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R&D Systems recombinant human cxcl5 protein
Recombinant Human Cxcl5 Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+human+cxcl5/pm40568729-431-0-22?v=R%26D+Systems
Average 93 stars, based on 9 article reviews

recombinant human cxcl5 protein - by Bioz Stars, 2026-07

93/100 stars

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a,b, Bulk RNA-seq was performed on the liver of mice fed CD or HFD for 10 weeks ( n = 3 per group). Top 10 enriched cytokine and chemokine-related GO terms ( a ) and heatmap of top 5 cytokine/chemokine DEGs based on fold change ( b ) in the liver of mice on HFD compared to mice on CD for 10 weeks. padj, adjusted p-value. Log2FC, log2FoldChange. c, Images of immunofluorescence staining of CXCL5 and Ly6G on the liver of mice fed HFD or CD for 10 weeks. DIC channel showed SBB-stained lipofuscin (black patches). Scale bar, 20 µm. d,e, dHL-60 were incubated with 50 ng/mL CXCL5 for 24 hours and stimulated with 4 µM ionomycin for 4 hours ( d ) or 1 hour ( e ). d, Representative images of NETs from dHL-60 (top) and quantification of NETs (%) (below). Yellow arrows indicate examples of NETs. n = 5 per group. Scale bar, 100 µm. e, Representative images of H4Cit high cells from dHL-60 (top) and quantification of H4Cit high cells (%) (bottom). n = 3 per group. Scale bar, 100 µm. f, dHL-60 were incubated with 50 ng/mL CXCL5 for 24 hours and stimulated with 50 µM oleic acid (OA) for 4 hours. Representative images of NETs (top) and quantification of NETs (%) (bottom). Yellow arrows indicate examples of NETs. n = 5 per group. Scale bar, 100 µm. US, unstimulated. Iono, ionomycin. Statistical comparisons were made using two-tailed unpaired t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001. Data are mean ± s.e.m.

Journal: bioRxiv

Article Title: Tirzepatide reduces diet-induced senescence and NETosis-mediated liver fibrosis in mice

doi: 10.1101/2025.05.27.656284

Figure Lengend Snippet: a,b, Bulk RNA-seq was performed on the liver of mice fed CD or HFD for 10 weeks ( n = 3 per group). Top 10 enriched cytokine and chemokine-related GO terms ( a ) and heatmap of top 5 cytokine/chemokine DEGs based on fold change ( b ) in the liver of mice on HFD compared to mice on CD for 10 weeks. padj, adjusted p-value. Log2FC, log2FoldChange. c, Images of immunofluorescence staining of CXCL5 and Ly6G on the liver of mice fed HFD or CD for 10 weeks. DIC channel showed SBB-stained lipofuscin (black patches). Scale bar, 20 µm. d,e, dHL-60 were incubated with 50 ng/mL CXCL5 for 24 hours and stimulated with 4 µM ionomycin for 4 hours ( d ) or 1 hour ( e ). d, Representative images of NETs from dHL-60 (top) and quantification of NETs (%) (below). Yellow arrows indicate examples of NETs. n = 5 per group. Scale bar, 100 µm. e, Representative images of H4Cit high cells from dHL-60 (top) and quantification of H4Cit high cells (%) (bottom). n = 3 per group. Scale bar, 100 µm. f, dHL-60 were incubated with 50 ng/mL CXCL5 for 24 hours and stimulated with 50 µM oleic acid (OA) for 4 hours. Representative images of NETs (top) and quantification of NETs (%) (bottom). Yellow arrows indicate examples of NETs. n = 5 per group. Scale bar, 100 µm. US, unstimulated. Iono, ionomycin. Statistical comparisons were made using two-tailed unpaired t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001. Data are mean ± s.e.m.

Article Snippet: To examine the effect of CXCL5 on NETosis, HL-60 were differentiated with DMSO for 6 days, and then exposed to 50 ng/mL recombinant human CXCL5 (PeproTech, 300-22) for 24 hours.

Techniques: RNA Sequencing, Immunofluorescence, Staining, Incubation, Two Tailed Test

a-g, Male Padi4 fl/fl (Control) and Vav1-Cre Padi4 fl/fl (KO) fed CD or HFD for 10 weeks ( a ). b, Single-plane confocal images of neutrophils and NETs in the liver of control and KO mice fed HFD for 10 weeks. DIC channel showed SBB-stained lipofuscin (black patches). Scale bar, 5 µm. c, Representative images of liver interstitial fibrosis revealed by Masson’s trichrome (left) and quantification of area of interstitial fibrosis (%) (right) in the liver sections. Left to right on the bar chart, n = 5, 3, 6, 8. Scale bar, 50 µm. d, Representative images of lipofuscin stained by SBB (left) and quantification of area of SBB (%) (right) in the liver. Left to right on the bar chart, n = 5, 4, 6, 8. Scale bar, 50 µm. e, Representative Western blots (left) and quantification (right) of CXCL5 levels in the liver. Ribosomal protein lateral stalk subunit P0 (RPLP0) served as the loading control. Left to right on the bar chart, n = 4, 3, 6, 4. f, Representative images of H&E-stained liver sections from control and KO mice on HFD for 10 weeks, showing hepatocyte ballooning (green arrows), steatosis (black arrows) and immune cell infiltration (yellow squares). Scale bar, 50 µm. g, Representative images of ORO-stained liver sections (left) and quantification of area of ORO (%) (right). Left to right on the bar chart, n = 5, 4, 5, 6. Scale bar, 50 µm. Statistical comparisons were made using two-tailed unpaired t-test ( c,d,g ) or Mann-Whitney test ( e ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. Data are mean ± s.e.m.

Journal: bioRxiv

Article Title: Tirzepatide reduces diet-induced senescence and NETosis-mediated liver fibrosis in mice

doi: 10.1101/2025.05.27.656284

Figure Lengend Snippet: a-g, Male Padi4 fl/fl (Control) and Vav1-Cre Padi4 fl/fl (KO) fed CD or HFD for 10 weeks ( a ). b, Single-plane confocal images of neutrophils and NETs in the liver of control and KO mice fed HFD for 10 weeks. DIC channel showed SBB-stained lipofuscin (black patches). Scale bar, 5 µm. c, Representative images of liver interstitial fibrosis revealed by Masson’s trichrome (left) and quantification of area of interstitial fibrosis (%) (right) in the liver sections. Left to right on the bar chart, n = 5, 3, 6, 8. Scale bar, 50 µm. d, Representative images of lipofuscin stained by SBB (left) and quantification of area of SBB (%) (right) in the liver. Left to right on the bar chart, n = 5, 4, 6, 8. Scale bar, 50 µm. e, Representative Western blots (left) and quantification (right) of CXCL5 levels in the liver. Ribosomal protein lateral stalk subunit P0 (RPLP0) served as the loading control. Left to right on the bar chart, n = 4, 3, 6, 4. f, Representative images of H&E-stained liver sections from control and KO mice on HFD for 10 weeks, showing hepatocyte ballooning (green arrows), steatosis (black arrows) and immune cell infiltration (yellow squares). Scale bar, 50 µm. g, Representative images of ORO-stained liver sections (left) and quantification of area of ORO (%) (right). Left to right on the bar chart, n = 5, 4, 5, 6. Scale bar, 50 µm. Statistical comparisons were made using two-tailed unpaired t-test ( c,d,g ) or Mann-Whitney test ( e ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. Data are mean ± s.e.m.

Article Snippet: To examine the effect of CXCL5 on NETosis, HL-60 were differentiated with DMSO for 6 days, and then exposed to 50 ng/mL recombinant human CXCL5 (PeproTech, 300-22) for 24 hours.

Techniques: Control, Staining, Western Blot, Two Tailed Test, MANN-WHITNEY

a-g, Male C57BL/6J mice were fed CD or HFD with concurrent treatment of TZP or vehicle for 10 weeks ( a ). b, Representative images of liver interstitial fibrosis revealed by Masson’s trichrome (left) and quantification of area of interstitial fibrosis (%) (middle) on FFPE liver sections. Level of liver fibrosis assessed by hydroxyproline content (µg per g liver) (right). For quantification of area of interstitial fibrosis (%), left to right on the bar chart, n = 9, 9, 10, 9. For hydroxyproline assay, left to right on the bar chart, n = 15, 13, 13, 15. Scale bar, 50 µm. c, Representative images of lipofuscin stained by SBB (left) and quantification of area of SBB (%) (right). Left to right on the bar chart, n = 9, 10, 10, 9. Scale bar, 50 µm. d, Single-plane confocal images of hepatocytes (ASGR1 + ) (left) and quantification of lipofuscin-laden hepatocytes (%) (right) in the liver sections. n = 5 per group. Scale bar, 20 µm. e, Representative Western blots (top) and quantification (bottom) of CXCL5 levels in the liver. RPLP0 served as the loading control. Left to right on the bar chart, n = 6, 6, 6, 8. f, Immunofluorescence staining for neutrophil recruitment (Ly6G + cells) in the liver (left) and quantification of neutrophils in 12 high-power fields (right). Left to right on the bar chart, n = 5, 5, 7, 7. Scale bar, 50 µm. Statistical comparisons were made using two-tailed unpaired t-test ( b-middle,c,d,e,f ) or Mann-Whitney test ( b-right ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. Data are mean ± s.e.m. g, Single-plane confocal images of neutrophils and NETs in the liver sections from HFD-fed mice treated with vehicle or TZP. Top, yellow arrows indicate NETs; bottom, non-NETing neutrophils. Regions outlined by white dotted boxes are magnified on the immediate right. Scale bars, 5 µm.

Journal: bioRxiv

Article Title: Tirzepatide reduces diet-induced senescence and NETosis-mediated liver fibrosis in mice

doi: 10.1101/2025.05.27.656284

Figure Lengend Snippet: a-g, Male C57BL/6J mice were fed CD or HFD with concurrent treatment of TZP or vehicle for 10 weeks ( a ). b, Representative images of liver interstitial fibrosis revealed by Masson’s trichrome (left) and quantification of area of interstitial fibrosis (%) (middle) on FFPE liver sections. Level of liver fibrosis assessed by hydroxyproline content (µg per g liver) (right). For quantification of area of interstitial fibrosis (%), left to right on the bar chart, n = 9, 9, 10, 9. For hydroxyproline assay, left to right on the bar chart, n = 15, 13, 13, 15. Scale bar, 50 µm. c, Representative images of lipofuscin stained by SBB (left) and quantification of area of SBB (%) (right). Left to right on the bar chart, n = 9, 10, 10, 9. Scale bar, 50 µm. d, Single-plane confocal images of hepatocytes (ASGR1 + ) (left) and quantification of lipofuscin-laden hepatocytes (%) (right) in the liver sections. n = 5 per group. Scale bar, 20 µm. e, Representative Western blots (top) and quantification (bottom) of CXCL5 levels in the liver. RPLP0 served as the loading control. Left to right on the bar chart, n = 6, 6, 6, 8. f, Immunofluorescence staining for neutrophil recruitment (Ly6G + cells) in the liver (left) and quantification of neutrophils in 12 high-power fields (right). Left to right on the bar chart, n = 5, 5, 7, 7. Scale bar, 50 µm. Statistical comparisons were made using two-tailed unpaired t-test ( b-middle,c,d,e,f ) or Mann-Whitney test ( b-right ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. Data are mean ± s.e.m. g, Single-plane confocal images of neutrophils and NETs in the liver sections from HFD-fed mice treated with vehicle or TZP. Top, yellow arrows indicate NETs; bottom, non-NETing neutrophils. Regions outlined by white dotted boxes are magnified on the immediate right. Scale bars, 5 µm.

Article Snippet: To examine the effect of CXCL5 on NETosis, HL-60 were differentiated with DMSO for 6 days, and then exposed to 50 ng/mL recombinant human CXCL5 (PeproTech, 300-22) for 24 hours.

Techniques: Hydroxyproline Assay, Staining, Western Blot, Control, Immunofluorescence, Two Tailed Test, MANN-WHITNEY

a-k, Male C57BL/6J mice were fed CD or HFD for 7 weeks, followed by treatment with TZP or vehicle for the subsequent 10 weeks along with the respective diets ( a ), and bulk RNA-seq was performed on the liver tissue from these mice ( n = 3 per group) ( g-k ). b, Representative images of liver interstitial fibrosis revealed by Masson’s trichrome (left) and quantification of area of interstitial fibrosis (%) (middle) on the FFPE liver sections. Level of liver fibrosis assessed by hydroxyproline content (µg per g liver) (right). For quantification of area of interstitial fibrosis (%), left to right on the bar chart, n = 8, 6, 8, 7. For hydroxyproline assay, left to right on the bar chart, n = 3, 5, 5, 5. Scale bar, 50 µm. c, Representative images of lipofuscin stained by SBB (left) and quantification of area of SBB (%) (right) of the liver sections. Left to right on the bar chart, n = 7, 7, 8, 8. Scale bar, 50 µm. d, Representative Western blots (top) and quantification of CXCL5 levels (bottom) in the liver. RPLP0 served as the loading control. Left to right on the bar chart, n = 4, 4, 5, 5. e, Immunofluorescence staining for neutrophil recruitment (Ly6G + cells) in the liver (left) and quantification of neutrophils in 12 high-power fields (right). Left to right on the bar chart, n = 5, 5, 8, 8. Scale bar, 50 µm. Statistical comparisons were made using two-tailed unpaired t-test ( b,d,e ) or Mann-Whitney test ( c ). * P < 0.05, ** P < 0.01 and *** P < 0.001. Data are mean ± s.e.m. f, Single-plane confocal images of neutrophils and NETs in the liver of HFD-fed mice treated with vehicle or TZP. Top, yellow arrows indicate NETs; bottom, non-NETing neutrophils. Regions outlined by the white dotted boxes are magnified on the immediate right. Scale bars, 5 µm (left) and 2 µm (right). g, Principal component analysis of the bulk RNA-seq data from the liver of mice with different treatments. h, Top 20 enriched GO terms based on adjusted p-value in the liver of mice fed HFD with and without TZP treatment. i-k, Heatmap of DEGs in fibrosis-related pathways (extracellular matrix organization, transforming growth factor beta receptor signaling pathway, fibroblast proliferation, collagen biosynthetic process, wound healing) ( i ), lipofuscin-related pathways (autophagosome-lysosome fusion, response to oxidative stress, cellular response to oxidative stress, intrinsic apoptotic signaling pathway in response to oxidative stress, lipid oxidation, macroautophagy, TOR signaling) ( j ), and neutrophil-related pathways (neutrophil activation, neutrophil chemotaxis, neutrophil migration) ( k ).

Journal: bioRxiv

Article Title: Tirzepatide reduces diet-induced senescence and NETosis-mediated liver fibrosis in mice

doi: 10.1101/2025.05.27.656284

Figure Lengend Snippet: a-k, Male C57BL/6J mice were fed CD or HFD for 7 weeks, followed by treatment with TZP or vehicle for the subsequent 10 weeks along with the respective diets ( a ), and bulk RNA-seq was performed on the liver tissue from these mice ( n = 3 per group) ( g-k ). b, Representative images of liver interstitial fibrosis revealed by Masson’s trichrome (left) and quantification of area of interstitial fibrosis (%) (middle) on the FFPE liver sections. Level of liver fibrosis assessed by hydroxyproline content (µg per g liver) (right). For quantification of area of interstitial fibrosis (%), left to right on the bar chart, n = 8, 6, 8, 7. For hydroxyproline assay, left to right on the bar chart, n = 3, 5, 5, 5. Scale bar, 50 µm. c, Representative images of lipofuscin stained by SBB (left) and quantification of area of SBB (%) (right) of the liver sections. Left to right on the bar chart, n = 7, 7, 8, 8. Scale bar, 50 µm. d, Representative Western blots (top) and quantification of CXCL5 levels (bottom) in the liver. RPLP0 served as the loading control. Left to right on the bar chart, n = 4, 4, 5, 5. e, Immunofluorescence staining for neutrophil recruitment (Ly6G + cells) in the liver (left) and quantification of neutrophils in 12 high-power fields (right). Left to right on the bar chart, n = 5, 5, 8, 8. Scale bar, 50 µm. Statistical comparisons were made using two-tailed unpaired t-test ( b,d,e ) or Mann-Whitney test ( c ). * P < 0.05, ** P < 0.01 and *** P < 0.001. Data are mean ± s.e.m. f, Single-plane confocal images of neutrophils and NETs in the liver of HFD-fed mice treated with vehicle or TZP. Top, yellow arrows indicate NETs; bottom, non-NETing neutrophils. Regions outlined by the white dotted boxes are magnified on the immediate right. Scale bars, 5 µm (left) and 2 µm (right). g, Principal component analysis of the bulk RNA-seq data from the liver of mice with different treatments. h, Top 20 enriched GO terms based on adjusted p-value in the liver of mice fed HFD with and without TZP treatment. i-k, Heatmap of DEGs in fibrosis-related pathways (extracellular matrix organization, transforming growth factor beta receptor signaling pathway, fibroblast proliferation, collagen biosynthetic process, wound healing) ( i ), lipofuscin-related pathways (autophagosome-lysosome fusion, response to oxidative stress, cellular response to oxidative stress, intrinsic apoptotic signaling pathway in response to oxidative stress, lipid oxidation, macroautophagy, TOR signaling) ( j ), and neutrophil-related pathways (neutrophil activation, neutrophil chemotaxis, neutrophil migration) ( k ).

Article Snippet: To examine the effect of CXCL5 on NETosis, HL-60 were differentiated with DMSO for 6 days, and then exposed to 50 ng/mL recombinant human CXCL5 (PeproTech, 300-22) for 24 hours.

Techniques: RNA Sequencing, Hydroxyproline Assay, Staining, Western Blot, Control, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Activation Assay, Chemotaxis Assay, Migration

Choline-deficient L-amino acid-defined high-fat diet (CDAHFD) induces lipofuscin deposition in the lysosomes of hepatocytes. Lipofuscin-containing cells produce higher levels of CXCL5, which in turn enhances NET formation, eventually leading to liver fibrosis. Intercepting NETosis through direct inhibition of peptidylarginine deiminase 4 (PAD4) or through decreasing hepatic lipofuscin load by TZP treatment both effectively reduce liver fibrosis. TZP treatment provides additional therapeutic benefits by lessening hepatic cellular senescence. The illustration was created with BioRender.com.

Journal: bioRxiv

Article Title: Tirzepatide reduces diet-induced senescence and NETosis-mediated liver fibrosis in mice

doi: 10.1101/2025.05.27.656284

Figure Lengend Snippet: Choline-deficient L-amino acid-defined high-fat diet (CDAHFD) induces lipofuscin deposition in the lysosomes of hepatocytes. Lipofuscin-containing cells produce higher levels of CXCL5, which in turn enhances NET formation, eventually leading to liver fibrosis. Intercepting NETosis through direct inhibition of peptidylarginine deiminase 4 (PAD4) or through decreasing hepatic lipofuscin load by TZP treatment both effectively reduce liver fibrosis. TZP treatment provides additional therapeutic benefits by lessening hepatic cellular senescence. The illustration was created with BioRender.com.

Article Snippet: To examine the effect of CXCL5 on NETosis, HL-60 were differentiated with DMSO for 6 days, and then exposed to 50 ng/mL recombinant human CXCL5 (PeproTech, 300-22) for 24 hours.

Techniques: Inhibition

(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.

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

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.

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

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).

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

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.

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

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

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

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).

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

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.

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

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.

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

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