Journal: bioRxiv

Article Title: Endocrine-Adapted Pituitary Macrophages Regulate Gonadotropin Secretion through CXCL5-CXCR2-MAPK Signaling

doi: 10.64898/2026.08.12.744557

Figure Lengend Snippet: ( A ) LH secretion following 12- or 30-hour pre-stimulation with 100 ng/mL CXCL5 and subsequent 30-minute stimulation with 10 nM GnRH is presented as MFI. ( B - C ) Corresponding GnRH receptor (GnRHR) and CXCR2 surface expressions were measured by flow cytometry and expressed as MFI. ( D ) LβT2 cells were pre-treated with anti-CXCR2 antibody (1:250 dilution) for 30 minutes prior to 12-hour BMDM-cultured media treatment, with the antibody maintained at a final dilution of 1:500. LβT2 cells were co-cultured with BMDM-conditioned media following the density to volume ratio of 2 gonadotropes per 1 macrophage. LH concentration in culture supernatants was measured by Luminex. ( E ) Phospho-protein array analysis using a mouse MAPK signaling pathway kit. LβT2 cells were treated with 100 ng/mL CXCL5 for 30 minutes without prior serum starvation of cells.

Article Snippet: Antibody-printed membranes from the JAK/STAT and MAPK Phospho Antibody Array kits (RayBiotech, Cat. No. AAM-JAKSTAT-1-4, Cat. No. AAH-MAPK-1-4) were incubated with normalized cell lysates overnight at 4°C, followed by sequential incubation with biotinylated detection antibodies and HRP-conjugated streptavidin as recommended by the manufacturer.

Techniques: Flow Cytometry, Cell Culture, Concentration Assay, Luminex, Protein Array

Journal: Head & Neck

Article Title: CCL26 ‐Mediated Modulation of Endothelial Secretome by Hypoxia‐Induced Tumor‐Derived Exosomes Enhances Metastatic Progression in Head and Neck Cancer

doi: 10.1002/hed.70259

Figure Lengend Snippet: Transport of TDExs to HUVECs and alteration of HUVECs‐derived secretome protein profiles. (A) PKH67 staining of HUVECs exposed to exosomes to demonstrate the successful inclusion of microvesicles by the endothelial cells. DAPI, a cell membrane permeable dye, is used to label the nuclear DNA of cells. PKH67 is used to label exosomes to track transfer of their cargo to the recipient cells. (B) Experimental workflow of proteomic analysis of proteins secreted by secretomes derived from HUVECs treated with TDExs from HNSCC cells. workflow are created using BioRender (RRID:SCR_018361) ( https://biorender.com ). TDExs derived from HNSCC cells induced the expression of cytokine proteins in HUVECs. A human cytokine protein array was performed using HUVECs‐derived secretomes cultured with normoxic and hypoxic induced (C) Detroit‐562 and (D) FaDu cells derived exosomes for 48 h. C6 and C7 Array panel images were analyzed using ImageJ after background subtraction. The framed spots in panels C6 array indicate the target chemokine CCL26. Representative array blots are shown after an exposure time of 5 min. Data are presented as mean ± SD. Statistical significance was determined using FDR‐adjusted q values (Benjamini–Krieger–Yekutieli, q < 0.05). Mean ± standard error of mean (SEM) is shown * p < 0.05, ** p < 0.01, t ‐test, *** p < 0.001. All experiments were performed in at least triplicate ( n = 3). [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Protein profiling of 120 cytokines in the HUVECs‐derived secretomes treated with niTDExs and hiTDExs derived from HNSCC cells was performed using the RayBio C‐Series Human Cytokine Antibody Array C6 & C7 Kit (RayBiotech, AAH‐CYT‐1000‐2), according to the manufacturer's instructions.

Techniques: Derivative Assay, Staining, Membrane, Expressing, Protein Array, Cell Culture

Journal: Head & Neck

Article Title: CCL26 ‐Mediated Modulation of Endothelial Secretome by Hypoxia‐Induced Tumor‐Derived Exosomes Enhances Metastatic Progression in Head and Neck Cancer

doi: 10.1002/hed.70259

Figure Lengend Snippet: Changed protein profile in HUVECs‐derived secretome via hiTDExs and validation of CCL26 chemokine abundance change among the changed proteins in the secretome. Detroit‐562 cells (A) C6 Array, (B) C7 Array, FaDu cells (C) C6 Array, (D) C7 Array bar graphs show the average signal intensities of the spots on the array panels and include expression level changes. The framed areas in the C6 array bar graphs indicate the target chemokine CCL26. Relative CCL26 level in secretome samples obtained from HUVECs treated with normoxic and hypoxic exosomes derived from (E) Cal 27, (F) Detroit‐562 and (G) FaDu cells. Relative CCL26 level in normoxic and hypoxic exosome lysates collected from serum‐free culture media of (H) Cal 27, (I) Detroit‐562 and (J) FaDu cells treated with or without CoCl 2 for 48 h. (K–M) Relative CCL26 level in HUVECs lysates treated with niTDExs and hiTDEXs for 48 h. Data were plotted as mean ± standard error of the mean and significance of differences were statistically performed using one‐way ANOVA where appropriate. Mean ± standard error of mean (SEM) is shown * p < 0.05, ** p < 0.01, t ‐test, *** p < 0.001, one‐way ANOVA test. All experiments were performed in at least triplicate ( n = 3). [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Protein profiling of 120 cytokines in the HUVECs‐derived secretomes treated with niTDExs and hiTDExs derived from HNSCC cells was performed using the RayBio C‐Series Human Cytokine Antibody Array C6 & C7 Kit (RayBiotech, AAH‐CYT‐1000‐2), according to the manufacturer's instructions.

Techniques: Derivative Assay, Biomarker Discovery, Expressing