Journal: Advanced Science

Article Title: Vinburnine Sensitizes Radiotherapy Efficacy in Nasopharyngeal Carcinoma by Triggering Pyroptosis and Immune Responses via Activation of EDAR‐NFκB Pathway

doi: 10.1002/advs.202506139

Figure Lengend Snippet: Vinburnine promotes IR‐EDAR‐NFκB‐induced apoptosis and pyroptosis. A) Reactome analysis of upregulated differential genes after 5µ m Vin / 2Gy IR treatment for 48h. B) Heatmap of differential gene expression in transcriptomics. C) qPCR of EDAR mRNA in NPC cells treated with 5µ m Vin / 2Gy IR for 48h ( n = 3). D) Protein expression of EDAR in the cell membrane/cytoplasm after being treated with 5µ m Vin / 2Gy IR for 48h. E) Western blotting detected the protein expression levels of the NFκB pathway (p65/p50) and pyroptosis index (GSDME/N‐GSDME/ Cleaved‐Caspase3) after 5µ m Vin / 2Gy IR treatment for 48h. Upon activation of the NFκB signaling pathway, its downstream signal, Caspase3, undergoes cleavage. This cleavage then cuts GSDME to form N‐GSDME, ultimately resulting in the pyroptosis of the cells. F) The binding of p65 to the EDAR promoter in the treated SUNE1 cells was detected by ChIP assay. G) The CB‐Dock2 website predicts the structural complex of vinburnine bound with the EDAR protein. Vinburnine is colored green; EDAR is colored grey and yellow. H) SPR technology proved that EDAR is the target of vinburnine. I) The Co‐IP experiment confirmed that after treatment with 5µ m Vin+2Gy IR, EDAR formed more protein complexes with EDARADD/TRAF6. Multiple samples were presented using mean ± standard deviation (SD). C) Statistical analysis with Two‐way ANOVA was used to analyze the statistical differences among multiple groups. F) Statistical analysis with One‐way ANOVA was used to analyze the statistical differences among multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns for non‐significant.

Article Snippet: Subsequently, western blotting confirmed that EDAR (Proteintech, China) formed protein complexes with EDARADD (Abclone, China) and TRAF6 (Immunoway, USA).

Techniques: Gene Expression, Expressing, Membrane, Western Blot, Activation Assay, Binding Assay, Co-Immunoprecipitation Assay, Standard Deviation

Journal: Advanced Science

Article Title: Vinburnine Sensitizes Radiotherapy Efficacy in Nasopharyngeal Carcinoma by Triggering Pyroptosis and Immune Responses via Activation of EDAR‐NFκB Pathway

doi: 10.1002/advs.202506139

Figure Lengend Snippet: Knocking down EDAR suppresses the radiosensitization effect of vinburnine. A) The cytotoxic effect of 5uM Vin/2Gy IR on EDAR‐knockdown cells was assessed using the CCK‐8 assay ( n = 5). B) Following EDAR knockdown, cells were treated with 5uM Vin/2Gy IR. Colony formation was evaluated by crystal violet staining (left) and the number of colonies was quantified (right) ( n = 3). C–E) Flow cytometry detected the apoptosis/ROS levels/mitochondrial membrane potential of the Vin±IR‐treated cells after EDAR knockdown ( n = 3). F) After EDAR knockdown, western blotting detected the protein expression (p65/p50/GSDME/N‐GSDME/Cleaved‐Caspase3) in the treated group. Multiple samples were presented using mean ± standard deviation (SD). A–E) Statistical analysis with Two‐way ANOVA was used to analyze the statistical differences among multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns for non‐significant.

Article Snippet: Subsequently, western blotting confirmed that EDAR (Proteintech, China) formed protein complexes with EDARADD (Abclone, China) and TRAF6 (Immunoway, USA).

Techniques: Knockdown, CCK-8 Assay, Staining, Flow Cytometry, Membrane, Western Blot, Expressing, Standard Deviation

Journal: Advanced Science

Article Title: Vinburnine Sensitizes Radiotherapy Efficacy in Nasopharyngeal Carcinoma by Triggering Pyroptosis and Immune Responses via Activation of EDAR‐NFκB Pathway

doi: 10.1002/advs.202506139

Figure Lengend Snippet: Vinburnine promotes IR‐EDAR‐NFκB‐induced apoptosis and pyroptosis. A) Reactome analysis of upregulated differential genes after 5µ m Vin / 2Gy IR treatment for 48h. B) Heatmap of differential gene expression in transcriptomics. C) qPCR of EDAR mRNA in NPC cells treated with 5µ m Vin / 2Gy IR for 48h ( n = 3). D) Protein expression of EDAR in the cell membrane/cytoplasm after being treated with 5µ m Vin / 2Gy IR for 48h. E) Western blotting detected the protein expression levels of the NFκB pathway (p65/p50) and pyroptosis index (GSDME/N‐GSDME/ Cleaved‐Caspase3) after 5µ m Vin / 2Gy IR treatment for 48h. Upon activation of the NFκB signaling pathway, its downstream signal, Caspase3, undergoes cleavage. This cleavage then cuts GSDME to form N‐GSDME, ultimately resulting in the pyroptosis of the cells. F) The binding of p65 to the EDAR promoter in the treated SUNE1 cells was detected by ChIP assay. G) The CB‐Dock2 website predicts the structural complex of vinburnine bound with the EDAR protein. Vinburnine is colored green; EDAR is colored grey and yellow. H) SPR technology proved that EDAR is the target of vinburnine. I) The Co‐IP experiment confirmed that after treatment with 5µ m Vin+2Gy IR, EDAR formed more protein complexes with EDARADD/TRAF6. Multiple samples were presented using mean ± standard deviation (SD). C) Statistical analysis with Two‐way ANOVA was used to analyze the statistical differences among multiple groups. F) Statistical analysis with One‐way ANOVA was used to analyze the statistical differences among multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns for non‐significant.

Article Snippet: EDAR knockdown lentiviral plasmids (GeneChem, China) were packaged with PSPAX2 and PMD2G in 293T cells.

Techniques: Gene Expression, Expressing, Membrane, Western Blot, Activation Assay, Binding Assay, Co-Immunoprecipitation Assay, Standard Deviation

Journal: Advanced Science

Article Title: Vinburnine Sensitizes Radiotherapy Efficacy in Nasopharyngeal Carcinoma by Triggering Pyroptosis and Immune Responses via Activation of EDAR‐NFκB Pathway

doi: 10.1002/advs.202506139

Figure Lengend Snippet: Knocking down EDAR suppresses the radiosensitization effect of vinburnine. A) The cytotoxic effect of 5uM Vin/2Gy IR on EDAR‐knockdown cells was assessed using the CCK‐8 assay ( n = 5). B) Following EDAR knockdown, cells were treated with 5uM Vin/2Gy IR. Colony formation was evaluated by crystal violet staining (left) and the number of colonies was quantified (right) ( n = 3). C–E) Flow cytometry detected the apoptosis/ROS levels/mitochondrial membrane potential of the Vin±IR‐treated cells after EDAR knockdown ( n = 3). F) After EDAR knockdown, western blotting detected the protein expression (p65/p50/GSDME/N‐GSDME/Cleaved‐Caspase3) in the treated group. Multiple samples were presented using mean ± standard deviation (SD). A–E) Statistical analysis with Two‐way ANOVA was used to analyze the statistical differences among multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns for non‐significant.

Article Snippet: EDAR knockdown lentiviral plasmids (GeneChem, China) were packaged with PSPAX2 and PMD2G in 293T cells.

Techniques: Knockdown, CCK-8 Assay, Staining, Flow Cytometry, Membrane, Western Blot, Expressing, Standard Deviation

Journal: Advanced Science

Article Title: Vinburnine Sensitizes Radiotherapy Efficacy in Nasopharyngeal Carcinoma by Triggering Pyroptosis and Immune Responses via Activation of EDAR‐NFκB Pathway

doi: 10.1002/advs.202506139

Figure Lengend Snippet: Vin+IR activates T cell immunity through the EDAR‐NFκB‐CCL5/CX3CL1 pathway. A) The mRNA expression of CCL5 and CX3CL1 in SUNE1 after 5µ m Vin/ 2Gy IR treatment ( n = 3). B) Diagram of human T cell‐related experiments. C). After 24h of 5µ m Vin / 2Gy IR treatment, 1×10^4 SUNE1 was co‐cultured with 2.5×10^4 T cells for 48h. The suspended T cells were washed away with PBS, and the viability of tumor cells was detected by CCK8 ( n = 5). D) T‐cell chemotaxis assay. The supernatant from SUNE1 cells treated with Vin/IR was collected and placed in the lower chamber of the transwell. T cells were then placed in the upper chamber of the transwell. After 48h, the liquid from the lower chamber was collected for T‐cell counting ( n = 3). E) T cells were co‐cultured with treated SUNE1 cell supernatant for 48h, and CD45 + CD3 + CD8 + GZMB + T cells were detected by flow cytometry ( n = 3). F) The ChIP assay was conducted to detect the binding of p65 to the CCL5 and CX3CL1 promoters following treatment with 5µ m Vin / 2Gy IR for 48h ( n = 3). G) Following sh‐EDAR or NFκBi pretreatment, the mRNA expression of CCL5 and CX3CL1 in Vin/IR‐treated SUNE1 was detected using qRT‐PCR ( n = 3). H–J) After sh‐EDAR or NFκBi pretreatment, the proliferation of SUNE1 cells treated with 5µ m Vin / 2Gy IR and co‐cultured with T cells, the number of chemotactic T cells in the supernatant of Vin/IR‐treated SUNE1 cells, and the proportion of CD45 + CD3 + CD8 + GZMB + T cells were measured ( n = 3). K) EDAR and CD8 multiplex immunofluorescence staining was performed on nasopharyngeal carcinoma tissue microarrays. Comparison of mean fluorescence intensity of EDAR in tissues with recurrence ( n = 39) and without recurrence ( n = 45) in NPC patients. L) Correlation between EDAR and CD8 expression in human NPC tissues ( n = 84). M) The patients were divided into EDAR low‐expression ( n = 21) and EDAR high‐expression ( n = 21) groups based on the quartile method. Survival analysis evaluated the association between EDAR expression levels and patient survival. Multiple samples were presented using mean ± standard deviation (SD). A, D–F) Statistical analysis with One‐way ANOVA was used to analyze the statistical differences among multiple groups. C, G–J) Statistical analysis with Two‐way ANOVA was used to analyze the statistical differences among multiple groups. K) The Mann‐Whitney test was used to analyze the expression of EDAR in patients with recurrence and those without recurrence. L) The correlation between EDAR and CD8 expression in tissue microarrays was analyzed using Pearson's and simple linear regression. M) Survival analysis (Kaplan‐Meier) was utilized to examine the correlation between EDAR expression and the prognosis of patients diagnosed with nasopharyngeal carcinoma. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns for non‐significant.

Article Snippet: EDAR knockdown lentiviral plasmids (GeneChem, China) were packaged with PSPAX2 and PMD2G in 293T cells.

Techniques: Expressing, Cell Culture, Chemotaxis Assay, Cell Counting, Flow Cytometry, Binding Assay, Quantitative RT-PCR, Multiplex Assay, Immunofluorescence, Staining, Comparison, Fluorescence, Standard Deviation, MANN-WHITNEY

Journal: eLife

Article Title: Mesenchymal Meis2 controls whisker development independently from trigeminal sensory innervation

doi: 10.7554/eLife.100854

Figure Lengend Snippet: ( A ) Micro-computed tomography (micro-CT) images through the snout of E15.5 control and Meis2 cKO mice showing abnormal whisker phenotype in the mutant. Arrows show some escaper whisker follicles (WFs). ( B ) Bright-field images of control and Meis2 cKO mice at E18.5. ( C ) Two examples of 100 μm frozen sections of snouts from E18.5 control and Meis2 cKO stained with DAPI (blue) and EDAR (red) showing only a few whiskers in the mutant. In contrast, EDAR-labeled hair follicles appeared to be normal in the mutant. Arrows show some escaper WFs. Scale bar: 1 mm.

Article Snippet: Primary antibodies used: SOX9 (Merck Sigma, AB5535), TRKA (R&D Systems, AF1056), MEIS2 (GeneScript, custom), TUJ1 (R&D Systems, MAB1195), EDAR (R&D Systems, AF745), Lef1 (Cell Signaling, C12A5), beta-galactosidase (Abcam, ab9361), SOX2 (R&D Systems, MAB2018), FOXD1 (Abcam, AB129324).

Techniques: Micro-CT, Control, Whisker Assay, Mutagenesis, Staining, Labeling

Journal: eLife

Article Title: Mesenchymal Meis2 controls whisker development independently from trigeminal sensory innervation

doi: 10.7554/eLife.100854

Figure Lengend Snippet: ( A ) Triple immunostaining of 100 μm sections shows the absence of TG nerve projections (TUJ1+, TRKA+) and normal WF (SOX9+) in Neurog1 -/- mice. Arrows in black and white images show examples of invagination of whisker placodes labeled by SOX9 antibody. Scale bar: 500 μm. ( B ) Whole-mount immunostaining of WFs with SOX9 and EDAR antibodies showing normal WF morphology and patterning in mutants at embryonic day 12.5 (E12.5) (top) and 13.5 (bottom). Scale bars: 300 μm. ( C ) EDAR staining of 10 μm sections at E12.5 showing normal initiation of placode formation in Neurog1 mutants. Scale bar: 150 μm. ( D ) Micro-computed tomography (micro-CT) images reveal normally developed whiskers (top) at E17.5 in mutants while TGs are lacking (bottom, arrows).

Article Snippet: Primary antibodies used: SOX9 (Merck Sigma, AB5535), TRKA (R&D Systems, AF1056), MEIS2 (GeneScript, custom), TUJ1 (R&D Systems, MAB1195), EDAR (R&D Systems, AF745), Lef1 (Cell Signaling, C12A5), beta-galactosidase (Abcam, ab9361), SOX2 (R&D Systems, MAB2018), FOXD1 (Abcam, AB129324).

Techniques: Triple Immunostaining, Whisker Assay, Labeling, Immunostaining, Staining, Micro-CT

Journal: eLife

Article Title: Mesenchymal Meis2 controls whisker development independently from trigeminal sensory innervation

doi: 10.7554/eLife.100854

Figure Lengend Snippet: ( A ) Whole-mount in situ hybridization of Shh mRNA documenting loss of WFs in mutants. Arrow shows an escaper whisker. ( B ) Whole-mount immunostaining of WFs with SOX9 and EDAR antibodies showing absence of WFs in Meis2 cKO at embryonic day 12.5 (E12.5). Two examples for each genotype are shown. Scale bar: 300 μm. ( C ) EDAR staining of 10 μm sections showing placode formation arrest in mutants. Scale bar: 150 μm.

Article Snippet: Primary antibodies used: SOX9 (Merck Sigma, AB5535), TRKA (R&D Systems, AF1056), MEIS2 (GeneScript, custom), TUJ1 (R&D Systems, MAB1195), EDAR (R&D Systems, AF745), Lef1 (Cell Signaling, C12A5), beta-galactosidase (Abcam, ab9361), SOX2 (R&D Systems, MAB2018), FOXD1 (Abcam, AB129324).

Techniques: In Situ Hybridization, Whisker Assay, Immunostaining, Staining

Journal: eLife

Article Title: Mesenchymal Meis2 controls whisker development independently from trigeminal sensory innervation

doi: 10.7554/eLife.100854

Figure Lengend Snippet: ( A ) Three examples of 10 μm FFPE sections stained with EDAR antibody showing affected expression of EDAR in Meis2 cKO mice. Arrows indicate EDAR-positive sites which are observed less frequently in the mutant snout. Scale bar: 500 μm. ( B ) Two representative images of normally developed escaper whisker follicles (WFs) in the mutants by LEF1 staining. LEF1 expression is similar in just forming placodes (top) and invaginated (bottom). Additionally, LEF1 expression shows a decline in the DC regions compared to peri-DC. Scale bar: 30 μm. ( C ) Three representative micrographs of LEF1-stained 10 μm FFPE snout sections showing normal expression of LEF1 in the dermis but the decreased number of LEF1+ placodes in the epithelium of the mutant snout. Scale bar: 500 μm. ( D ) Quantification of Lef1+ placode count and Lef1 fluorescence intensities in placodes, DCs, or interfollicular upper dermis. Data are presented as mean ± sem, ***p=0.0002. ( E ) In situ hybridization HCR-FISH using probes for Axin2 and Wnt10b in control snout at embryonic day 12.5 (E12.5) showing placodal expression of Wnt10b (green) and Axin2 (arrows, red) as well as widespread expression of Axin2 in the dermis. Scale bar: 60 μm.

Article Snippet: Primary antibodies used: SOX9 (Merck Sigma, AB5535), TRKA (R&D Systems, AF1056), MEIS2 (GeneScript, custom), TUJ1 (R&D Systems, MAB1195), EDAR (R&D Systems, AF745), Lef1 (Cell Signaling, C12A5), beta-galactosidase (Abcam, ab9361), SOX2 (R&D Systems, MAB2018), FOXD1 (Abcam, AB129324).

Techniques: Staining, Expressing, Mutagenesis, Whisker Assay, Fluorescence, In Situ Hybridization, Control

Journal: eLife

Article Title: Mesenchymal Meis2 controls whisker development independently from trigeminal sensory innervation

doi: 10.7554/eLife.100854

Figure Lengend Snippet: ( A ) Whole-mount immunostaining of FOXD1, TUJ1, and SOX9 of heads from controls and Foxd1 -null mutants at embryonic day 13.5 (E13.5) showing normal formation of WFs in mutants in which FOXD1 signal disappears. Normal WF development is also reflected in normal WF innervation represented by TUJ1 staining. Scale bars: 500 μm. ( B ) Whole-mount immunostaining of EDAR confirmed normal placode (Pc) appearance in Foxd1 -null mutants. Scale bars: 500 μm.

Article Snippet: Primary antibodies used: SOX9 (Merck Sigma, AB5535), TRKA (R&D Systems, AF1056), MEIS2 (GeneScript, custom), TUJ1 (R&D Systems, MAB1195), EDAR (R&D Systems, AF745), Lef1 (Cell Signaling, C12A5), beta-galactosidase (Abcam, ab9361), SOX2 (R&D Systems, MAB2018), FOXD1 (Abcam, AB129324).

Techniques: Immunostaining, Staining

Journal: JCI Insight

Article Title: A single-cell atlas of normal and KRAS G12D -malformed lymphatic vessels

doi: 10.1172/jci.insight.185181

Figure Lengend Snippet: ( A ) Schematics of the Prox1-CreER T2 and Kras LSL-G12D alleles. ( B ) Schematic showing when mice received tamoxifen (50 μg; p.o.). Tissues were collected when mice were 21 days old. ( C ) Low- and high-magnification views of Vegfr3-positive lymphatic vessels in 100-μm-thick lung sections from control and Kras G12D mice. The high-magnification images are of the boxed regions. ( D ) The diameter of lymphatic vessels was significantly greater in Kras G12D mice (25.65 ± 1.070 μm; n = 5) than in control (Ctrl) mice (14.31 ± 0.9752 μm; n = 5). Data are presented as mean ± SEM. **** P < 0.0001 by 2-tailed, unpaired Student’s t test. Scale bars: 500 μm (low magnification) and 50 μm (high magnification).

Article Snippet: We used a goat anti-Vegfr3 antibody (R&D Systems, AF745; 1:250).

Techniques: Control