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: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

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: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

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 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: Purified human LC3B protein was purchased from MedChemExpress ® (HY- P70909 ).

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

Journal: Endocrinology

Article Title: Deficiency of C1QL1 reduced murine ovarian follicle reserve through intraovarian and endocrine control.

doi: 10.1210/endocr/bqac048

Figure Lengend Snippet: Figure 5. Deficiency of C1QL1 promoted apoptosis and inhibited autophagy in the ovary. (A) Representative images of TUNEL staining of ovarian sections. The green signals indicate representative cells with positive TUNEL staining, which was mainly detected in the large atretic follicles. (B) The intensity of TUNEL-positive signals was shown in charts (n = 4 per group). (C) Representative images of immunofluorescence staining of ovarian sections with LC3B antibody. LC3B-positive signals were found in granulosa cells and oocytes and were weakened in C1QL1-deficient ovaries. (D) The intensity of LC3B-positive signals are shown (n = 4 per group). (E) Apoptosis- and autophagy-related proteins in the ovaries were analyzed by Western blotting. Representative blots are shown in the left panel, and the amount of protein normalized to β-tubulin, which is presented as mean ± SEM, is shown in the right panel. n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significance.

Article Snippet: The sections and granulosa cells were stained with anti-rabbit LC3B antibody and Alexa Fluor 594-conjugated secondary antibody and mounted with DAPI (Boster).

Techniques: TUNEL Assay, Staining, Immunofluorescence, Western Blot