Journal: Bioengineered

Article Title: Neutrophils correlate with hypoxia microenvironment and promote progression of non-small-cell lung cancer

doi: 10.1080/21655979.2021.1987820

Figure Lengend Snippet: CXCL6 is the critical chemokine induced by hypoxic NSCLC cell to recruit TANs derived from NSCLC tissues. ( A ) Quantification of neutrophil migration as assessed by transwell assays; ( B ) and ( C ) Expression of CXCL6 in hypoxic or normoxic NSCLC cells was examined by real-time PCR and ELISA; ( D ) Quantification of neutrophil migration as assessed by transwell assays

Article Snippet: According to the manufacturer’s instructions, the level of CXCL6 was measured using the Human CXCL6 ELISA Kit (Abnova).

Techniques: Derivative Assay, Migration, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Bioengineered

Article Title: Neutrophils correlate with hypoxia microenvironment and promote progression of non-small-cell lung cancer

doi: 10.1080/21655979.2021.1987820

Figure Lengend Snippet: TANs derived from NSCLC tissues promote NSCLC cells proliferation, migration and invasion. ( A ), ( B ) and ( C ): NSCLC cells cocultured with TANs or alone were subjected to colony formation, wound healing, and transwell invasion assays; ( D ) Schematic illustration of the crosstalk between CXCL6-overexpressing NSCLC cells and TANs in the TME

Article Snippet: According to the manufacturer’s instructions, the level of CXCL6 was measured using the Human CXCL6 ELISA Kit (Abnova).

Techniques: Derivative Assay, Migration

Journal: Materials Today Bio

Article Title: CXCL chemokines-mediated communication between macrophages and BMSCs on titanium surface promotes osteogenesis via the actin cytoskeleton pathway

doi: 10.1016/j.mtbio.2023.100816

Figure Lengend Snippet: Primers used for qRT-PCR.

Article Snippet: Proteins secreted by the two types of cells in the co-culture model were detected using CXCL3 and PTN ELISA Kits from JiangLai Biological (Shanghai, China) and CXCL6 and CXCL14 ELISA Kits from Cloud-Clone Corp (Wuhan, China).

Techniques: Sequencing

Journal: Materials Today Bio

Article Title: CXCL chemokines-mediated communication between macrophages and BMSCs on titanium surface promotes osteogenesis via the actin cytoskeleton pathway

doi: 10.1016/j.mtbio.2023.100816

Figure Lengend Snippet: Dynamic interactions between BMMs and BMSCs in the co-culture system. Under co-culture conditions, a reciprocal interplay unfolds between BMMs and BMSCs, driven by distinct signaling mechanisms. BMMs initiate changes in BMSCs by upregulating the secretion of chemokines and engaging chemokine receptors on BMSC surfaces. Conversely, BMSCs exert influence on BMMs by heightening PTN secretion and interacting with the Sdc3 receptors on BMM surfaces. (A) Temporal evolution of chemokine family-related gene expression in BMMs at mRNA levels in separate culture and co-culture systems. (B) Alterations in the expression of chemokine family-related gene receptors in BMSCs at the mRNA level in separate and co-culture systems. (C) Time-dependent shifts in PTN mRNA expression. (D) Temporal changes in PTN receptor Sdc3 mRNA expression on macrophages. (E, F) Protein expression profiles of CXCL3, CXCL6, CXCL14, and PTN over time in separate and co-culture systems. (G, H) Dynamics of protein expression for chemokine receptors CCR1 and CXCR2, and PTN receptor Sdc3, over time in separate and co-culture systems. (I) Time-course variations in CXCL3, CXCL6, CXCL14, and PTN levels in cell supernatant observed using ELISA in separate and co-culture systems. Data are presented as mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. mRNA and protein expression were normalized to those of the separate culture system on day 1.

Article Snippet: Proteins secreted by the two types of cells in the co-culture model were detected using CXCL3 and PTN ELISA Kits from JiangLai Biological (Shanghai, China) and CXCL6 and CXCL14 ELISA Kits from Cloud-Clone Corp (Wuhan, China).

Techniques: Co-Culture Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Inflammation-Driven Secretion Potential Is Upregulated in Osteoarthritic Fibroblast-Like Synoviocytes

doi: 10.3390/ijms231911817

Figure Lengend Snippet: Chemokine secretion levels by HFLS and HFLS-OA stimulated with TNFα or LPS measured using the ELISA method. Upregulation of chemokine level secretion measured in media using the ELISA method. Cells were stimulated for 24 h with 10 ng/mL of TNFα ( A , C , E ) or LPS ( B , D , F ). Afterward, the media were collected, and CXCL6 ( A , B ), CXCL10 ( C , D ), and CXCL16 ( E , F ) levels were determined. Data are presented as pg/mL ± SEM and analyzed with two-way ANOVA followed by Tukey’s post hoc test. ** 0.01 > p > 0.001; *** p < 0.001 vs. control group. ## 0.01 > p > 0.001; ### p < 0.001 HFLS vs. HFLS-OA stimulated groups. HFLS, human fibroblast-like synoviocytes; HFLS-OA, osteoarthritic human fibroblast-like synoviocytes; TNFα, tumor necrosis factor alpha; LPS, lipopolysaccharide; ELISA, enzyme linked immunosorbent assay; CXCL6, chemokine (C-X-C motif) ligand 6; CXCL10, chemokine (C-X-C motif) ligand 10; CXCL16, chemokine (C-X-C motif) ligand 16.

Article Snippet: The protein levels of the CXCL6 (Human GCP2 (Granulocyte Chemotactic Protein 2) ELISA Kit, ELK Biotechnology, Wuhan, China), CXCL10 (Human IP-10/CXCL10 ELISA Kit, Elabscience, Wuhan, China), CXCL16 (Human CXCL16 ELISA Kit, Elabscience, Wuhan, China), ANGPTL1 (Human ANGPTL1 ELISA Kit, ELK Biotechnology, Wuhan, China), FGF5 (Human FGF5 ELISA Kit, ELK Biotechnology, Wuhan, China) and IGF-2 (Human IGF-2 ELISA Kit, Elabscience, Wuhan, China) were measured using commercially available enzyme-linked immunosorbent assay kits according to the manufacturers’ instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: The Therapeutic Treatment with the GAG-Binding Chemokine Fragment CXCL9(74–103) Attenuates Neutrophilic Inflammation and Lung Dysfunction during Klebsiella pneumoniae Infection in Mice

doi: 10.3390/ijms23116246

Figure Lengend Snippet: The treatment with CXCL9(74–103) decreases lung inflammation after LPS instillation. Mice were instilled with LPS (25 ug/mouse) or saline (Ctrl), and after 6 h, mice were treated intravenously with 100 μL of CXCL9(74–103) 1 mg/mL or vehicle (PBS). After 18 h, mice were euthanized, and the number of ( A ) total leukocytes, ( B ) neutrophils, and ( C ) mononuclear cells was evaluated in bronchoalveolar lavage fluid (BALF). ( D ) Levels of IL-1β and the chemokines ( E ) CXCL1, ( F ) CXCL2, ( G ) CXCL6, and ( H ) total protein concentrations were also evaluated in the BALF. Black points = control; red points = vehicle; blue points = CXCL9(74–103). Data are shown as the median from one representative out of three independent experiments. * p < 0.05 when compared to control group; # p < 0.05 when compared with the vehicle group; n = 5 mice per group.

Article Snippet: The cytokine IL-1β (cat. no. DY401-5) and the chemokines CXCL1 (cat. no. DY453-05), CXCL2 (cat. no. DY452-05) and CXCL6 (cat. no. MX000) were measured from the BALF supernatants by ELISA following the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Techniques: Saline, Control

Journal: International Journal of Molecular Sciences

Article Title: The Therapeutic Treatment with the GAG-Binding Chemokine Fragment CXCL9(74–103) Attenuates Neutrophilic Inflammation and Lung Dysfunction during Klebsiella pneumoniae Infection in Mice

doi: 10.3390/ijms23116246

Figure Lengend Snippet: The treatment with CXCL9(74–103) decreased inflammation after pneumonia induced by Klebsiella pneumoniae . Mice were infected with K. pneumoniae (1 × 10 6 CFU/mouse) by intratracheal injection, and after 6 h, mice were treated intravenously with 100 μL of CXCL9(74–103) at 1 mg/mL or vehicle (PBS). Control mice received a saline injection into the trachea. Mice were euthanized 24 h after intratracheal challenge, and ( A ) the number of total leukocytes, ( B ) neutrophils, and ( C ) mononuclear cells were evaluated in BALF. ( D ) Levels of IL-1β, and of the chemokines ( E ) CXCL1, ( F ) CXCL2, and ( G ) CXCL6, as well as ( H ) total protein were measured, in the BALF. ( I ) CFU counts in BALF and ( J ) CFU counts in the lung tissue. Black points = control; red points = vehicle; blue points = 6 h CXCL9(74–103). Data are shown as the median from one representative out of three independent experiments. * p < 0.05 when compared to control (Ctrl) group; # p < 0.05 when compared with the vehicle (v) group; n = 5 mice per group. BALF—Bronchoalveolar fluid.

Article Snippet: The cytokine IL-1β (cat. no. DY401-5) and the chemokines CXCL1 (cat. no. DY453-05), CXCL2 (cat. no. DY452-05) and CXCL6 (cat. no. MX000) were measured from the BALF supernatants by ELISA following the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Techniques: Infection, Injection, Control, Saline

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Oligonucleotide primer sets for real-time PCR.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Expressions of CXCL6, CXCR1, and CXCR2 in osteosarcoma (OS) cells. The mRNA expressions of CXCL6 (A) , CXCR1 (B) , and CXCR2 (C) in multiple OS cell lines were evaluated by real-time PCR. The protein levels of CXCL6 (D) , CXCR1 (E) , and CXCR2 (F) in multiple OS cell lines were detected by western blot assay. (G–I) The protein quantification histograms were shown.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Anti-CXCL6 antibody inhibited the migration, invasion and EMT of OS cells. OS cells were cultured for 72 h, then incubated with anti-CXCL6 antibody (10 μg/mL) or a control antibody (IgG, 10 μg/mL) for another 24 h. (A) The level of CXCL6 in the supernatant fluid of cultured MG63 and 143B cells was detected by ELISA. (B) The migration of MG63 and 143B cells was evaluated by Transwell assay (no matrigel). Scal bar = 100 μm. (C,D) The number of migrated cells was shown. (E) The invasion of MG63 and 143B cells was assessed by Transwell assay (matrigel). Scal bar = 100 μm. (F,G) The number of invasive cells was shown. The expressions of E-cadherin (H) and N-cadherin (I) in MG63 and 143B cells were determined by immunofluorescence assay. Scal bar = 50 μm. (J) The protein levels of E-cadherin, N-cadherin, and Snail were evaluated by western blot assay. (K–P) The protein quantification histograms were shown. (Q–S) The mRNA expression of E-cadherin, N-cadherin, and Snail was detected by real-time PCR. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, versus the OS cell group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Migration, Cell Culture, Incubation, Control, Enzyme-linked Immunosorbent Assay, Transwell Assay, Immunofluorescence, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Recombinant human (rh) CXCL6 facilitated the migration and invasion of OS cells. OS cells were treated with 100 ng/ml rhCXCL6 for 24 h. (A) The migration of SaOS-2 and U2OS cells was detected by Transwell assay (no matrigel). Scal bar = 100 μm. (B,C) The number of migrated cells was shown. (D) The invasion of SaOS-2 and U2OS cells was determined by Transwell assay (matrigel). Scal bar = 100 μm. (E,F) The number of invasive cells was shown. ∗∗ P < 0.01, ∗∗∗ P < 0.001, versus the OS cell group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Recombinant, Migration, Transwell Assay

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: CXCL6/CXCR2 axis contributed to migration and invasion of OS cells. After transfection with siRNAs for 24 h, OS cells were treated with 100 ng/ml rhCXCL6 for 24 h. The mRNA expression of CXCR2 in SaOS-2 (A) and U2OS (B) cells was detected by real-time PCR. The protein expression of CXCR2 in SaOS-2 (C) and U2OS (D) cells was assessed by western blot assay. The protein quantification histograms were shown. (E) The migration of SaOS-2 and U2OS cells was detected by Transwell assay (no matrigel). Scal bar = 100 μm. (F,G) The number of migrated cells was shown. (H) The invasion of SaOS-2 and U2OS cells was determined by Transwell assay (matrigel). Scal bar = 100 μm. (I,J) The number of invasive cells was shown. (K) The protein levels of MMP9 and Snail in SaOS-2 and U2OS cells were detected by western blot assay. (L–O) The protein quantification histograms were shown. (P) MMP-9 activity in the supernatant fluid of cultured SaOS-2 and U2OS cells was determined by gelatin zymography method. (Q,R) The quantification histograms were shown. ∗∗ P < 0.01, ∗∗∗ P < 0.001, versus the SaOS-2+NC or U2OS+NC group. # P < 0.05, ## P < 0.01, ### P < 0.001, versus the rhCXCL6+NC group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Migration, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transwell Assay, Activity Assay, Cell Culture, Zymography

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Effect of CXCL6/CXCR2 axis on E-cadherin and N-cadherin expressions. The expressions of E-cadherin (A) and N-cadherin (B) in SaOS-2 and U2OS cells with different treatments were determined by immunofluorescence assay. Scal bar = 50 μm.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: PI3K/AKT and β-catenin signaling pathways participated in the regulation of migration, invasion and EMT by CXCL6/CXCR2 axis in OS cells. After transfection with siRNAs for 24 h, OS cells were treated with 100 ng/ml rhCXCL6 for 24 h. (A) The protein levels of p-AKT, AKT, and nuclear β-catenin in SaOS-2 and U2OS cells were detected by western blot assay. β-actin and Histone H3 were used as loading controls. (B–E) The protein quantification histograms were shown. OS cells were pre-treated with 50 μM LY294002 or 10 μM XAV939 for 1 h, then treated with 100 ng/ml rhCXCL6 for 24 h. (F) The migration of SaOS-2 and U2OS cells was detected by Transwell assay (no matrigel). Scal bar = 100 μm. (G,H) The number of migrated cells was shown. (I) The invasion of SaOS-2 and U2OS cells was determined by Transwell assay (matrigel). Scal bar = 100 μm. (J,K) The number of invasive cells was shown. (L) The protein levels of E-cadherin, N-cadherin, Snail, and MMP9 in SaOS-2 and U2OS cells were detected by western blot assay. (M–T) The protein quantification histograms were shown. (U) MMP-9 activity in the supernatant fluid of cultured SaOS-2 and U2OS cells was assessed by gelatin zymography assay. (V,W) The quantification histograms were shown. ∗∗∗ P < 0.001, versus the OS cell or OS cell +NC group. # P < 0.05, ## P < 0.01, ### P < 0.001, versus the rhCXCL6+NC or rhCXCL6 group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Protein-Protein interactions, Migration, Transfection, Western Blot, Transwell Assay, Activity Assay, Cell Culture, Zymography Assay

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Effect of CXCL6/CXCR2 axis on OS tumor growth and pulmonary metastasis in vivo . U2OS cells were infected with lentivirus expressing CXCL6 or NC. The mRNA (A) and protein level (B) of CXCL6 in U2OS cells were determined by real-time PCR and western blot assay. (C) After the injection of LV-CXCL6 or LV-NC U2OS cells for 21 days, the representative images of nude mice and their removed xenograft tumors were shown. (D) The tumor volume was calculated and the tumor growth-curve was shown. (E) The weight of xenograft tumors was shown. (F) The expression of PCNA in tumor tissues was detected by immunohistochemical staining. Scal bar = 50 μm. (G) The percentage of PCNA positive cells were calculated and shown. (H) The protein levels of p-AKT, AKT, and nuclear β-catenin in tumor tissues were detected by western blot assay. β-actin and Histone H3 were used as loading controls. (I,J) The protein quantification histograms were shown. (K) Representative images of the lung of nude mice. (L) The number of lung metastatic nodes was quantified. ∗∗∗ P < 0.001, versus the LV-NC group. ## P < 0.01, ### P < 0.001, versus the LV-CXCL6 group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: In Vivo, Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Immunohistochemical staining, Staining