human skin fibroblast cell line ws1  (ATCC)

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: Identification and characterization of eccDNA in rat skin induced by ionizing radiation. A) Flowchart for eccDNA purification and sequencing ( n = 4 for each group). B) Quantification of unique eccDNA. C) Distribution of unique eccDNA lengths. D) Overlap of eccDNA across sample groups. E) Genomic origins of eccDNA. F) Identification of three eccDNAs using PCR and Sanger sequencing, with circle 17:44148731‐48208624 (4059.8 Kb) present in all samples (CE: crude eccDNA, EE: exonuclease‐treated eccDNA). G) Flowchart of semiquantitative analysis of PCR by gel electrophoresis on the eccDNA gene. H) PCR detected five genes on circle 17:44148731‐48208624 . I) Flowchart for eccDNA pre‐treatment of rats with radiation‐induced skin injury ( n = 3 for each group). J) Vps41 protein expression in rat skin 3 days post eccDNA transfection. K) Skin damage photos at 8, 40, and 65 days post‐irradiation in eccDNA‐pre‐treated rats (4 µg injection; scale bar: 1 cm). L) Radiation damage scores and affected areas in eccDNA pre‐treated rats. M,N) Immunofluorescence and analysis of inflammatory factors (IL‐6, IL‐10, TNF‐α) in irradiated skin of eccDNA‐pre‐treated rats (scale bar: 100 µm). O) Inflammatory cytokine array detection in eccDNA‐treated WS1 cells ( n = 5 per group). P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons, one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons, and limma's empirical Bayes moderated t‐statistics for high‐throughput expression data. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Purification, Sequencing, Nucleic Acid Electrophoresis, Expressing, Transfection, Irradiation, Injection, Immunofluorescence, MANN-WHITNEY, High Throughput Screening Assay

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: eccDNA drives increased VPS41 expression. A) Diagram of Circle 17:44148731‐48208624 in rat skin. B) Validation of the PS enzyme for linear DNA removal. C) Standard curve for semiquantitative analysis of PCR by gel electrophoresis on the eccDNA gene. D) PCR analysis of RALA and VPS41 amplification in HaCaT cells post‐irradiation. E) Gel electrophoresis shows VPS41 copy number on gDNA remains unchanged after irradiation. F) Elevated mRNA and protein expression of VPS41 in HaCaT cells after irradiation. G) Vps41 protein expression in various tissues after 4 Gy (X‐ray) total body irradiation in rats. H) Vps41 expression (Log 2 Fold Change) from scRNA‐Seq data of irradiated rat skin ( n = 4 per group). I) Relative expression levels of Vps41 (Log 2 Fold Change) in different cell types in scRNA‐Seq data of irradiated rat skin. J) Immunohistochemical analysis of VPS41 expression in a patient with clinical radiation‐induced skin injury (scale bar: 100 µm). K) Significant upregulation of VPS41 mRNA levels after transfection of HaCaT cells‐derived eccDNA into WS1 cells. L) Increased VPS41 expression after HaCaT cells‐derived eccDNA transfection into HEK‐293T cells. M) PCR analysis suggests intact gene expression elements for VPS41 on eccDNA. N) Nuclear‐cytoplasmic separation and semiquantitative analysis of PCR by gel electrophoresis reveal VPS41 gene localization on eccDNA. O) Apoptosis rate of WS1 cells transfected with total eccDNA after irradiation. P) Apoptosis rate of WS1 cells transfected with purified eccDNA after irradiation. Q) UVB and paclitaxel treatment effects on VPS41 expression in skin cells (HaCaT and WS1). R) Semiquantitative analysis of PCR by gel electrophoresis of DNA damage inducers and inhibitors on eccDNA VPS41 amplification in HaCaT cells. Treatments include IR (6 Gy), UVB (20 mJ/cm 2 ), etoposide (2 µ m ), paclitaxel (20 n m ), and cisplatin (2 µ m ). P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Biomarker Discovery, Nucleic Acid Electrophoresis, Amplification, Irradiation, Immunohistochemical staining, Transfection, Derivative Assay, Gene Expression, Purification, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: VPS41 upregulation confers radioprotective effects at the cellular level. A) VPS41 protein localization after EGFP‐VPS41 plasmid transfection in HaCaT and WS1 cells (Hoechst: blue, EGFP: green, LysoTracker: red; scale bar: 20 µm). B) Western blot showing VPS41 plasmid overexpression efficiency in HaCaT and WS1 cells. C) ROS levels in HaCaT cells post‐irradiation after VPS41 plasmid transfection. D,E) Effect of VPS41 plasmid transfection on γH2AX levels in HaCaT cells after irradiation (scale bar: 20 µm). F) Cell viability in HaCaT and WS1 cells post‐irradiation with VPS41 plasmid transfection. G) LDH release in HaCaT and WS1 cells following irradiation and VPS41 plasmid transfection. H) Colony formation rate in HaCaT cells post‐irradiation with VPS41 plasmid. I) Reduced apoptosis in HaCaT and WS1 cells post‐irradiation after VPS41 plasmid transfection. J) Western blot showing shVPS41 knockdown efficiency in HaCaT and WS1 cells. K) Decreased irradiated cell viability after shVPS41 infection in HaCaT and WS1 cells. L) Increased LDH release in irradiated HaCaT and WS1 cells post‐shVPS41 infection. M) Elevated apoptosis rate in irradiated HaCaT and WS1 cells after shVPS41 infection. N) Reduced colony formation in irradiated HaCaT cells after shVPS41 infection. P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Plasmid Preparation, Transfection, Western Blot, Over Expression, Irradiation, Knockdown, Infection, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: Therapeutic effects of AAV‐Vps41 on radiation‐induced skin injuries in rats. A) Flowchart for skin radiation injury in rats pre‐treated with AAV‐Vps41 for 16 days ( n = 4 per group). B) Increased Vps41 protein expression in rat skin one month post AAV‐Vps41 infection. C) Photographs of skin radiation injury in AAV‐Vps41 pre‐treated rats on days 12, 44, and 72 post‐irradiation (scale bar: 1 cm). D) Radiation injury score statistics in AAV‐Vps41 pre‐treated rats. E) Analysis of the area of skin radiation injury in AAV‐Vps41‐treated rats. F) HE staining showing tissue resistance to ionizing radiation in AAV‐Vps41 pre‐treated rats (45 Gy for 72 days), scale bar 250 µm. G,H) Immunofluorescence detection and analysis of IL‐6, IL‐10, TNF‐α in irradiated skin of AAV‐Vps41 pre‐treated rats (scale bar: 100 µm). I) Schematic diagram of inflammatory factor chip detection in AAV‐Vps41 pre‐treated WS1 cells. J) GO classification of inflammatory factor chip results in AAV‐Vps41 pre‐treated WS1 cells. K) Heat Map of inflammatory factor chip results in AAV‐Vps41 pre‐treated WS1 cells. P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons, Fisher's exact test for GO enrichment analysis, and limma's empirical Bayes moderated t‐statistics for protein expression data. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Infection, Irradiation, Staining, Immunofluorescence, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: VPS41 negatively regulates KAI1 expression through the lysosomal pathway to confer resistance to apoptosis. A) Flowchart for screening VPS41 interaction proteins via differential protein analysis and mass spectrometry after VPS41 upregulation post‐irradiation. B) Volcano plot showing proteomic analysis (VPS41 vs Vector). C) Electron microscopy analysis reveals inhibited apoptosis progression in cells with upregulated VPS41 after irradiation (scale bar: 5 µm). D) Intersection of differential proteins identified four candidates: ISG15, KAI1, IFT20, and ATPAF1. E) Co‐localization of VPS41‐EGFP and KAI1‐BFP plasmids in WS1 and HaCaT cells assessed by confocal microscopy (scale bar: 20 µm). F) Immunoprecipitation confirms VPS41 binds KAI1. G) PNGase F treatment has minimal effect on VPS41‐KAI1 interaction. H) Western Blot shows upregulation of VPS41 decreases KAI1 expression, suppressing apoptosis, while VPS41 downregulation increases KAI1 expression and enhances apoptosis. I) VPS41 and KAI1 expression changes in HaCaT cells treated with CQ (20µ m ) or MG‐132 (20 µ m ) combined with X‐ray (10 Gy). J) Analysis of KAI1 decay rate after CHX (300 µ m ) treatment and X‐ray (10 Gy) in HaCaT cells. K) Effect of VPS41 knockdown and eccDNA transfection on apoptosis rates in irradiated cells with or without KAI1 overexpression. L) Apoptosis testing shows KAI1 reverses VPS41‐mediated radiation resistance. M) LDH measurement assesses the role of KAI1 in reversing VPS41‐mediated radiation resistance. P values were calculated using different statistical methods based on data type: unpaired two‐tailed t test for differential protein analysis and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified. [Correction added on 28 April 2025, after first online publication: figure 5 is updated in this version].

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Mass Spectrometry, Irradiation, Plasmid Preparation, Electron Microscopy, Confocal Microscopy, Immunoprecipitation, Western Blot, Knockdown, Transfection, Over Expression, Two Tailed Test

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: The interaction between VPS41 and KAI1 is critical for the radioprotection of VPS41. A) AlphaFold 3 prediction of structural domains for VPS41 and KAI1. B) IP experiments validate interaction domains between truncated VPS41 and KAI1 after transfection of various VPS41 truncation plasmids into HEK‐293T cells. C) IP experiments verify interaction domains between truncated KAI1 and VPS41 after transfection of KAI1 truncation plasmids into HEK‐293T cells. D) Co‐transfection of VPS41_WT‐EGFP and truncated variants with KAI1‐BFP in WS1 cells, followed by confocal microscopy to assess co‐localization (scale bar: 20 µm). E) The interaction between VPS41 and KAI1 remains unaffected by CQ treatment, which inhibits endosome and lysosome fusion. F) Apoptosis assays investigate the effects of truncated VPS41 on radiation‐induced apoptosis in HEK‐293T cells. G) Apoptosis assays assess the impact of different KAI1 truncation variants on radiation‐induced apoptosis in HEK‐293T cells. H) AlphaFold 3.0 predicts interaction sites of VPS41‐1‐286 and KAI1‐Δ111‐228. I) Schematic diagram of peptide array experiment. J) ECL imaging results of KAI‐Δ111‐228 peptide array. K) Peptide array and AlphaFold 3.0 analyze protein binding sites. L) Conservation of KAI1 binding peptide containing K263 among species. P values were calculated using one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Transfection, Cotransfection, Confocal Microscopy, Peptide Microarray, Imaging, Protein Binding, Binding Assay

Journal: Antibiotics

Article Title: Development and Characterization of Azithromycin-Loaded Microemulsions: A Promising Tool for the Treatment of Bacterial Skin Infections

doi: 10.3390/antibiotics11081040

Figure Lengend Snippet: Fibroblasts viability (%) after 24 h of incubation at 37 °C with loaded microemulsions and AZT solution at different drug concentrations. The values denote the mean ± S.D. (n = 4). All treatments were analyzed against control (Ctrl), *** p < 0.001.

Article Snippet: Biocompatibility was tested by MTT assay on WS1, a human fibroblast cell line (American Type Culture Collection, ATCC, Manassas, VA, USA).

Techniques: Incubation, Control