Journal: PLoS ONE

Article Title: Necroptosis in pancreatic cancer promotes cancer cell migration and invasion by release of CXCL5

doi: 10.1371/journal.pone.0228015

Figure Lengend Snippet: (a) Images of human cytokine antibody array (120 targets) of conditioned medium derived from AsPC-1 treated with necroptotic stimuli or DMSO (control). Boxes: positive controls; circles: CXCL5, MIP-3α and IL-8. (b) Signals were quantified relative to CM-control. (c) Human cytokine antibody array of conditioned medium from PANC-1, which was treated with apoptotic stimuli or DMSO (control). (d) Signals relative to CM-control. (e, f) Analysis of mRNA expression of chemokine receptors, CXCR2 and CCR6 by qRT-PCR. Results are shown relative to gene expression in non-cancerous HPDE cells after normalization against 18S rRNA. (g) Western blot analysis of CXCR2 in human pancreatic cancer cells and in HPDE. (h) Concentration of CXCL5 in conditioned medium from AsPC-1 or BxPC-3, which were treated with TSZ ± nec-1 or DMSO (control), and measured by ELISA. Graphs show mean ± SE. * P < 0.05; ** P <0.01.

Article Snippet: After 12 hours’ incubation, CM was collected, and CXCL5 was quantified using a Human CXCL5 Quantikine ELISA Kit (R & D Systems Europe Ltd, Oxford, UK).

Techniques: Ab Array, Derivative Assay, Control, Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Necroptosis in pancreatic cancer promotes cancer cell migration and invasion by release of CXCL5

doi: 10.1371/journal.pone.0228015

Figure Lengend Snippet: PC cell motility was evaluated by Transwell migration and Transwell-Matrigel invasion assays. PC cells pretreated with rh CXCL5 (10 ng/ml) or vehicle for 12 hours. (a) Representative images of migrated cells. scale bars = 100 μm. (b) Quantitative data of migrated cells. (c) Quantitative data of invaded cells. Graphs show mean ± SE. * P < 0.05; ** P <0.01.

Article Snippet: After 12 hours’ incubation, CM was collected, and CXCL5 was quantified using a Human CXCL5 Quantikine ELISA Kit (R & D Systems Europe Ltd, Oxford, UK).

Techniques: Migration

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Schematic of the protocol to compare the effects of conditioned media (CM) from CCD-18Co human normal colon tissue fibroblasts (NF CM), primary human colon cancer-associated fibroblasts (CAF CM), and human HCT 116 colon cancer cell-stimulated CAF CM (CAF CCM) on C2C12 myotube wasting. B Myosin heavy chain 2 (MYH2) immunostaining of C2C12 myotubes cultured in normal myotube differentiation media (DM) and treated with NF CM, CAF CM, and CAF CCM for 72 h (scale bar = 150 μm). C Calculation of mean myotube diameter. D Western blot analysis of MYH2 and atrogin-1 expression in the treated myotubes. E Densitometry of MYH2 and atrogin-1 expression relative to GAPDH. F Cytokine array analysis CAF CM, CAF CCM, and HCT 116 cancer cell CM. Red boxes and numbers indicate upregulated cytokines in the CAF CCM compared to CAF CM. G Quantification of the fold-change for the upregulated cytokines. H ELISA-based detection of CXCL5 in NF CM, CAF CM, CAF CCM, and HCT 116 cancer cell CM. The CAF CM and CCM values are the mean obtained from three sources of CAF: two derived from patients and one provided commercially. All experiments were conducted 3 times independently and the values are indicated as the mean ± SD. For C and E : * = p < 0.05 and *** = p < 0.001 compared to DM. For H : ** = p < 0.01 compared to CAF CM

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Immunostaining, Cell Culture, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Myosin heavy chain 2 (MYH2) immunostaining of C2C12 myotubes cultured in normal differentiation media (DM), DM plus 10 ng/mL CXCL5, or DM plus 20 ng/mL CXCL5 for 72 h. (scale bar = 150 μm) B Calculation of mean myotube diameter. C Western blot analysis of atrogin-1 and MuRF-1 expression in the treated myotubes. D Densitometry of atrogin-1 and MuRF-1 expression relative to GAPDH. E Western blot analysis of total extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 in myotubes treated with CAF CCM or 20 ng/mL CXCL5 for 0.5 h and 2 h. F Densitometry of phosphorylated ERK1/2 relative to ERK1/2. G MHY2 immunostaining of C2C12 myotubes cultured for 72 h as follows: (1) Normal differentiation media (DM), (2) DM:CAF CCM (1:1) plus vehicle (0.1% DMSO), (3) DM:CAF CCM (1:1) plus 0.5 μg/mL CXCL5 neutralizing antibody, and (4) DM:CAF CCM (1:1) plus 22 nM SB225005 for 96 h (scale bar = 150 μm). H Calculation of mean myotube diameter. All experiments were conducted 3 times independently and the values are indicated as the mean ± SD. For B , D , F and H : * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 indicate significantly increased compared to untreated. For H : # = p < 0.05 and ## = p < 0.01 indicates significantly increased compared to the DM:CCM (1:1) group

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Immunostaining, Cell Culture, Western Blot, Expressing

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A IVIS imaging of NOD-SCID mice 3 weeks post-xenograft with 1 × 10 6 human HCT 116 luc2 colon cancer cells, or 1 × 10 6 HCT 116 luc2 cancer cells plus 2 × 10 6 human colon CAF. B Mean total flux detected from the tumor at the end point (ns = not significant). C Dissected tumors and tumor mass at the 3 week endpoint. D Tumor free body weight (B.W) at the 3 week end point. E Representative images of H&E stained gastrocnemius muscle (scale bar = 50 μm). F Calculation of the myofiber cross sectional area. G qPCR analysis of CXCL1, 2, 3, 5, 6, 7, and 8 (known CXCR2 ligands) and IL-6 in the dissected tumor. H IVIS imaging of NOD-SCID mice at 3 weeks post-xenograft with human HCT 116 luc2 cancer cells plus human colon CAF, with or without CXCL5 neutralization (CXCL5 Neu Ab). I Mean total flux detected from the tumor at the 3 week end point. J Dissected tumors and tumor mass. K Tumor free body weight (B.W) at the 3 week end point. L Hanging tolerance in the treated mice. M Tibialis anterior (TA) muscle mass. N Representative H&E staining of the TA muscle (scale bar = 100 µm). O Calculation of the myofiber cross sectional area. P CXCR2 immunostaining of the TA muscle (scale bar = 100 µm). Q Proportion of CXCR2 positive myofibers. 5 mice per group were used for the experiments and the values are indicated as the mean ± SEM. For D and F * = p < 0.05 and ** = p < 0.01 indicate significantly decreased compared to PBS treated mice. For G , * = p < 0.05 indicates significantly increased compared to HCT 116 injected mice. For I – Q 5–7 mice per group were used for the experiments and the values are indicated as the mean ± SEM. * = p < 0.05 and ** = p < 0.01 indicate significantly decreased compared to vehicle alone. # = p < 0.05 and ## = p < 0.01 indicate significantly increased compared to HCT 116 plus CAF

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Imaging, Staining, Neutralization, Immunostaining, Injection

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Heat map of genes showing differential expression between the tibialis anterior (TA) muscles of NOD-SCID mice treated as follows: (1) No xenograft (Normal); (2) Xenograft with HCT 116 human cancer cells plus CAF (HCT 116 + CAF); (3) Xenograft with HCT 116 human cancer cells plus CAF, followed by treatment with a CXCL5 neutralizing antibody (Neu Ab). B KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis for the HCT-116 + CAF compared with Neu Ab treatment groups. C Gene ontology (GO) functional analysis for the HCT-116 + CAF group compared with Neu Ab treatment group. D Western blot analysis of PI3K-AKT and ERK1/2 phosphorylation in the dissected TA muscle from vehicle alone, HCT 116 + CAF, and Neu Ab-treated groups. E Densitometry of PI3K-AKT and ERK1/2 phosphorylation relative to PI3K-AKT and ERK1/2. F Heat map for genes showing differential expression between (1) HCT 116 + CAF xenograft compared to vehicle alone (HCT-116 + CAF/Normal), and (2) HCT 116 + CAF xenograft treated with a CXCL5 neutralizing antibody compared to HCT 116 + CAF xenograft alone (Neu Ab/HCT 116 + CAF). G qPCR analysis of the differentially expressed genes, in addition to atrogin-1 and MuRF-1 in the TA muscles. H Western blot analysis of PI3K-AKT phosphorylation in CAF CCM treated myotubes. I Densitometry of PI3K-AKT phosphorylation relative to PI3K-AKT. J Protein synthesis rate as determined by western blot analysis of puromycin incorporation (SUnSET assay). K Densitometry of puromycin incorporation normalized by GAPDH expression. For E and G : 4–5 mice per group were used for the experiments and the values are indicated as the mean ± SEM. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 indicate significantly increased or decreased compared to vehicle alone-injected mice (Normal). # = p < 0.05, ## = p < 0.01 and ### = p < 0.001 indicate significantly decreased compared to HCT 116 + CAF. For I and K : All values are indicated as the mean ± SD. * = p < 0.05 and *** = p < 0.001 indicate significantly decreased compared to vehicle alone. # = p < 0.05 indicates significantly increased compared to CAF CCM treatment

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Quantitative Proteomics, Muscles, Functional Assay, Western Blot, Phospho-proteomics, Expressing, Injection

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Schematic of the protocol to investigate CXCL5 neutralization in a model of cytokine-induced skeletal muscle wasting. C57BL6/J mice were treated with 40 ng/kg CXCL5 and 80 ng/kg IL-6 for 4 weeks with or without 120 μg/kg CXCL5 neutralizing antibody. 120 μg/kg IgG1 was used as control. B Body weight at the end point of experiment. C Quadriceps muscle mass. D Gastrocnemius muscle mass. E Tibialis anterior (TA) muscle mass. F Laminin staining of the TA muscle (scale bar = 150 µm). G TA myofiber cross sectional area. H CXCR2 staining of the TA muscle (scale bar = 150 µm). I The proportion of CXCR2 positive fibers in TA muscle. J Western blot analysis of atrogin-1, MuRF-1 and CXCR2 expression in the TA muscle. K Densitometry of atrogin-1, MuRF-1 and CXCR2 expression normalized by the expression of GAPDH. For B – I, 7 mice per group were used for the experiments and for J – K, 4 mice per group were used for the experiments and the analysis was carried out two times. The values were indicated as the mean ± SEM. * = p < 0.05 and ** = p < 0.01 indicate significantly increased or decreased compared to IgG1 control. # = p < 0.05 and ## = p < 0.01 indicate significantly decreased compared to CXCL5 + IL-6 + IgG1

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Neutralization, Control, Staining, Western Blot, Expressing

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Representative MYH2-stained images of human donor myotubes cultured as follows: (1) Differentiation media (DM) for 72 h; (2) Treatment with human HCT 116 colon cancer cell-stimulated CAF CM (CAF CCM) for 72 h; (3) Treatment with CAF CCM and CXCL5 neutralizing antibody (CAF CCM + Neu Ab) for 72 h (scale bar = 100 μm). B Calculation of mean myotube diameter. C Western blot analysis of MYH2 and atrogin-1 expression. D Densitometry of MYH2 and atrogin-1 expression relative to α–tubulin. E Immunohistochemical analysis of CXCL5 and vimentin expression in tumor-stromal and normal tissues obtained from a colon carcinoma patient. White arrows indicate overlapping CXCL5 and vimentin immunostaining. Quantification of CXCL5 fluorescence in the tumor-stromal tissues is also shown (** = p < 0.01 compared to normal). All experiments were conducted 3 times independently and the values were indicated as the mean ± SD. For B and D : * = p < 0.05 indicate significantly increased or decreased compared to DM alone. # = p < 0.05 and ## = p < 0.01 indicate significantly increased or decreased compared to CAF CCM. G Working model of the role of CAF in cancer cachexia progression. Molecular crosstalk between cancer cells and CAF in the tumor microenvironment induces the secretion of chemokine CXCL5 by CAF. CXCL5 activates muscle atrophy signaling, as shown by decreased PI3K-AKT and ERK phosphorylation and upregulation of the key atrogenes, atrogin-1 and MuRF-1, causing the skeletal muscle loss observed in cancer cachexia

Article Snippet: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

Techniques: Staining, Cell Culture, Western Blot, Expressing, Immunohistochemical staining, Immunostaining, Fluorescence, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 1. CXCL5 protein expression is concordant with prostate cancer progression. Shown are representative panels from a hematox- ylin and eosin–stained, high-density tissue microarray probed with antibody against CXCL5, as follows: (A) Benign glands demonstrating weak staining. (B) PCa (Gleason sum 3 + 3) demonstrating weak staining. (C) PCa (Gleason sum 4 + 4) demonstrating moderate to strong staining. (D) Hormone refractory METs demonstrating strong staining. (E) PCa demonstrating moderate to strong staining asso- ciated with stromal inflammatory component (yellow arrows point to areas of inflammation). (F) Benign glands demonstrating strongly staining luminal secretions (black arrows). Original magnifications, ×100. Panel E has been enlarged further, ×4, to illustrate the area of inflammatory infiltrate concomitant with CXCL5 protein expression. (G) Boxplot depicting median product score distributions of protein expression levels for benign glands, malignant glands from PCa, and malignant areas from METs and P values associated with the statistical evaluation of these distributions.

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Expressing, Staining, Microarray

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 2. Nontransformed and transformed prostate epithelial cells express the CXCL5 receptor and endogenously secrete CXCL5. (A) Immunoblot analysis of protein lysates prepared from transformed PC3 and LNCaP, and nontransformed N15C6 and BPH-1 prostate epithelial cells probed with antibodies specific for the CXCL5 receptor, CXCR2, and loading control, β-actin. Pri- mary antibody concentrations used were 1:1000 for CXCR2 and 1:5000 for β-actin. (B) Protein levels (pg/ml) of CXCL5 present in media conditioned by transformed LNCaP and PC3 or nontrans- formed N15C6 or BPH-1 cells prostate epithelial cells were deter- mined by ELISA. The graph shows the pg/ml CXCL5 detected plotted on a logarithmic scale (y axis).

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Transformation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 4. CXCL5-stimulated proliferative and invasive responses. (A) N15C6 (light gray bars) or BPH-1 (dark gray bars) nontransformed prostate epithelial cells proliferated to significantly higher levels when grown for 72 hours in SF media supplemented with 10 pM CXCL5 than those grown in SF alone (*P < .001). Preincubation of the cells for 1 hour with 1 μg/ml antibody against CXCR2, the receptor for CXCL5, followed by supplementation with CXCL5 and maintenance of growth in CXCL5 + anti-CXCR2–containing media significantly ablated the proliferative response (#P < .001). In contrast, cellular growth after preincubation with an antibody against an unrelated chemokine receptor, CXCR4, followed by supplementation with CXCL5 and maintenance of growth in CXCL5 + anti-CXCR4–containing media was similar to that observed for non–pretreated cells grown in CXCL5-supplemented media and was significantly higher than that in SF alone (*P < .001). All data are shown normalized to growth in unsupplemented SF, which was set at one-fold. (B) N15C6 (LEFT) or LNCaP (RIGHT) cells were grown in SF media (untreated, UnT) or SF media supplemented with 10 pM CXCL5 for N15C6 or 100 pM CXCL5 for LNCaP (treated, T) for the times indicated. The cells were then harvested and assessed for nucleosomal DNA fragmentation. The fraction of cells exhibiting apoptosis plotted on the y axis was calculated as the difference in absorbance measured at 405 nm and at the reference wavelength of 490 nm after adjusting for background absorbance at both wavelengths. No significant differences in the fraction of cells exhibiting apoptosis were observed between treated and untreated cells at any time point, demonstrating that CXCL5 does not promote antiapoptotic responses in these cells. (C) Fifteen thousand each of N15C6 (black bars) or PC3 (gray bars) cells were plated onto Matrigel-coated membranes and were exposed to complete media or complete media supplemented with 20 nM CXCL5 for 24 hours. After 24 hours, the cells that migrated and invaded through the Matrigel were stained and counted. N15C6 cells did not demonstrate an invasive response to treatment with CXCL5. However, approximately six-fold more PC3 cells migrated through the synthetic basement membrane, Matrigel, in response to 20 nM CXCL5 compared to vehicle (control, set at one-fold) (*P < .05). PC3 cell invasion through the Matrigel in response to CXCL5 was significantly inhibited by pretreatment with 1 μg/ml blocking antibody (anti- CXCR2) (#P < .05) but not by pretreatment with nonspecific antibody (anti-CXCR4) (*P < .05).

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Staining, Membrane, Control, Blocking Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 5. CXCL5 activates MAPK signaling in nontransformed N15C6 prostate epithelial cells. Nontransformed N15C6 cells rapidly and transiently phosphorylated ERK 1/2 and STAT3 when treated with either subnanomolar (10 or 100 pM) or nanomolar (1 nM) levels of CXCL5, whereas NF-κB subunit activation was evident only after treatment with 1 nM CXCL5. Primary antibody concentrations used were 1:500 for phospho-ERK, 1:500 for phospho-65 (NF-κB), 1:1000 for phospho-STAT3, 1:1000 for total ERK, 1:1000 for total p65, and 1:2000 for total STAT3. A total of 20 μg of protein lysate was electrophoresed per well. Immunoblots are shown on the left, and corresponding densitometric evaluations of the same blots are shown on the right. Phosphorylation relative to total protein quantitated from the immunoblot is shown in the densitometric plots as phospho/total protein.

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 6. CXCL5 activates both MAPK and PI3K signaling in transformed LNCaP prostate epithelial cells. Transformed LNCaP cells rapidly and transiently phosphorylated both ERK 1/2 and the p65 subunit of NF-κB on treatment with subnanomolar (10 or 100 pM) levels of CXCL5. Immunoblots are shown in the top panel, and corresponding densitometric evaluations of the same blots are shown in the bottom panel. Phosphorylation relative to total protein quantitated from the immunoblot is shown in the densitometric plots as phospho/total protein. A total of 100 μg of protein lysate was electrophoresed per well. Primary antibody concentrations used were as described for Figure 5.

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Transformation Assay, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 7. CXCL5 stimulates a transcriptional response in both nontransformed and transformed prostate epithelial cells. Quantitative real-time PCR of RNA purified from N15C6 cells (left) or LNCaP cells (right) treated with subnanomolar CXCL5 as shown demonstrates rapid and robust transcription of the EGR1 gene significantly higher than levels obtained at time 0 (set at one-fold) (*P < .05). Data shown are averaged from three or more separate experiments per time point per concentration of CXCL5 examined.

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Transformation Assay, Real-time Polymerase Chain Reaction, Purification, Concentration Assay

Journal: Cardiovascular diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus.

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: Fig. 2 Treatment with CXCL5 neutralizing antibody upregulated VEGF/SDF-1 expression and promoted angiogenesis in late-EPCs from non-DM subjects and HAECs under the HG conditions. The network formation and migration abilities were improved after the administration of CXCL5 mAb in EPCs from non-DM subjects (n = 3; A, B). Western blotting and statistical analyses of VEGF and SDF-1 in EPCs from non-DM subjects (n = 3; C). The network formation and migration abilities were improved after the administration of CXCL5 mAb in HAECs (n = 3; D, E). Western blotting and statistical analyses of VEGF and SDF-1 in HAECs (n = 3; F). CXCL5 C-X-C motif chemokine ligand 5, EPC endothelial progenitor cell, HG high glucose, HAEC human aortic endothelial cell, mAb,monoclonal antibody, SDF-1 stromal cell-derived factor 1, VEGF vascular endothelial growth factor. N represents the number of independent experiments on different days and in different experimental runs. The Mann–Whitney U test was used to determine statistically significant differences. *p < 0.05, **p < 0.01

Article Snippet: Some cells were treated with CXCL5 monoclonal antibody (1 or 10 μg/ mL; R&D Systems, MAB-254, Minneapolis, MN, USA) or recombinant human CXCL5 protein (1 or 10 ng/mL; R&D Systems, 254-XB, Minneapolis, MN, USA).

Techniques: Expressing, Migration, Western Blot, Derivative Assay, MANN-WHITNEY

Journal: Cardiovascular diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus.

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: Fig. 7 Summary of beneficial effects of CXCL5 suppression in diabetic vasculopathy. CXCL5 Chemokine C-X-C motif ligand 5, CXCR2 Chemokine C-X-C motif receptor 2, EPC endothelial progenitor cell, ERK extracellular signal-regulated kinase, DM diabetes mellitus, IL interleukin, SDF-1 stromal cell-derived factor 1, TNF-α tumor necrosis factor-α, VEGF vascular endothelial growth factor

Article Snippet: Some cells were treated with CXCL5 monoclonal antibody (1 or 10 μg/ mL; R&D Systems, MAB-254, Minneapolis, MN, USA) or recombinant human CXCL5 protein (1 or 10 ng/mL; R&D Systems, 254-XB, Minneapolis, MN, USA).

Techniques: Derivative Assay

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: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 1. CXCL5 protein expression is concordant with prostate cancer progression. Shown are representative panels from a hematox- ylin and eosin–stained, high-density tissue microarray probed with antibody against CXCL5, as follows: (A) Benign glands demonstrating weak staining. (B) PCa (Gleason sum 3 + 3) demonstrating weak staining. (C) PCa (Gleason sum 4 + 4) demonstrating moderate to strong staining. (D) Hormone refractory METs demonstrating strong staining. (E) PCa demonstrating moderate to strong staining asso- ciated with stromal inflammatory component (yellow arrows point to areas of inflammation). (F) Benign glands demonstrating strongly staining luminal secretions (black arrows). Original magnifications, ×100. Panel E has been enlarged further, ×4, to illustrate the area of inflammatory infiltrate concomitant with CXCL5 protein expression. (G) Boxplot depicting median product score distributions of protein expression levels for benign glands, malignant glands from PCa, and malignant areas from METs and P values associated with the statistical evaluation of these distributions.

Article Snippet: The TMA was then incubated overnight with primary CXCL5 antibody (Human CXCL5/ENA-78 MAb clone 33160; R&D Systems, Minneapolis, MN) at a dilution of 1:25.

Techniques: Expressing, Staining, Microarray

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 2. Nontransformed and transformed prostate epithelial cells express the CXCL5 receptor and endogenously secrete CXCL5. (A) Immunoblot analysis of protein lysates prepared from transformed PC3 and LNCaP, and nontransformed N15C6 and BPH-1 prostate epithelial cells probed with antibodies specific for the CXCL5 receptor, CXCR2, and loading control, β-actin. Pri- mary antibody concentrations used were 1:1000 for CXCR2 and 1:5000 for β-actin. (B) Protein levels (pg/ml) of CXCL5 present in media conditioned by transformed LNCaP and PC3 or nontrans- formed N15C6 or BPH-1 cells prostate epithelial cells were deter- mined by ELISA. The graph shows the pg/ml CXCL5 detected plotted on a logarithmic scale (y axis).

Article Snippet: The TMA was then incubated overnight with primary CXCL5 antibody (Human CXCL5/ENA-78 MAb clone 33160; R&D Systems, Minneapolis, MN) at a dilution of 1:25.

Techniques: Transformation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 4. CXCL5-stimulated proliferative and invasive responses. (A) N15C6 (light gray bars) or BPH-1 (dark gray bars) nontransformed prostate epithelial cells proliferated to significantly higher levels when grown for 72 hours in SF media supplemented with 10 pM CXCL5 than those grown in SF alone (*P < .001). Preincubation of the cells for 1 hour with 1 μg/ml antibody against CXCR2, the receptor for CXCL5, followed by supplementation with CXCL5 and maintenance of growth in CXCL5 + anti-CXCR2–containing media significantly ablated the proliferative response (#P < .001). In contrast, cellular growth after preincubation with an antibody against an unrelated chemokine receptor, CXCR4, followed by supplementation with CXCL5 and maintenance of growth in CXCL5 + anti-CXCR4–containing media was similar to that observed for non–pretreated cells grown in CXCL5-supplemented media and was significantly higher than that in SF alone (*P < .001). All data are shown normalized to growth in unsupplemented SF, which was set at one-fold. (B) N15C6 (LEFT) or LNCaP (RIGHT) cells were grown in SF media (untreated, UnT) or SF media supplemented with 10 pM CXCL5 for N15C6 or 100 pM CXCL5 for LNCaP (treated, T) for the times indicated. The cells were then harvested and assessed for nucleosomal DNA fragmentation. The fraction of cells exhibiting apoptosis plotted on the y axis was calculated as the difference in absorbance measured at 405 nm and at the reference wavelength of 490 nm after adjusting for background absorbance at both wavelengths. No significant differences in the fraction of cells exhibiting apoptosis were observed between treated and untreated cells at any time point, demonstrating that CXCL5 does not promote antiapoptotic responses in these cells. (C) Fifteen thousand each of N15C6 (black bars) or PC3 (gray bars) cells were plated onto Matrigel-coated membranes and were exposed to complete media or complete media supplemented with 20 nM CXCL5 for 24 hours. After 24 hours, the cells that migrated and invaded through the Matrigel were stained and counted. N15C6 cells did not demonstrate an invasive response to treatment with CXCL5. However, approximately six-fold more PC3 cells migrated through the synthetic basement membrane, Matrigel, in response to 20 nM CXCL5 compared to vehicle (control, set at one-fold) (*P < .05). PC3 cell invasion through the Matrigel in response to CXCL5 was significantly inhibited by pretreatment with 1 μg/ml blocking antibody (anti- CXCR2) (#P < .05) but not by pretreatment with nonspecific antibody (anti-CXCR4) (*P < .05).

Article Snippet: The TMA was then incubated overnight with primary CXCL5 antibody (Human CXCL5/ENA-78 MAb clone 33160; R&D Systems, Minneapolis, MN) at a dilution of 1:25.

Techniques: Staining, Membrane, Control, Blocking Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 5. CXCL5 activates MAPK signaling in nontransformed N15C6 prostate epithelial cells. Nontransformed N15C6 cells rapidly and transiently phosphorylated ERK 1/2 and STAT3 when treated with either subnanomolar (10 or 100 pM) or nanomolar (1 nM) levels of CXCL5, whereas NF-κB subunit activation was evident only after treatment with 1 nM CXCL5. Primary antibody concentrations used were 1:500 for phospho-ERK, 1:500 for phospho-65 (NF-κB), 1:1000 for phospho-STAT3, 1:1000 for total ERK, 1:1000 for total p65, and 1:2000 for total STAT3. A total of 20 μg of protein lysate was electrophoresed per well. Immunoblots are shown on the left, and corresponding densitometric evaluations of the same blots are shown on the right. Phosphorylation relative to total protein quantitated from the immunoblot is shown in the densitometric plots as phospho/total protein.

Article Snippet: The TMA was then incubated overnight with primary CXCL5 antibody (Human CXCL5/ENA-78 MAb clone 33160; R&D Systems, Minneapolis, MN) at a dilution of 1:25.

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 6. CXCL5 activates both MAPK and PI3K signaling in transformed LNCaP prostate epithelial cells. Transformed LNCaP cells rapidly and transiently phosphorylated both ERK 1/2 and the p65 subunit of NF-κB on treatment with subnanomolar (10 or 100 pM) levels of CXCL5. Immunoblots are shown in the top panel, and corresponding densitometric evaluations of the same blots are shown in the bottom panel. Phosphorylation relative to total protein quantitated from the immunoblot is shown in the densitometric plots as phospho/total protein. A total of 100 μg of protein lysate was electrophoresed per well. Primary antibody concentrations used were as described for Figure 5.

Article Snippet: The TMA was then incubated overnight with primary CXCL5 antibody (Human CXCL5/ENA-78 MAb clone 33160; R&D Systems, Minneapolis, MN) at a dilution of 1:25.

Techniques: Transformation Assay, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 7. CXCL5 stimulates a transcriptional response in both nontransformed and transformed prostate epithelial cells. Quantitative real-time PCR of RNA purified from N15C6 cells (left) or LNCaP cells (right) treated with subnanomolar CXCL5 as shown demonstrates rapid and robust transcription of the EGR1 gene significantly higher than levels obtained at time 0 (set at one-fold) (*P < .05). Data shown are averaged from three or more separate experiments per time point per concentration of CXCL5 examined.

Article Snippet: The TMA was then incubated overnight with primary CXCL5 antibody (Human CXCL5/ENA-78 MAb clone 33160; R&D Systems, Minneapolis, MN) at a dilution of 1:25.

Techniques: Transformation Assay, Real-time Polymerase Chain Reaction, Purification, Concentration Assay