human fibroblast cell line ws1  (ATCC)

Journal: Molecular Biomedicine

Article Title: miR-338-3p promotes radiation recall like dermatitis by suppressing pleiotrophin via the PI3K/Akt/Bcl2 pathway

doi: 10.1186/s43556-026-00424-5

Figure Lengend Snippet: Construction and validation of the RRLD model in vitro. a Study protocol for the construction and validation of the RRLD model in vitro. b Cell viability of different HaCaT and WS1 cells, as well as primary rat epidermal cells treated with EPI or 5‑Fu after irradiation, as assessed by CCK‑8 assay ( n = 6). c Representative fluorescence microscopy images of reactive oxygen species (ROS) in HaCaT cells treated with EPI or 5‑Fu after irradiation using DCFH‑DA ( n = 3). Scale bar = 100 µm. d Flow cytometry analysis of apoptosis rates in HaCaT cells across different treatment groups and f corresponding quantitative analysis of apoptosis rates ( n = 4). e Flow cytometry analysis of apoptosis rates in WS1 cells across different treatment groups and g corresponding quantitative analysis of apoptosis rates ( n = 4). h Expression levels of apoptosis‑related proteins in different treatment groups (β-actin was the loading control, n = 3) and corresponding quantification of relative Bax/Bcl2 ratio in ( i ) HaCaT and j WS1 cells. Results are presented as mean ± SD. HaCaT cells were treated with 0.05 µM EPI (1 µM 5-Fu), and WS1 and primary rat epidermal cells were treated with 0.05 µM EPI (5 µM 5-Fu) for in vitro RRLD models. For comparisons between multiple groups, one-way ANOVA followed by post-hoc multiple-comparison tests was used. *** P < 0.001

Article Snippet: The human skin fibroblast cell line WS1 was obtained from the American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, In Vitro, Irradiation, Fluorescence, Microscopy, Flow Cytometry, Expressing, Control, Comparison

Journal: Molecular Biomedicine

Article Title: miR-338-3p promotes radiation recall like dermatitis by suppressing pleiotrophin via the PI3K/Akt/Bcl2 pathway

doi: 10.1186/s43556-026-00424-5

Figure Lengend Snippet: Identification of key regulatory miRNAs and target genes involved in RRLD by mRNA and miRNA sequencing. a Schematic overview of the study design, including transcriptome (mRNA) and miRNA sequencing followed by integrated bioinformatic analysis. b Venn diagram showing differentially expressed miRNAs among treatment groups ( n = 3). c Venn diagram showing differentially expressed mRNAs among treatment groups ( n = 3). d Relative expression levels of 13 candidate miRNAs in rat skin tissues: comparison between IR and RRLD (EPI or 5‑Fu) groups, as measured by qRT‑PCR (IR: n = 3; RRLD: n = 6). e Relative expression levels of the same candidate miRNAs in human peripheral blood serum samples (RT vs. RRD) measured by qRT‑PCR (IR: n = 17; RRD: n = 8). RT: Radiotherapy; RRD: radiation recall dermatitis. f Relative expression of miR‑338‑3p in HaCaT cells under different treatments ( n = 3). g Relative expression of miR‑338‑3p in WS1 cells under different treatments ( n = 3). h Venn diagram showing that both the human and rat 3′‑UTRs of the candidate genes PTN and TTK harbor predicted binding sites for miR‑338‑3p. i Relative expression levels of PTN and TTK mRNAs in HaCaT cells under different treatments ( n = 3). j Relative expression levels of PTN and TTK mRNAs in WS1 cells under different treatments ( n = 3). k Serum levels of PTN (pg/mL) in HNSCC RT and RRD groups determined by ELISA assay ( n = 3). HNSCC: head and neck squamous cell carcinoma; NPC: Nasopharyngeal carcinoma. Results are presented as mean ± SD. 0.05 µg/µL EPI (10 µg/µL 5-Fu) was used for the in vivo RRLD model. HaCaT cells were treated with 0.05 µM EPI (1 µM 5-Fu), and WS1 was treated with 0.05 µM EPI (5 µM 5-Fu) for in vitro RRLD models. For comparisons between two groups, Student’s t-test was used. For comparisons between multiple groups, one-way ANOVA followed by post-hoc multiple-comparison tests was used. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant

Article Snippet: The human skin fibroblast cell line WS1 was obtained from the American Type Culture Collection (ATCC).

Techniques: Sequencing, Expressing, Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, In Vivo, In Vitro

Journal: Molecular Biomedicine

Article Title: miR-338-3p promotes radiation recall like dermatitis by suppressing pleiotrophin via the PI3K/Akt/Bcl2 pathway

doi: 10.1186/s43556-026-00424-5

Figure Lengend Snippet: Regulatory role of miR-338-3p in RRLD models. a Relative miR-338-3p expression levels after transfecting with miR-338-3p mimics ( n = 3). b Relative miR-338-3p expression levels after transfecting with miR-338-3p inhibitor ( n = 3). c Effect of miR-338-3p overexpression on radiosensitivity of HaCaT cells evaluated by colony-formation assay (plating efficiency normalized to negative control, n = 3). d Effect of miR-338-3p knockdown on radiosensitivity of HaCaT cells by colony-formation assay ( n = 3). e Flow cytometry analysis of apoptosis in the RRLD model of HaCaT cells with miR-338-3p mimics and f corresponding quantitative analysis of apoptosis rates ( n = 4). g Flow cytometry analysis of apoptosis in WS1 cells with miR-338-3p mimics and h corresponding quantitative analysis of apoptosis rates ( n = 4). i Study protocol for evaluating effects of miR-338-3p knockdown in RRLD rats. j Representative photographs of rat hindlimb skin showing effects of miR-338-3p knockdown by antagomir in the RRLD model and k corresponding quantitative skin injury scores ( n = 4). l Representative hematoxylin–eosin (H&E) stained images of rat hindlimb skin illustrating effects of miR-338-3p knockdown on histopathology (Epidermal thickness is indicated by area within the white dashed lines, Scar bar = 100 µm) and m corresponding quantitative measurements of epidermal thickness from H&E sections ( n = 4). Results are presented as mean ± SD. 10 µg/µL 5-Fu was used for the in vivo RRLD model. HaCaT cells were treated with 1 µM 5-Fu, and WS1 was treated with 5 µM 5-Fu for in vitro RRLD models. For comparisons between two groups, Student’s t-test was used. ** P < 0.01; *** P < 0.001. NC: negative control

Article Snippet: The human skin fibroblast cell line WS1 was obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Over Expression, Colony Assay, Negative Control, Knockdown, Flow Cytometry, Staining, Histopathology, In Vivo, In Vitro

Journal: Molecular Biomedicine

Article Title: miR-338-3p promotes radiation recall like dermatitis by suppressing pleiotrophin via the PI3K/Akt/Bcl2 pathway

doi: 10.1186/s43556-026-00424-5

Figure Lengend Snippet: Regulatory role of PTN in RRLD models. a PTN protein expression levels in PTN overexpression or PTN knockdown groups via adenoviral transduction by western blotting (β-actin was the loading control, n = 3) and b corresponding quantification of relative PTN protein expression levels in HaCaT and WS1 cells. c Effect of PTN overexpression (OE) on radiosensitivity of HaCaT cells assessed by colony‑formation assay ( n = 3). d Effect of PTN knockdown on radiosensitivity of HaCaT cells assessed by colony‑formation assay ( n = 3). e Apoptosis rate of HaCaT cells with PTN OE and f corresponding quantitative analysis of apoptosis rates ( n = 4). g Apoptosis rate of WS1 cells with PTN overexpression and h corresponding quantitative analysis of apoptosis rates ( n = 4). i Study protocol for evaluating effects of PTN OE in RRLD rats. j Representative photographs of rat hindlimb skin showing the effects of PTN overexpression (Ad‑PTN OE) in the in vivo RRLD model and k corresponding quantitative skin‑injury scores ( n = 4). l Representative hematoxylin–eosin (H&E) stained images of rat hindlimb skin following PTN overexpression (Epidermal thickness is indicated by area within the white dashed lines, Scar bar = 100 µm) and m corresponding quantitative measurements of epidermal thickness from H&E sections ( n = 4). 10 µg/µL 5-Fu was used for the in vivo RRLD model. HaCaT cells were treated with 1 µM 5-Fu, and WS1 was treated with 5 µM 5-Fu for in vitro RRLD models. Results are presented as mean ± SD. For comparisons between two groups, Student’s t-test was used. ** P < 0.01; *** P < 0.001. NC, negative control; Ad, adenovirus; ns, not significant

Article Snippet: The human skin fibroblast cell line WS1 was obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Over Expression, Knockdown, Transduction, Western Blot, Control, In Vivo, Staining, In Vitro, Negative Control

Journal: Molecular Biomedicine

Article Title: miR-338-3p promotes radiation recall like dermatitis by suppressing pleiotrophin via the PI3K/Akt/Bcl2 pathway

doi: 10.1186/s43556-026-00424-5

Figure Lengend Snippet: miR‑338‑3p participates in the process of RRLD by targeting PTN. a Predicted miR‑338‑3p binding site sequences in the 3′‑UTR of human and rat PTN and Dual‑luciferase reporter assay in WS1 cells co‑transfected with miR‑338‑3p mimics or negative control (NC) and wild‑type (WT) or mutant (MUT) PTN‑3′‑UTR plasmids and b corresponding relative luciferase activity ( n = 4). c The mRNA expression levels of PTN in HaCaT and WS1 cells after transfecting with miR‑338‑3p mimics or ( d ) inhibitors ( n = 3). e The protein expression levels of PTN in HaCaT cells after transfecting with miR‑338‑3p mimics and g corresponding relative PTN protein expression (β-actin as the loading control; n = 3). f The protein expression levels of PTN in HaCaT cells after transfecting with miR‑338‑3p inhibitors and h corresponding relative PTN protein expression (β-actin as the loading control; n = 3). i Representative immunohistochemistry (IHC) images of PTN in non-RRLD rat skin tissue from the miRNA agomir NC group and miR-338-3p agomir group, with ( k ) corresponding immunoreactive scores (blue arrow indicates PTN immunoactivity, n = 4). j Representative IHC images of PTN in non-RRLD rat skin tissue from the miRNA antagomir NC group and miR-338-3p antagomir group, with ( l ) corresponding immunoreactive scores (blue arrow indicates PTN immunoactivity, n = 4). m Flow cytometry analysis of apoptosis in HaCaT and WS1 cells in the RRLD model under different treatments: miRNA mimics NC + Ad‑NC, miR‑338‑3p mimics + Ad‑NC, miRNA mimics NC + Ad‑PTN OE, and miR‑338‑3p mimics + Ad‑PTN OE and corresponding quantitative analysis of apoptosis rates in ( n ) HaCaT and ( o ) WS1 cells ( n = 4). Results are presented as mean ± SD. 10 µg/µL 5-Fu was used for the in vivo RRLD model. HaCaT cells were treated with 1 µM 5-Fu, and WS1 was treated with 5 µM 5-Fu for in vitro RRLD models. For comparisons between two groups, Student’s t-test was used. For comparisons between multiple groups, one-way ANOVA followed by post-hoc multiple-comparison tests was used. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. Ad, adenovirus; NC, negative control; OE, overexpression

Article Snippet: The human skin fibroblast cell line WS1 was obtained from the American Type Culture Collection (ATCC).

Techniques: Binding Assay, Reporter Assay, Negative Control, Mutagenesis, Luciferase, Activity Assay, Expressing, Control, Immunohistochemistry, Flow Cytometry, In Vivo, In Vitro, Comparison, Over Expression

Journal: Molecular Biomedicine

Article Title: miR-338-3p promotes radiation recall like dermatitis by suppressing pleiotrophin via the PI3K/Akt/Bcl2 pathway

doi: 10.1186/s43556-026-00424-5

Figure Lengend Snippet: miR-338-3p targets PTN to induce apoptosis via the PI3K/Akt/Bcl2 signaling pathway. a Representative images of RRLD rat hindlimb skin showing effects of miR‑338‑3p overexpression with or without PTN overexpression in the in vivo RRLD model ( n = 4). b Representative hematoxylin–eosin (H&E) images of RRLD rat hindlimb skin illustrating histopathological changes under the same treatments (Epidermal thickness is indicated by area within the white dashed lines, Scale bar = 100 µm) and c corresponding quantitative skin‑injury scores and d epidermal thickness from H&E images ( n = 4). e Western blot analysis of PTN protein and proteins in the PI3K/Akt/Bcl2 pathway in RRLD (HaCaT) and RRLD (WS1) transfected with miR‑338‑3p inhibitor or mimics, and f corresponding relative protein expression levels of markers in RRLD HaCaT and WS1 cells (β-actin as the loading control; n = 3). g Scheme of miR-338-3p targeting PTN to induce the apoptosis of RRLD in rats. Results are presented as mean ± SD. 10 µg/µL 5-Fu was used for the in vivo RRLD model. HaCaT cells were treated with 1 µM 5-Fu, and WS1 was treated with 5 µM 5-Fu for in vitro RRLD models. For comparisons between two groups, Student’s t-test was used. For comparisons between multiple groups, one-way ANOVA followed by post-hoc multiple-comparison tests was used. * P < 0.05; ** P < 0.01; *** P < 0.001. Ad, adenovirus; NC, negative control; OE, overexpression

Article Snippet: The human skin fibroblast cell line WS1 was obtained from the American Type Culture Collection (ATCC).

Techniques: Over Expression, In Vivo, Western Blot, Transfection, Expressing, Control, In Vitro, Comparison, Negative Control

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