Journal: Cell Death & Disease

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

doi: 10.1038/s41419-019-1448-x

Figure Lengend Snippet: a , b Culture supernatants collected from MSCs on day 3 were analyzed using a Human XL Cytokine Array Kit. Several cytokines with differential expression were found in the culture supernatants of ASMSCs, including CXCL5, ANG, GROα, and THBS1. c Expression levels of these cytokines in ASMSCs and HDMSCs were confirmed by qRT-PCR. d Western blot analysis was performed to detect the protein level of CXCL5 in ASMSCs compared with HDMSCs. e Quantitative data for western blot analyses are shown. f CXCL5 secreted from HDMSCs and ASMSCs was measured by ELISA on day 3. Data are presented as the mean ± SD ( n = 30). The results represent three independent experiments. *, p < 0.05; HDMSCs, mesenchymal stem cells from healthy donors; ASMSCs, mesenchymal stem cells from patients with ankylosing spondylitis; ANG, angiogenin; THBS1, thrombospondin-1

Article Snippet: The level of CXCL5 protein in the cell culture supernatant was quantitated using Human CXCL5 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

doi: 10.1038/s41419-019-1448-x

Figure Lengend Snippet: CD14 + monocytes were cultured with HDMSCs or ASMSCs after knocking down CXCL5 in the presence of M-CSF and RANKL. a Secretion of CXCL5 from MSCs was detected by ELISA on day 3 after transfection with sh-CXCL5. b Representative images of TRAP staining (× 100). c The number of TRAP + osteoclasts in each well is shown. d Representative images of F-actin assays (× 200). e Representative images of bone resorption assays (× 200). f Pit formation on each slide was assessed. Data are presented as the mean ± SD ( n = 30). The results represent three independent experiments. *, p < 0.05; HDMSCs, mesenchymal stem cells from healthy donors; ASMSCs, mesenchymal stem cells from patients with ankylosing spondylitis

Article Snippet: The level of CXCL5 protein in the cell culture supernatant was quantitated using Human CXCL5 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Staining

Journal: Cell Death & Disease

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

doi: 10.1038/s41419-019-1448-x

Figure Lengend Snippet: a Representative images of TRAP staining (× 100), F-actin and bone resorption assays (× 200) of osteoclasts treated with the indicated doses of CXCL5 under osteoclastogenic conditions. b The number of TRAP + osteoclasts in each well on day 9 is shown. c Pit formation on each slide on day 15 was assessed. d mRNA expression levels of TRAP, CTSK, and NFATc1 were determined by qRT-PCR on day 9. e Protein levels of TRAP, CTSK, and NFATc1 were determined by western blot analysis on day 9. f Quantitative data for TRAP, CTSK, and NFATc1 protein levels determined by western blot analyses are shown. g Activation of signaling pathways involved in osteoclastogenesis was determined by western blot analyses on day 9. h Quantitative data for activation of signaling pathways determined by western blot analyses are shown. Data are presented as the mean ± SD ( n = 18). The results represent three independent experiments. *, p < 0.05

Article Snippet: The level of CXCL5 protein in the cell culture supernatant was quantitated using Human CXCL5 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot, Activation Assay, Protein-Protein interactions

Journal: Cell Death & Disease

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

doi: 10.1038/s41419-019-1448-x

Figure Lengend Snippet: a Bioinformatics analysis in three databases (TargetScan, miRBase, and miRDB) to identify possible miRNAs targeting CXCL5. Seven miRNAs with the highest scores for targeting CXCL5 are shown. b Expression levels of miRNAs in HDMSCs and ASMSCs were determined by qRT-PCR. c Expression levels of miR-4284 in HDMSCs and ASMSCs after treatment with miR-4284 inhibitor or mimic. d Expression levels of CXCL5 in HDMSCs and ASMSCs after treatment with miR-4284 inhibitor or mimic. e Possible binding sites between miR-4284 and CXCL5 are shown, and a mutant (MUT) CXCL5 site was constructed. f Binding sites were confirmed by luciferase assays. The miR-4284 mimic decreased the luciferase activity of wild-type (WT) CXCL5 but had no impact on MUT CXCL5. Data are presented as the mean ± SD ( n = 30). The results represent three independent experiments. *, p < 0.05; HDMSCs, mesenchymal stem cells from healthy donors; ASMSCs, mesenchymal stem cells from patients with ankylosing spondylitis

Article Snippet: The level of CXCL5 protein in the cell culture supernatant was quantitated using Human CXCL5 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Mutagenesis, Construct, Luciferase, Activity 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: 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