Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor microenvironment biomarkers predicting pathological response to neoadjuvant chemoimmunotherapy in locally advanced esophageal squamous cell carcinoma: post-hoc analysis of a single center, phase 2 study

doi: 10.1136/jitc-2024-008942

Figure Lengend Snippet: Neutrophil-enriched tumor microenvironment phenotype in non-pCR patients. (A) Differential gene expression analysis based on pCR in cohort 1 (fold change >1.5, p<0.05). Red points on the right side represent highly expressed genes in pCR, while blue points on the left represent highly expressed genes in non-pCR. Dot size indicates the absolute value of the fold change and p value. ( B ) Results of pathway enrichment under different therapeutic responses in cohort 1. ( C ) Gene Set Enrichment Analysis (GSEA) of differentially expressed genes based on Gene Ontology Cellular Component (GO-CC) biological process enrichment. (D–E) Enrichment results of neutrophil infiltration and neutrophil phenotype-related genes between the non-pCR and pCR groups within cohort 1 obtained using different methods. (F–G) Representative multiplex immunofluorescence images of patients in cohort 2. Nuclei (DAPI, blue), PANCK (green), CD66b (orange), CD15 (pink), ARG1 (yellow). (H–I) Proportion of different cells in patients with different therapeutic responses in cohort 2. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively. pCR, pathological complete response; TANs, tumor-associated neutrophils; TAN1, antitumoral TAN; TAN2, protumoral TAN.

Article Snippet: Staining for DAPI (TG470SN, TissueGnostics), panCK (zm-0069, ZSGB-BIO), CD66b (ab300122, Abcam), CD15 (ab172729, Abcam), ARG1 (ab133543, Abcam), INHBB (ab69286, Abcam), and HSPA2 (ab108416, Abcam) was conducted to characterize the TME and molecular features of responders and non-responders.

Techniques: Gene Expression, Multiplex Assay, Immunofluorescence

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: BBB maturation stages in the postnatal cerebral cortex based on glial configuration. (A) 3D images of AQP4, Iba1, lectin, and DAPI staining in the rat cerebral cortex at P1–30 are shown. Scale bar indicates 30 μm. AQP4 gradually accumulated in the blood vessels and localized to the blood vessels after P15. The number of Iba1+ microglia significantly increased after P10 and peaked at P15, changing their shape from ameboid to ramified. (B) Temporal changes in biotin permeability are corrected to a maximum value of 100%. Typical images of P4 and P15 rat brain cortical regions stained with streptavidin are shown. A solid line was drawn perpendicular to the blood vessel to quantify the fluorescence, which is indicated by the dashed line, and the red fluorescence value was measured, as shown in the upper right graph. The traces show the average of 70–76 transverse vessel lines for the respective postnatal ages. The maximum fluorescence value was set at 100, and the sum of the fluorescence values 20 μm to the left and to the right was calculated. The peak became sharper as the age increased. Astrocyte and microglial coverage rates were corrected to a P30 value of 100%. BBB formation and maturation in rats are classified into three phases based on biotin leakage and astrocyte and microglial contact with blood vessels . The phases are as follows: the “immature stage” when biotin permeability is still high; the “organic formation stage” when glial structures around blood vessels are formed; and the “completion stage” when the structures around blood vessels become stable and the morphology of microglia changes to a ramified type. Abbreviations: AQP4: aquaporin 4; BBB: blood–brain barrier; DAPI: 4′,6-diamidino-2-phenylindole; Iba1: ionized calcium binding adapter protein 1; P: postnatal day.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Staining, Permeability, Fluorescence, Binding Assay

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Expression of blood vessel developmental markers in the postnatal cerebral cortex. (A) Images of CD31, CD34, CD146, agrin, and Tie2 co-stained with lectin and DAPI in the rat cerebral cortex at P1–30 are shown. Scale bar indicates 50 μm. CD31 and Tie2 signals gradually increased in blood vessels, whereas CD34, CD146, and agrin signals gradually decreased. (B) The graphs show temporal changes in the percentage of marker+ area relative to the total vessel area. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 Images were obtained from the cerebral cortical regions of three rat pups of each age with a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01. vs. P1 value, while # p < 0.05, ## p < 0.01 vs. P10 value. (C) Graph summarizing results for B and B. Changes in biotin permeability, CD34, CD146, and agrin were corrected to a P1 value of 100%, and the astrocyte coverage rate, microglial coverage rate, CD31, and Tie2 were corrected to a P30 value of 100%. The grey zone represents the organic formation term (P4P15). Abbreviations: CD31, cluster of differentiation 31 or platelet endothelial cell adhesion molecule 1; CD146, melanoma cell adhesion molecule; DAPI: 4′,6-diamidino-2-phenylindole; P, postnatal day; Tie2, TEK receptor tyrosine kinase; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy, Permeability

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Analysis of vascular TJ protein expression in the postnatal cerebral cortex. (A) Images of claudin-5, occludin, and ZO-1 co-stained with lectin and DAPI in the rat cerebral cortex at P1–30. Scale bar indicates 100 μm. Claudin-5 and occludin signals increased in blood vessels during development, whereas ZO-1 signals remained unchanged. (B) The graphs show changes in the percentage of marker+ area of the total vessel area at P4, P15, and P30. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 images were obtained from the cerebral cortical regions of three rat pups from each age group using a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01 vs. P4 value, and # p < 0.05 vs. P15 value. DAPI: 4′,6-diamidino-2-phenylindole; P, postnatal day; TJ, tight junction; ZO-1: zonula occludens-1; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Analysis of vascular expression of transporters and receptor proteins in the postnatal cerebral cortex. (A) Images of P-gp, BCRP, Glut1, and TfR co-stained with lectin and DAPI in the rat cerebral cortex at P1–30. Scale bar indicates 100 μm. The arrowhead shows the co-localization areas of the marker and lectin. P-gp and BCRP signals gradually increased in the blood vessels during development, whereas Glut1 and TfR signals were high in the blood vessels at P1 and remained unchanged. (B) The graphs show changes in the percentage of marker+ area of the total vessel area at P4, P15, and P30. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 images were obtained from the cerebral cortical regions of three rat pups from each age group using a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01 vs. P4 value. Abbreviations: BCRP: breast cancer resistance protein; DAPI: 4′,6-diamidino-2-phenylindole; Glut1: glucose transporter type 1; P: postnatal day; P-gp: P-glycoprotein; TfR: transferrin receptor; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

doi: 10.1111/jcmm.13707

Figure Lengend Snippet: AFAP1‐AS1 enhances cisplatin resistance in laryngeal carcinoma cells. A, HEp‐2 was treated with 4 μmol L −1 cisplatin. Expression of AFAP1‐AS1 was analysed at various times (0, 6, 12, 18, 24, and 30 h) by qRT‐PCR. * P < .05, ** P < .01, compared with 0 h. B, AFAP1‐AS1 silenced HEp‐2 cells were cultured in 96‐well plates. Cell viability was analysed by CCK8 assay under treatment with various concentration of cisplatin (0, 2, 4, 8, 16 and 32 μmol L −1 ). ** P < .01, compared with control siRNA transfected cells. C, Apoptosis assays in AFAP1‐AS1 silenced HEp‐2 cells under 8 μmol L −1 cisplatin treatment. ** P < .01, compared with control siRNA transfected cells. D, Cisplatin‐resistant HEp‐2 cell lines (HEp‐2/R) were established. Cell viability assays were performed in HEp‐2 and HEp‐2/R cells under various concentrations of cisplatin treatment. * P < .05, ** P < .01, compared with HEp‐2 cells. E, Expression of AFAP1‐AS1 in HEp‐2 and HEp‐2/R cells was analysed by qRT‐PCR. ** P < .01, compared with HEp‐2 cells

Article Snippet: We conducted nuclear DAPI staining to access cell apoptosis using Cell Apoptosis DAPI Detection Kit (GenScript, Piscataway, NJ) according to manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, CCK-8 Assay, Concentration Assay, Control, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

doi: 10.1111/jcmm.13707

Figure Lengend Snippet: miR‐320a reduces stemness and cisplatin resistance in laryngeal carcinoma cells. A, HEp‐2 cell morphology of parental cells and stemness‐enriched cell spheres (left) and corresponding miR‐320a expression (right). ** P < .01, compared with parental cells. B, Expression of miR‐320a in miR‐320 overexpression HEp‐2 cells by qRT‐PCR. *** P < .001, compared with control miRNA transfected cells. C, Expression of stemness‐associated genes in miR‐320a overexpression HEp‐2 cells. Gene expression was analysed by qRT‐PCR. * P < .05, ** P < .01, compared with control miRNA transfected cells. D, Number of tumour spheres in miR‐320a overexpression HEp‐2 cells. ** P < .01 compared with control miRNA transfected cells. E, miR‐320a overexpression HEp‐2 cells were cultured in 96‐well plates. Cell viability was analysed using CCK8 assays under various concentrations of cisplatin (0, 2, 4, 8, 16 and 32 μmol L −1 ). ** P < .01, compared with control miRNA transfected cells. F, Apoptosis assay in miR‐320a overexpression HEp‐2 cells under 8 μmol L −1 cisplatin treatment. ** P < .01, compared with control miRNA transfected cells. G, Expression of miR‐320a in HEp‐2 and HEp‐2/R cells was analysed by qRT‐PCR. ** P < .01, compared with HEp‐2 cells

Article Snippet: We conducted nuclear DAPI staining to access cell apoptosis using Cell Apoptosis DAPI Detection Kit (GenScript, Piscataway, NJ) according to manufacturer's instructions.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Control, Transfection, Gene Expression, Cell Culture, Apoptosis Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

doi: 10.1111/jcmm.13707

Figure Lengend Snippet: AFAP1‐AS1 regulates laryngeal carcinoma cells through miR‐320a/ RBPJ. A, Expression of RBPJ mRNA in AFAP1‐AS1 silenced, miR‐320a inhibition (miR‐320a‐in) and AFAP1‐AS1 silenced plus miR‐320a inhibition HEp‐2 cells by qRT‐PCR. * P < .05, compared with control cells. B, Expression of RBPJ protein in AFAP1‐AS1 silenced, miR‐320a inhibition and AFAP1‐AS1 silenced plus miR‐320a inhibition HEp‐2 cells by Western blot. C, Expression of RBPJ mRNA in AFAP1‐AS1 silenced, RBPJ and AFAP1‐AS1 silenced plus RBPJ HEp‐2 cells by qRT‐PCR. * P < .05, compared with control cells. D, Expression of RBPJ mRNA in AFAP1‐AS1 silenced, RBPJ and AFAP1‐AS1 silenced plus RBPJ HEp‐2 cells by Western blot. E, Number of tumour spheres in AFAP1‐AS1 silenced, RBPJ and AFAP1‐AS1 silenced plus RBPJ HEp‐2 cells. * P < .05 compared with control cells. F, Apoptosis assay in AFAP1‐AS1 silenced, RBPJ and AFAP1‐AS1 silenced plus RBPJ HEp‐2 cells under 8 μmol L −1 cisplatin treatment. * P < .05, compared with control cells

Article Snippet: We conducted nuclear DAPI staining to access cell apoptosis using Cell Apoptosis DAPI Detection Kit (GenScript, Piscataway, NJ) according to manufacturer's instructions.

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Control, Western Blot, Apoptosis Assay