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: Cellular and Molecular Life Sciences

Article Title: Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

doi: 10.1007/s00018-022-04488-8

Figure Lengend Snippet: Experimental setup and results of colocalization analyses of septin-3 (SEPT3) due treatments. A Embryos were excised from three different dams to create biological triplicates of cultures. Embryonic cortices were isolated and homogenized to prepare cortical primary neuronal cell cultures. Two wells per culture were exposed to treatments (either AUTEN-67 or CCCP), two wells per culture were used as control. Cells were immunostained against SEPT3, LC3B and PINK1. Three images were captured and analyzed per well for every treatment/control condition. B – D Percentage comparison of colocalizing signals between treated and control cells. Means are marked with horizontal lines with values; boxes represent standard error of the mean (SEM). Flags indicate standard deviation (SD). In the figure legend, results are presented as mean ± SEM; SD. The p -values are of independent two-tailed Student’s t -test. B LC3B colocalizing SEPT3 in AUTEN-67 treatments, 13.01% ± 1.21%; 5.14%. Controls: 9.26% ± 1.1%; 4.68%. Significance: p = 0.0283. C LC3B colocalizing SEPT3, CCCP treatments: 10.49% ± 1.53%; 6.50%. Controls: 4.01% ± 1.06%; 4.38%. Significance: p = 0.0016. D PINK1 colocalizing SEPT3, CCCP treatment: 8.07% ± 1.52%; 6.46%. Controls: 8.65% ± 1.74%; 7.19%. Significance: p = 0.80

Article Snippet: Human recombinant LC3B and GABARAPL2 (Novus Biologicals NBP1-50960 and UL-420-500, respectively) probes were labeled with Alexa Fluor 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Isolation, Control, Comparison, Standard Deviation, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

doi: 10.1007/s00018-022-04488-8

Figure Lengend Snippet: Protein–protein interaction measurements of septin-3 (SEPT3) and Atg8 homologs. A Microscale thermophoresis dose–response curves of LC3B-SEPT3 (yellow) and GABARAPL2-SEPT3 interactions (blue). X axis represents SEPT3 concentrations on a log scale, while Y axis reflects MST dose–response, normalized to baseline (Fnorm). Corresponding K D values and K D confidence are displayed. B Spectral shift fluorescence response of LC3B-SEPT3 (yellow) and GABARAPL2-SEPT3 (blue). C Fluorescence polarization direct titration curves of LC3B-SEPT3 (marked yellow) and GABARAPL2-SEPT3 (marked blue) interactions. X axis represents SEPT3 concentrations on a linear scale, while Y axis reflects polarization

Article Snippet: Human recombinant LC3B and GABARAPL2 (Novus Biologicals NBP1-50960 and UL-420-500, respectively) probes were labeled with Alexa Fluor 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Microscale Thermophoresis, Fluorescence, Titration

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

doi: 10.1007/s00018-022-04488-8

Figure Lengend Snippet: Trp fluorescence of septin-3. A Structure and sequence of septin-3, marking positions of tryptophans. Core LIR4 and corresponding W284 is marked light-blue, while LIR1 W139 is marked dark-blue. Non-LIR W93 is marked orange. B Trp fluorescence spectra of septin-3 (orange), septin-3-LC3B complex (turquoise), septin-3 after adding 200 nM NaI (grey), septin-3 in complex with LC3B after adding 200 nM NaI (pink)

Article Snippet: Human recombinant LC3B and GABARAPL2 (Novus Biologicals NBP1-50960 and UL-420-500, respectively) probes were labeled with Alexa Fluor 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Fluorescence, Sequencing

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

doi: 10.1007/s00018-022-04488-8

Figure Lengend Snippet: Confocal microscopy images of primary neuronal cells. A Representative image of enriched LC3B positive particles in the cell body of an AUTEN-67 treated primary neuron indicate enhanced autophagy compared to control. B Overlapping spots of fluorescent signals along the axons (left) and in the cell body (right). C Enlarged and resolved images of overlapping signals from B, with observable LC3B and septin-3 (SEPT3) colocalization, where SEPT3’s colocalization with PINK1 is minimal. However, more complex colocalization patterns of LC3B–PINK1–SEPT3 can be observed from other parts of the sample ( D )

Article Snippet: Human recombinant LC3B and GABARAPL2 (Novus Biologicals NBP1-50960 and UL-420-500, respectively) probes were labeled with Alexa Fluor 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Confocal Microscopy, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

doi: 10.1007/s00018-022-04488-8

Figure Lengend Snippet: Western blot analysis of LC3B, septin-3 and p62 protein level changes in primary neurons presented as mean ± SEM due to treatments, with corresponding bands. A Effect of 100 μM AUTEN-67 treatment compared to untreated controls: significant changes in levels of LC3B-II ( p = 0.0006), septin-3 ( p = 0.014) and p62 ( p = 0.010) can be observed. B Effect of 100 μM AUTEN-67, 100 nM Bafilomycin A1 treatment compared to untreated controls: significant changes in levels of LC3B-II ( p = 0.034), septin-3 ( p = 0.043) and p62 ( p = 0.026) can be observed. C Effect of 100 μM AUTEN-67, 10 nM 3-MA treatment compared to controls: no significant change in protein levels can be observed ( p = 0.586 for LC3B-II, p = 0.231 for septin-3 and p = 0.0468 for p62)

Article Snippet: Human recombinant LC3B and GABARAPL2 (Novus Biologicals NBP1-50960 and UL-420-500, respectively) probes were labeled with Alexa Fluor 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

doi: 10.1007/s00018-022-04488-8

Figure Lengend Snippet: TEM images of immunostained cortical samples. A 10 nm colloidal gold stained septin-3 localizing to mitochondria (M). B Septin-3 located at double-membrane and multivesicular body-like (MVB) structures. C Septin-3 located on LC3B positive autophagic vesicles with double staining of septin-3 (orange arrows) and LC3B (blue arrows), using 10 nm and 6 nm colloidal gold, respectively

Article Snippet: Human recombinant LC3B and GABARAPL2 (Novus Biologicals NBP1-50960 and UL-420-500, respectively) probes were labeled with Alexa Fluor 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Staining, Membrane, Double Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

doi: 10.1007/s00018-022-04488-8

Figure Lengend Snippet: Theories of septin-3 function. A Hypothetical roles of septin-3 in synaptic/neuronal autophagy. (1) Septin-3 acting as an adapter between autophagophore-bound LC3B and selective presynaptic cargo escorting it for autophagic degradation. (2) Septin-3 binding to partner motor protein myosin 1B could play role in autophagosome-positioning, or autolysosome biogenesis, since MYO1B is located on lysosomes. (3) TRκB is located on late endosomes and signaling endosomes. Septin-3 is reported binding TRκB. Septin-3 therefore could play a role in autophagosome‒late endosome interaction. (4) CDC10, orthologue of septin-3 in yeast localizes to endosomes and autophagosomes and is assumed to play role in membrane-transport to autophagosome. This assumption stands for septin-3 in mammalian neurons. B Possible connections of septin-3 accumulation, neuro-immunological pruning and autophagy

Article Snippet: Human recombinant LC3B and GABARAPL2 (Novus Biologicals NBP1-50960 and UL-420-500, respectively) probes were labeled with Alexa Fluor 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Binding Assay, Membrane