Journal: Oncology Letters

Article Title: CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells

doi: 10.3892/ol.2017.7236

Figure Lengend Snippet: Primer sequences used for reverse transcription-polymerase chain reaction.

Article Snippet: The secretion levels of CXCL5 were determined by ELISA using Human CXCL5 Elisa kit (cat. no. EK0728; Wuhan Boster Biological Technology, Ltd.), according to the manufacturer's protocol.

Techniques: Sequencing

Journal: Oncology Letters

Article Title: CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells

doi: 10.3892/ol.2017.7236

Figure Lengend Snippet: Reverse transcription-polymerase chain reaction conditions for each primer set.

Article Snippet: The secretion levels of CXCL5 were determined by ELISA using Human CXCL5 Elisa kit (cat. no. EK0728; Wuhan Boster Biological Technology, Ltd.), according to the manufacturer's protocol.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells

doi: 10.3892/ol.2017.7236

Figure Lengend Snippet: CXCL5 and its receptor CXCR2 are expressed by parental, HepG2-NEG and HepG2-A1113 cells. (A) CXCL5 mRNA was upregulated in HepG2-A1113 cells compared with HepG2-parental or HepG2-NEG cells. The expression levels of CXCR2 (A) mRNA and (B) protein were not significantly different in all three cell lines. (C) However, the expression of CXCL5 secretory protein was upregulated in HepG2-A1113 cells compared with HepG2-parental or HepG2-NEG cells. *P<0.05. CXCL5, C-X-C motif chemokine ligand 5; CXCR2, C-X-C chemokine receptor type 2; HepG2-NEG, empty vector-transfected HepG2 cells; HepG2-A1113, CXCL5-transfected HepG2 cells.

Article Snippet: The secretion levels of CXCL5 were determined by ELISA using Human CXCL5 Elisa kit (cat. no. EK0728; Wuhan Boster Biological Technology, Ltd.), according to the manufacturer's protocol.

Techniques: Expressing, Plasmid Preparation, Transfection

Journal: Oncology Letters

Article Title: CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells

doi: 10.3892/ol.2017.7236

Figure Lengend Snippet: Exogenous CXCL5 expression contributes to the tumorigenesis of HepG2 cells in vitro. (A) Cell counting kit-8 assay showed significant growth promotion when HepG2 cells grew in medium supplemented with 20 ng/ml exogenous CXCL5 for 48, 72 or 96 h or 40 ng/ml exogenous CXCL5 for 72 or 96 h; 60 ng/ml exogenous CXCL5 significantly inhibited proliferation only after 96 h of treatment (*P<0.05 vs. 0, 40 or 60 ng/ml; &P<0.05 vs. 0 or 60 ng/ml; #P<0.05 vs. 0, 20 or 40 ng/ml). (B) Treatment with 60 ng/ml exogenous CXCL5 efficiently promoted colony formation of HepG2 cells after 12 days (*P<0.05). (C) Representative images indicated that exogenous CXCL5 at certain concentrations significantly enhanced the migration of HepG2 cells in a Transwell assay (*P<0.05 vs. 0, 20, 60 or 80 ng/ml; &P<0.05 vs. 0 or 20 ng/ml). CXCL5, C-X-C motif chemokine ligand 5.

Article Snippet: The secretion levels of CXCL5 were determined by ELISA using Human CXCL5 Elisa kit (cat. no. EK0728; Wuhan Boster Biological Technology, Ltd.), according to the manufacturer's protocol.

Techniques: Expressing, In Vitro, Cell Counting, Migration, Transwell Assay

Journal: Oncology Letters

Article Title: CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells

doi: 10.3892/ol.2017.7236

Figure Lengend Snippet: Overexpression of CXCL5 is positively associated with the oncogenic potential of HepG2 cells in vitro. (A) Growth, (B) colony formation and (C) migration assays showed the growth, colony formation and migration, respectively, of HepG2-A1113 cells were significantly increased compared with HepG2-parental or HepG2-NEG cells (*P<0.05). CXCL5, C-X-C motif chemokine ligand 5.

Article Snippet: The secretion levels of CXCL5 were determined by ELISA using Human CXCL5 Elisa kit (cat. no. EK0728; Wuhan Boster Biological Technology, Ltd.), according to the manufacturer's protocol.

Techniques: Over Expression, In Vitro, Migration

Journal: Oncology Letters

Article Title: CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells

doi: 10.3892/ol.2017.7236

Figure Lengend Snippet: Upregulation of CXCL5 in LX-2 cells is involved in the proliferation and migration of HepG2 cells by paracrine signaling. CXCL5 (A) mRNA and (B) secretory protein in LX-2 A1113 cells were significantly increased compared with parental and LX-2 NEG cells (*P<0.05). The (C) proliferation and (D) migration capacity of HepG2 cells treated with CM from LX-2 A1113 cells were increased compared with the cells treated with CM from LX-2 parental or LX-2 NEG cells (*P<0.05). CXCL5, C-X-C motif chemokine ligand 5; LX-2 A1113, CXCL5-transfected LX-2 cells; LX-2 NEG, empty vector-transfected LX-2 cells; CM, culture medium.

Article Snippet: The secretion levels of CXCL5 were determined by ELISA using Human CXCL5 Elisa kit (cat. no. EK0728; Wuhan Boster Biological Technology, Ltd.), according to the manufacturer's protocol.

Techniques: Migration, Transfection, Plasmid Preparation

Journal: Oncology Letters

Article Title: CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells

doi: 10.3892/ol.2017.7236

Figure Lengend Snippet: Overexpression of CXCL5 regulates the expression of genes in HepG2 cells. (A) In reverse transcription-polymerase chain reaction assays, downregulation of NDRG3, Bax and P53 mRNA in HepG2-A1113 cells were detected compared with HepG2-parental or HepG2-NEG cells. However, the levels of Bcl-2 and VEGF mRNA in HepG2-A1113 cells were increased. (B) Western blotting assays showed upregulation of IL-18, IL-1β and CSE proteins in HepG2-A1113 cells (*P<0.01; &P<0.05). Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; VEGF, vascular endothelial growth factor; IL, interleukin; CXCL5, C-X-C motif chemokine ligand 5; CSE, cystathionine-γ-lyase; HepG2-NEG, empty vector-transfected HepG2 cells; HepG2-A1113, CXCL5-transfected HepG2 cells.

Article Snippet: The secretion levels of CXCL5 were determined by ELISA using Human CXCL5 Elisa kit (cat. no. EK0728; Wuhan Boster Biological Technology, Ltd.), according to the manufacturer's protocol.

Techniques: Over Expression, Expressing, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection

Journal: Biomolecules

Article Title: Modulating the CXCR2 Signaling Axis Using Engineered Chemokine Fusion Proteins to Disrupt Myeloid Cell Infiltration in Pancreatic Cancer

doi: 10.3390/biom15050645

Figure Lengend Snippet: CXCR2 ligands are the most abundant soluble factors secreted by PDAC cells. ( A ) Cytokine arrays were used to identify relative concentrations of 105 secreted factors from four PDAC cell lines (BxPC-3, Capan-1, CFPAC-1, and PANC-1). Concentrations were determined relative to reference spots (A1,2,23,24 and J1,2). A representative cytokine array image using PANC-1 CM is shown. ( B ) Quantification of secreted factors that were upregulated in four out of four (gray) or three out of four (white) cell lines are shown. ( C ) CM from PDAC cell lines, HEK cells, and HPNE cells was analyzed by ELISA for CXCL8, CXCL5, and CXCL1 ( n = 2–10). ( D ) CM from cell lines established from PDAC mouse models Panc02 and KPC was analyzed by ELISA for mouse CXCL5 and mouse CXCL1 ( n = 2).

Article Snippet: Kits were as follows: Human CXCL1 (88-52122-22, Invitrogen, Waltham, MA, USA), Human CXCL5 (dy254-05, R & D Systems, Minneapolis, MN, USA), Human CXCL8 (88-8086-22, Invitrogen, Waltham, MA, USA), Mouse CXCL1 (P352782, R & D Systems, Minneapolis, MN, USA), and Mouse CXCL5 (MX000, R & D Systems, Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: Modulating the CXCR2 Signaling Axis Using Engineered Chemokine Fusion Proteins to Disrupt Myeloid Cell Infiltration in Pancreatic Cancer

doi: 10.3390/biom15050645

Figure Lengend Snippet: CXCR2 ligand production in PDAC cells is mutant-KRAS- and NFκB-dependent. ( A ) Western blot analysis of KRAS G12D or G12R mutant expression in inducible HPNE cells upon treatment with 1 μg/mL doxycycline for 48 h. ( B ) CM was collected from KRAS-mutant-inducible HPNE cells with or without treatment with doxycycline and analyzed by ELISA for CXCL5 and CXCL1. Statistical significance was determined using ordinary one-way ANOVA, and (-) DOX, G12D, and G12R were compared by Šídák’s multiple comparisons test (* p < 0.05, n = 3). ( C ) PANC-1 cells and CFPAC-1 cells were treated with a dose course of MRTX1133 for 30 h, and CM was collected and analyzed by ELISA for CXCL8 and CXCL1. Statistical significance was determined using Student’s t -test (* p < 0.05, n = 2). ( D ) PANC-1 cells expressing IκB-SR were treated with 10 ng/mL TNFα for 30 min. Cells were lysed and separated into the cytosolic and nuclear fractions. Western blotting analysis is shown. ( E ) CM was collected from PANC-1 cells expressing GFP or IκB-SR and analyzed by ELISA for CXCL1 and CXCL5. Statistical significance was determined using ordinary one-way ANOVA, and GFP and IkB-SR were compared by Šídák’s multiple comparisons test (* p < 0.05, n = 3). Original images of ( A , D ) can be found in .

Article Snippet: Kits were as follows: Human CXCL1 (88-52122-22, Invitrogen, Waltham, MA, USA), Human CXCL5 (dy254-05, R & D Systems, Minneapolis, MN, USA), Human CXCL8 (88-8086-22, Invitrogen, Waltham, MA, USA), Mouse CXCL1 (P352782, R & D Systems, Minneapolis, MN, USA), and Mouse CXCL5 (MX000, R & D Systems, Minneapolis, MN, USA).

Techniques: Mutagenesis, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: Modulating the CXCR2 Signaling Axis Using Engineered Chemokine Fusion Proteins to Disrupt Myeloid Cell Infiltration in Pancreatic Cancer

doi: 10.3390/biom15050645

Figure Lengend Snippet: Design and characterization of CXCR2 ligand fusion proteins. ( A ) Purified CXCL1, CXCL5, Fc, and fusion proteins CXCL1-Fc and CXCL5-Fc were analyzed by SDS-PAGE. Proteins were separated under reducing and non-reducing conditions and visualized using Coomassie Blue staining. ( B ) THP-1 cells were incubated on ice with proteins for 30 min, and binding was analyzed by flow cytometry using a FITC-conjugated anti-His tag antibody. Statistical significance was determined using Student’s t -test (* p < 0.05, n = 3). ( C ) HL60 and primary human neutrophils were incubated with proteins as in ( B ) and analyzed by flow cytometry, as in ( B ). ( D ) Binding of CXCL1-Fc and CXCL5-Fc to THP-1 cells was analyzed as in ( B ) across a range of concentrations. ( E ) THP-1 cells and HL60 cells were incubated with CXCL1 or CXCL1-Fc for the indicated times. Cell lysates were analyzed by Western blotting for p-ERK, total ERK, p-AKT, and total AKT (pan). ( F ) THP-1 cells were incubated with the indicated concentrations of CXCL1 or CXCL1-Fc for 5 min. Cell lysates were analyzed by Western blotting for p-ERK and total ERK. Original images of ( E , F ) can be found in .

Article Snippet: Kits were as follows: Human CXCL1 (88-52122-22, Invitrogen, Waltham, MA, USA), Human CXCL5 (dy254-05, R & D Systems, Minneapolis, MN, USA), Human CXCL8 (88-8086-22, Invitrogen, Waltham, MA, USA), Mouse CXCL1 (P352782, R & D Systems, Minneapolis, MN, USA), and Mouse CXCL5 (MX000, R & D Systems, Minneapolis, MN, USA).

Techniques: Purification, SDS Page, Staining, Incubation, Binding Assay, Flow Cytometry, Western Blot

Journal: EBioMedicine

Article Title: Interleukin-8 as a therapeutic target for chronic low back pain: Upregulation in human cerebrospinal fluid and pre-clinical validation with chronic reparixin in the SPARC-null mouse model

doi: 10.1016/j.ebiom.2019.04.032

Figure Lengend Snippet: The murine IL-8 analogue, CXCL5 (LIX), is elevated in intervertebral discs from male SPARC-null mice. The murine IL-8 analogues CXCL1 (KC, top row, A,C,E) and CXCL5 (LIX, bottom row, B,D,F) were measured by ELISA in male discs (1st column, A,B), male serum (2nd column, C,D) or female discs (3rd column, E,F) from SPARC-null and WT mice. No changes were observed in CXCL1 (KC). In contrast, CXCL5 (LIX) was upregulated in male mouse discs. Data is presented as mean ± SEM, n = 5–8, t -tests were used to analyze the data. * = p < 0.05.

Article Snippet: CXCL1 (KC) and CXCL5 (LIX) ELISA kits (RayBiotech, Cat. #s ELM-KC and ELM-LIX) were used to quantify concentrations in protein extracts from mouse discs and from serum collected from the same mice.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 regulates the expression and secretion of CXCL5 and CCL5 in bladder cancer. (A) Schematic of the transcriptome sequencing workflow following METTL3 knockdown in MB49 cells. (B) Heatmap of differentially expressed genes after METTL3 knockdown (criteria for differential genes: p<0.05, fold change >1.5 or <0.67. (C) Volcano plot of differentially expressed genes after METTL3 knockdown. (D) Network diagram of GO enrichment analysis of differentially expressed genes. (E) Chemokines with differential expression following METTL3 silencing. (F–G) Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of CXCL5 and CCL5 mRNA expression levels following METTL3 overexpression or knockdown in MB49 cells. (H) RT-qPCR analysis of CXCL5 and CCL5 mRNA expression levels in MB49 cells treated with DMSO or STM2457 for 72 hours. (I) ELISA of CXCL5 and CCL5 secretion levels in the culture supernatant of MB49 cell lines; ELISA of CXCL5 and CCL5 levels in (J) mouse tumor tissues and (K) in peripheral blood serum. RT-qPCR analysis of CXCL5 and CCL5 mRNA expression levels in (L) 5637 cells and (M) T24 cells treated with DMSO or STM2457 for 72 hours. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Secretion levels of CXCL5 and CCL5 were measured using the Mouse CXCL5 ELISA Kit (Elabscience, #E-EL-M0471) and the Mouse RANTES ELISA Kit (Elabscience, #E-EL-M0009), respectively.

Techniques: Expressing, Sequencing, Knockdown, Quantitative Proteomics, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 regulates CXCL5 expression through N6-methyladenosine (m6A) modification, thereby promoting myeloid-derived suppressor cell (MDSC) chemotaxis. (A) Schematic diagram of the workflow used for Methylated RNA Immunoprecipitation (MeRIP) sequencing and data analysis in METTL3-knockdown MB49 stable cell lines. (B) Bar chart showing the number of m6A modification sites identified in MeRIP sequencing results. (C) Venn diagram of downstream target gene screening for METTL3, intersecting chemokines significantly altered in RNA sequencing with those showing significant downregulation in m6A modification levels in m6A sequencing. (D) m6A peak map of CXCL5 mRNA modification sites. (E) Bar chart of MeRIP-qPCR results showing the m6A modification level of CXCL5 mRNA in MB49 cells after METTL3 knockdown. (F) RNA Immunoprecipitation (RIP) assay detecting the interaction between METTL3 and CXCL5 mRNA. (G) RNA degradation assay showing CXCL5 mRNA stability after silencing METTL3. (H) RNA degradation assay showing CXCL5 mRNA stability after treatment with METTL3 inhibitor STM2457 (2 µg/mL, 72 hours) in MB49 cells. (I) Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of IGF2BP1 and CXCL5 mRNA expression levels in MB49 cells after silencing IGF2BP1. (J) RT-qPCR analysis of IGF2BP2 and CXCL5 mRNA expression levels in MB49 cells after silencing IGF2BP2. (K) RT-qPCR analysis of METTL3, IGF2BP1, and CXCL5 mRNA expression levels in MB49 cells after overexpression of METTL3 and/or silencing of IGF2BP1. (L) Schematic of the animal experiment. (M) Images of bladder cancer tumors in mice. (N) Growth curves of bladder cancer tumors in mice. (O) Tumor weights of bladder cancer tumors in mice. ns, no significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Secretion levels of CXCL5 and CCL5 were measured using the Mouse CXCL5 ELISA Kit (Elabscience, #E-EL-M0471) and the Mouse RANTES ELISA Kit (Elabscience, #E-EL-M0009), respectively.

Techniques: Expressing, Modification, Derivative Assay, Chemotaxis Assay, Methylation, RNA Immunoprecipitation, Sequencing, Knockdown, Stable Transfection, RNA Sequencing, Degradation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression