goat serum in PBS and then stained with Wheat Germ Agglutinin (WGA) – CF-568 conjugate (Biotium, San Francisco, CA, USA, Cat No. 29077-1), primary antibody for p75 NTR -ECD (Cell Signaling Technology, Cat No. 8238), secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific; 1:1000.

Techniques: Clinical Proteomics, Membrane, Staining, Labeling, Immunostaining, Cell Culture

Journal: bioRxiv

Article Title: LM11a-31 Inhibits p75 Neurotrophin Receptor (p75 NTR ) Cleavage and is Neuroprotective in a Cell Culture Model of Parkinson’s Disease

doi: 10.1101/2024.09.10.612299

Figure Lengend Snippet: (A) Schematic depicting regulated intramembrane proteolysis of p75 NTR . The extracellular domain of the receptor is initially cleaved by an α-secretase, thereby yielding a p75 NTR -CTF of ∼24 kDa. Cleavage of the transmembrane region of the p75 NTR -CTF yields a p75 NTR -ICD fragment of ∼ 19kDa. (B) Representative immunoblot analysis of p75 NTR fragments in lysates of differentiated LUHMES cells that were exposed for 18h to vehicle solution or 10 µM 6-OHDA and cotreated with vehicle solution or 80 µM dynasore. Immunoblotting was performed using an antibody specific for the p75 NTR -ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. (C – E) Densitometric analysis of p75 NTR -CTF (C) , p75 NTR -ICD (D) , and p75 NTR -FL (E) from immunoblots described in 2B ( n = 7; c, Friedman test with Dunn’s post-hoc analysis; d and e, ANOVA with Tukey’s HSD). Abbreviations: Veh , vehicle; 6OH , 6-hydroxydopamine; Dyn , dynasore; A.U ., arbitrary units; *, p<0.05; **, p<0.01; n , number of experiments, each featuring an independent cell culture preparation.

Article Snippet: Fixed slides were blocked with 10% normal goat serum in PBS and then stained with Wheat Germ Agglutinin (WGA) – CF-568 conjugate (Biotium, San Francisco, CA, USA, Cat No. 29077-1), primary antibody for p75 NTR -ECD (Cell Signaling Technology, Cat No. 8238), secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific; 1:1000.

Techniques: Western Blot, Control, Cell Culture

Journal: bioRxiv

Article Title: LM11a-31 Inhibits p75 Neurotrophin Receptor (p75 NTR ) Cleavage and is Neuroprotective in a Cell Culture Model of Parkinson’s Disease

doi: 10.1101/2024.09.10.612299

Figure Lengend Snippet: (A) Representative immunoblot of p75 NTR fragments from lysates of differentiated LUHMES cells treated with vehicle solution or 10 µM 6-OHDA and cotreated with vehicle solution or 20 nM LM11a-31. Immunoblotting was performed using an antibody specific for the p75 NTR -ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. (B – D) Densitometric analysis of p75 NTR -CTF (B), p75 NTR -ICD (C), and p75 NTR -FL (D) from immunoblots described in 5A ( n = 5; ANOVA with Tukey’s HSD). Abbreviations: Veh , vehicle; 6OH , 6-hydroxydopamine; LM , LM11a-31; A.U ., arbitrary units; *, p<0.05; ns , not significant; n , number of experiments, each featuring an independent cell culture preparation.

Article Snippet: Fixed slides were blocked with 10% normal goat serum in PBS and then stained with Wheat Germ Agglutinin (WGA) – CF-568 conjugate (Biotium, San Francisco, CA, USA, Cat No. 29077-1), primary antibody for p75 NTR -ECD (Cell Signaling Technology, Cat No. 8238), secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific; 1:1000.

Techniques: Western Blot, Control, Cell Culture

Journal: bioRxiv

Article Title: LM11a-31 Inhibits p75 Neurotrophin Receptor (p75 NTR ) Cleavage and is Neuroprotective in a Cell Culture Model of Parkinson’s Disease

doi: 10.1101/2024.09.10.612299

Figure Lengend Snippet: The accumulation of reactive oxygen species (ROS) and oxidative damage in cells with oxidative stress triggers activation of JNK. This activation stimulates clathrin-mediated endocytosis of p75 NTR , increasing its susceptibility to cleavage by metalloproteases, such as TACE, and the γ-secretase complex in endosomes. Simultaneously, oxidative stress causes downregulation of Trk receptors, diminishing their antagonism of p75 NTR cleavage. Together, these processes result in accumulation of p75 NTR fragments, which contribute to downstream signaling pathways that promote neuronal death.

Article Snippet: Fixed slides were blocked with 10% normal goat serum in PBS and then stained with Wheat Germ Agglutinin (WGA) – CF-568 conjugate (Biotium, San Francisco, CA, USA, Cat No. 29077-1), primary antibody for p75 NTR -ECD (Cell Signaling Technology, Cat No. 8238), secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific; 1:1000.

Techniques: Activation Assay, Protein-Protein interactions