recombinant human cxcl5 protein  (R&D Systems)

Journal: Nature Communications

Article Title: IL-17A is increased in diabetic wounds and impairs keratinocyte function via histone demethylase JMJD3

doi: 10.1038/s41467-025-67456-3

Figure Lengend Snippet: a ChIP analysis of H3K27me3 or IgG at indicated promoters in keratinocytes with (white) or without (blue) IL-17A stimulation. Itga3: n = 5 (unstimulated), n = 4 (IL-17A-stimulated) technical replicates, p = 0.0034, Timp1 : n = 3 technical replicates, p = 0.0046, Ccl20 : n = 3 technical replicates, p = 0.0059, Cxcl1 : n = 3 technical replicates, p = 0.0020, Cxcl3 : n = 3 technical replicates, p = 0.0258, Cxcl5 : n = 3 technical replicates, p = 0.0174. n = 3 independent experiments. b qPCR analysis of keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (1 µM) (red). Itga3 : n = 4 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0017 (IL-17A vs. IL-17A and inhibitor), Timp1: n = 6 biological replicates, p < 0.0312 (DMSO vs. IL-17A), p = 0.0029 (IL-17A vs. IL-17A and inhibitor), Ccl20: n = 3 biological replicates, p = 0.0008 (DMSO vs. IL-17A), p = 0.0428 (IL-17A vs. IL-17A and inhibitor), Cxcl1 : n = 3 biological replicates, p = 0.0003 (DMSO vs. IL-17A), p = 0.0294 (IL-17A vs. IL-17A and inhibitor), Cxcl3 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0003 (IL-17A vs. IL-17A and inhibitor), Cxcl5 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0007 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. c Western blot of ITGA-3 expression in keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). Representative densitometry plot is shown. n = 3 independent experiments. d Protein quantification of lysates from keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). TIMP-1: n = 6 biological replicates, p = 0.0012 (DMSO vs. IL-17A), p = 0.0068 (IL-17A vs. IL-17A and inhibitor), CCL-20: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0199 (IL-17A vs. IL-17A and inhibitor), CXCL-1: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0158 (IL-17A vs. IL-17A and inhibitor), CXCL-3: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0167 (IL-17A vs. IL-17A and inhibitor), CXCL-5: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0005 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. e qPCR analysis of keratinocytes treated with a non-targeting control (siNTC) (white) or si Jmjd3 (gray). Jmjd3 : n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0192, Itga3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Timp1: n = 3 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0205, Ccl20: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0015, Cxcl1: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Cxcl3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, Cxcl5: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, n = 3 independent experiments. f qPCR analysis of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (yellow) keratinocytes. n = 3 biological replicates, Itga3 : p = 0.0184, Timp1 : p = 0.0044, Ccl20 : p = 0.0160, Cxcl1 : p = 0.0371, Cxcl3 : p = 0.0042. n = 3 independent experiments. g , h Scratch assays of primary murine ( n = 3 biological replicates, p < 0.0001 (48 h)) and N/TERT ( n = 3 biological replicates, p = 0.0340 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J4 (red). n = 3 independent experiments. i , j Scratch assays of primary murine ( n = 3 biological replicates, p = 0.0016 (48 h)) and N/TERT ( n = 6 biological replicates (IL-17A alone), n = 4 biological replicates (IL-17A and GSK-J1), p = 0.0223 (8 h), p < 0.0001 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J1 (red). n = 3 independent experiments. k Scratch assay of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (blue) keratinocytes. n = 3 biological replicates, p = 0.0198 (48 h). n = 3 independent experiments. Data were analyzed for variances, and 2-tailed Student’s t tests for ( a ), ( e ), ( f ) and 1-way ANOVA tests for ( b ), ( d ), ( g – k ) were performed. Data are presented as the mean ± SEM.

Article Snippet: After stimulation, cell free supernatant was collected and analyzed by the University of Michigan Immune Monitoring Shared Resource Core for CCL-20, CXCL-1, CXCL-5 or specific enzyme immunoassay kits for CXCL-3 (Boster Bio) and TIMP-1 (R&D Systems) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Wound Healing Assay

Journal: bioRxiv

Article Title: Immune-Epithelial Interactions via TGF-β Orchestrates Stem-Cell Niche Formation and Morphogenesis

doi: 10.1101/2025.05.22.655596

Figure Lengend Snippet: (a) Cytokine array profiling comparing co-cultures and epithelial-alone hydrogels shows elevated levels of IL-6, CXCL5, CCL2, TIMP-1, LIF, GRO-a (CXCL1), IL-8, IGFBP-1, IGFBP-2, VEGF-A, SDF-1a, and IGF-1in responder co-cultures with immune cells (n=2 patients). (b) Bar graph showing treatment of responder epithelial cells with individual (IL-6, CCL2, GRO-a (CXCL1), CXCL5, VEGF) or pooled recombinant cytokines at 2ng/ml. (n=8; ns, not significant, **p-value < 0.01, ***p-value < 0.001). (c) Bar graph showing treatment of responder epithelium with conditioned media from responder co-cultures (n=8, ns, not significant). (d) Flow cytometry analysis of patient single cells showing percent of CD45 immune cells out of all breast cells. (n=12; ns, not significant). (e) Bar graph and scatter plot showing percent of CD3 + CD4 - CD8 - double negative (DN) T cells out of all CD45 + immune cells (n=12; ***p-value < 0.001, two-tailed t-test). (f) Scatter plot showing percent of γδ T cells out of all DN T cells.

Article Snippet: For treatment with recombinant proteins IL-6 (PHC0066, Thermofisher,2ng/ml), CCL2 (279-MC-050/CF, R&D Systems, 2ng/ml), GRO-a (275-GR-010/CF, R&D Systems, 2ng/ml), CXCL5 (254-XB-025/CF, R&D Systems, 2ng/ml), VEGF (78073, Stem Cell Technologies, 2ng/ml), treatment began at timepoint 0 and continued for the entire culture period.

Techniques: Recombinant, Flow Cytometry, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 3. CXCL5 expression correlates positively with bone metastasis progression. (A) Serum samples were isolated from tumor-bearing mice [14 d after intracardiac (I.C.) injection, 3 × 104 cells/mouse], and protein-based arrays were analyzed as described by R&D Biosystems. Heatmap is presented. Cropped blot for CXCL5 is presented in the bottom panel. (B) CXCL5 level in serum from mice under different experimental conditions (sham/basal level vs. 14-d post-I.C. or postorthotopic implantation (1 × 104 cells/mouse). A mouse-specific CXCL5 enzyme-linked immunosorbent assay (ELISA) kit (R&D Biosystems) was used for determining CXCL5 levels. (C) Bone marrow (BM) cells were isolated from WT (BMWT) and mda-9−/− (BMKO) mice and stimulated with tumor cell–derived conditioned media for 12 h. Total cellular RNA was extracted, and qPCR was performed to detect mouse CXCL5 mRNA. Data are presented as fold-change relative to the unstimulated wild- type (BMWT) group. Different letters in two variables are statistically significant (P < 0.05). (D) CXCL5 levels in serum from tumor-bearing mice receiving “sham” (represented as “−“) or “WT” (represented as “+”) bone marrow (experiments described in Fig. 1E). *P < 0.01. (E) A total of 3 × 104 RM1-BM-Luc cells were injected by the intracardiac (I.C.) route into WT and mda-9−/− mice (schematically presented in the left panel). Fourteen days postinjection, total BM cells were isolated and stained for lineage-specific cell surface markers and intracellular CXCL5 expression to identify the specific cell population(s) that differentially respond to tumor cells. Percentages of CXCL5 expression in bone marrow–derived mesenchymal stromal cells: (CD45−CD90+) are presented. *P < 0.05. (F) Tumor-implanted bones (from animals in Fig. 2A) were immunostained with different antibodies as indicated, and representative photographs are presented.

Article Snippet: Human and mouse specific recombinant protein CXCL5, PDGF- AA, GM- CSF were procured from R&D Systems.

Techniques: Expressing, Isolation, Injection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 4. MDA-9 expression in tumor cells nonautonomously activates the Hippo pathway and induces CXCL5 in HS5 cells. (A) Bone metastasis development in athymic nude mice following intracardiac injection of PC-3ML control and mda-9 KO (PC-3MLmda-9 KO) clone (1 × 105 cells/mouse). Representative images 36 d after tumor cell implantation (Left). Bone metastases detected by BLI imaging. The percentage of mice with BLI-positive lesions is shown (Right). (B) HS5 cells were incubated with normalized (equal amount of total protein) tumor cell–derived condition media for 12 h. HS5-derived conditioned media were analyzed for CXCL5 levels using ELISA. (C) HS5 cells were transfected with a 1.5-kb CXCL5-Prom using a standard transfection protocol. Twelve hours posttransfection, cells were stimulated with conditioned media for an additional 12 h. Luciferase activity was measured and presented after normalizing with Renilla luciferase. (D) HS5 cells were incubated with tumor cell–derived conditioned media for 12 h and stained with fluorescently labeled anti-YAP. DAPI was used for nuclear staining. Translocation of YAP into the nucleus was monitored under a fluorescence microscope. (E) HS5 cells were incubated with tumor cell– (parental and its mda-9 knockout variant) derived conditioned media for 12 h, and cell lysates were analyzed for different proteins, as indicated. (F) MDA-9/Syntenin expression was knocked down in HS5 cells using an Adenovirus expressing shmda-9. Twenty-four hours postinfection, culture media were replaced with fresh media containing PC-3ML CM for an additional 12 h. Cell lysates were analyzed for the indicated proteins. Different letters in two variables are statistically significant (P < 0.05).

Article Snippet: Human and mouse specific recombinant protein CXCL5, PDGF- AA, GM- CSF were procured from R&D Systems.

Techniques: Expressing, Injection, Control, Imaging, Incubation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, Activity Assay, Staining, Labeling, Translocation Assay, Fluorescence, Microscopy, Knock-Out, Variant Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 6. Tumor cell–derived PDGF-AA induces CXCL5 expression in HS5 cells. (A) Normalized (based on equal amounts of total protein) conditioned media, isolated from parental and knockout variants, were subjected to protein-based growth factor arrays as described by Ray Biotech. Heatmap is presented. Densitometry analyses (n = 2, for corresponding proteins) were performed for each growth factor, and the data are presented only for three. (B) HS5 cells were treated for the indicated times with PDGF-AA, and CXCL5 protein expression was determined. (C) HS5 cells were transfected with a 1.5-kb CXCL5-Prom using a standard transfection protocol. Twelve hours posttransfection, cells were treated with PDGF-AA for an additional 12 h. Luciferase activity was measured and presented after normalizing with Renilla luciferase. (D) PC-3ML-derived conditioned media were incubated with control IgG or anti-PDGF-AA before treatment of HS5 cells for 12 h, and CXCL5 levels were measured in media. (E) Expression of PDGF-AA was determined in the indicated tumor cells and corresponding mda-9 knockout clones. (F) Cells were infected with an Adenovirus expressing control or mda-9 overexpressing construct for 24 h, and PDGF-AA expression in media was determined using ELISA. (G) HS5 cells were treated with PDGF-AA and analyzed for expression of the indicated proteins. (H) Tumor cells were cultured in the presence or absence of IKK2i for 12 h and analyzed for expression of the indicated proteins. Different letters in two variables are statistically significant (P < 0.05). (I) MDA-9 was knocked-into PC-3MLmda-9 KO cells and cultured for an additional 12 h in the presence or absence of IKK2i. PDGF-AA was determined using ELISA. *=P < 0.05.

Article Snippet: Human and mouse specific recombinant protein CXCL5, PDGF- AA, GM- CSF were procured from R&D Systems.

Techniques: Derivative Assay, Expressing, Isolation, Knock-Out, Transfection, Luciferase, Activity Assay, Incubation, Control, Clone Assay, Infection, Construct, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 5. Hippo pathway activates CXCL5 in HS5 cells. (A) Panel i Schematic representation of the experimental protocol used in panels ii–iv. Different overexpression/ siRNAs (GOF/LOF) constructs were transfected into the indicated cells, and after 24 h, tumor-derived condition medium was used to stimulate HS5 cells for an additional 12 h. Panels ii and iii represent CXCL5 expression in HS5-derived conditioned media, determined by ELISA. Panels iv and v show CXCL5 promoter activity. In this experiment, HS5 cells were cotransfected with both CXCL5-Prom and either shYAP1 or YAP1 OE vectors before treating with conditioned media. *=P <0.05. (B) YAP expression was analyzed in paraffin sections (tumor-bearing bones from the experiment performed in Fig. 2A). (C) HS5 cells were incubated with different fractions of PC-3ML-derived conditioned media for 24 h after initial transfection with a CXCL5-Prom. Luciferase activity was measured and presented after normalizing with Renilla luciferase. *P <0.05.

Article Snippet: Human and mouse specific recombinant protein CXCL5, PDGF- AA, GM- CSF were procured from R&D Systems.

Techniques: Over Expression, Construct, Transfection, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation, Luciferase

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 7. MDA-9 expression in HS5 regulates PDGF/PDGFRα signaling. (A) mda-9 expression was blunted, and 24 h postinfection, HS5 cells were stimulated with PDGF-AA for an additional 12 h. mRNA for CXCL5 expression was analyzed using qPCR. Different letters in two variables are statistically significant (P < 0.05). (B) HS5 cells were incubated with media supplemented with the indicated tumor cell–derived conditioned media for 12 h, and western blotting was done for phosho-PDGFRα and total PDGFR. (C) HS5 cells were incubated with media supplemented with the indicated tumor cell–derived conditioned media and PDGF- AA for 12 h, and western blotting was done for the indicated proteins. (D) BM-MSCs from WT and mda-9−/− mice were stimulated with mouse PDGF-AA, and CXCL5 expression was determined in the culture media. (E) phospho-PDGFR expression was analyzed in paraffin sections (tumor-bearing bone from the animal experiment, described in Fig. 2A). Different letters in two variables are statistically significant (P < 0.05).

Article Snippet: Human and mouse specific recombinant protein CXCL5, PDGF- AA, GM- CSF were procured from R&D Systems.

Techniques: Expressing, Incubation, Derivative Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 8. CXCL5 is expressed in both stroma and tumor cells. (A) Paraffin-embedded section from a patient with metastatic PC bone metastasis was immunostained with anti-CXCL5, and the expression in the stroma and tumor compartment is presented in the Inset. (B) H-Score was determined by an anatomic pathologist based on staining intensity, and values are presented. (C) PC3-ML cells were stained with CytoTrack™ Red and cocultured with of HS5 cells. After 24 h, cells were stained with CXCL5-FITC and analyzed by FACS [the left panel represents double staining (tumor cell-Red and CXCL5-FITC)]; the middle panel is for only CXCL5- FITC (only HS5). The right panel represents conditioned media that were collected from the above indicated PC3-ML and HS5 cocultured cells, and secretion of CXCL5 was measured by ELISA. (D) Human serum samples from the indicated patient groups were analyzed for PDGF-AA and CXCL5 (n = 20). Different letters in two variables are statistically significant (P < 0.05). *Statistically significant. **P < 0.001.

Article Snippet: Human and mouse specific recombinant protein CXCL5, PDGF- AA, GM- CSF were procured from R&D Systems.

Techniques: Expressing, Staining, Double Staining, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 9. Schematic of the proposed role of MDA-9 in BM- MSCs in creating a favorable environment for metastatic outgrowth through secretion of CXCL5 in response to tumor cell–derived growth factor, PDGF-AA.

Article Snippet: Human and mouse specific recombinant protein CXCL5, PDGF- AA, GM- CSF were procured from R&D Systems.

Techniques: Derivative Assay