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: 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

Journal: OncoTargets and therapy

Article Title: The clinical significance of CXCL5 in non-small cell lung cancer

doi: 10.2147/OTT.S148772

Figure Lengend Snippet: Representative images of CXCL5 expression by immunohistochemical staining. Notes: ( A ) Normal, ( B ) adenocarcinoma, ( C ) squamous carcinoma, ( D ) papillary cancer, ( E ) SCLC, ( F ) carcinoid, ( G ) bronchioloalveolar cancer, ( H ) giant cell carcinoma, ( I ) metastatic tumor, ( J ) quantitative score of CXCL5. Magnification 200×. Abbreviation: SCLC, small cell lung cancer.

Article Snippet: The ELISA kit for ENA78 (CXCL5) was purchased from Cloud-Clone Corp (Katy, TX, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: OncoTargets and therapy

Article Title: The clinical significance of CXCL5 in non-small cell lung cancer

doi: 10.2147/OTT.S148772

Figure Lengend Snippet: Association of CXCL5 expression with pathological parameters in adenocarcinoma by immunohistochemical staining. Notes: ( A ) Grade 1 vs 3, ( B ) T stage 1 vs 3, ( C ) TNM stage I vs III, ( D ) lymph node positive vs negative. Left panel: representative images, right panel: quantitative score.

Article Snippet: The ELISA kit for ENA78 (CXCL5) was purchased from Cloud-Clone Corp (Katy, TX, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: OncoTargets and therapy

Article Title: The clinical significance of CXCL5 in non-small cell lung cancer

doi: 10.2147/OTT.S148772

Figure Lengend Snippet: Prognostic value of CXCL5 in adenocarcinoma. Notes: ( A ) Kaplan–Meier curve of patients with low or high CXCL5 expression. Median level was used to divide patients into subgroups. ( B ) Multivariate analysis of clinical and pathological parameters. Abbreviations: Sig, significance; Exp, odd ratio value; B, beta; SE, standard error.

Article Snippet: The ELISA kit for ENA78 (CXCL5) was purchased from Cloud-Clone Corp (Katy, TX, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: The clinical significance of CXCL5 in non-small cell lung cancer

doi: 10.2147/OTT.S148772

Figure Lengend Snippet: Serum CXCL5 concentration detected by ELISA. Notes: ( A ) Standard curve of CXCL5 with OD value. ( B ) Peripheral serum CXCL5 in healthy volunteers and lung adenocarcinoma patients. Abbreviations: OD, optical density; N, normal.

Article Snippet: The ELISA kit for ENA78 (CXCL5) was purchased from Cloud-Clone Corp (Katy, TX, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: OncoTargets and therapy

Article Title: The clinical significance of CXCL5 in non-small cell lung cancer

doi: 10.2147/OTT.S148772

Figure Lengend Snippet: Forest plot of HR and OS of CXCL5. Notes: ( A ) Relative risk of CXCL5 mRNA expression to OS in NSCLC. OR value of CXCL5 in ADC vs SQC ( B ) stages III–IV vs I–II in NSCLC ( C ) and ADC ( D ), OR value of CXCL5 comparing N+ and N− patients in NSCLC ( E ) and ADC ( F ). Abbreviations: HR, hazard ratio; OR, odds ratio; OS, overall survival; NSCLC, non-small cell lung cancer; ADC, adenocarcinoma; SQC, squamous cell lung cancer ; N+, lymph node positive; N−, lymph node negative.

Article Snippet: The ELISA kit for ENA78 (CXCL5) was purchased from Cloud-Clone Corp (Katy, TX, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: The clinical significance of CXCL5 in non-small cell lung cancer

doi: 10.2147/OTT.S148772

Figure Lengend Snippet: Kaplan–Meier survival curve. Notes: Association between CXCL5 and OS ( A ), PPS ( B ), and PFS ( C ) in NSCLC patients. ( D ) Association of CXCL5 with PFS in patients with ADC. Abbreviations: HR, hazard ratio; OS, overall survival; PPS, post-progression survival; PFS, progression-free survival; ADC, adenocarcinoma; NSCLC, non-small cell lung cancer.

Article Snippet: The ELISA kit for ENA78 (CXCL5) was purchased from Cloud-Clone Corp (Katy, TX, USA).

Techniques:

Journal: bioRxiv

Article Title: Parkinson’s disease-derived α-Synuclein assemblies combined with TNFα and Prostaglandin E 2 induces a specific chronic-type inflammatory phenotype in microglial cells

doi: 10.1101/2023.11.30.569208

Figure Lengend Snippet: ( A ) Representative images showing CD25 immunostaining with Hoechst nuclear stain (blue) in non-stimulated microglial cells (NSC) and cells exposed to IL4 (10ng/mL), IFNγ (20ng/mL) or a combination of TNFα (800 UI/ml), PGE 2 (1µg/ml) and Pam3C (1µg/ml) (TPP stimulation). Scale bar: 60µm. ( B ) Quantification of CXCL5 and IL1α release by microglial cells exposed or not (NSC) to the indicated treatments. Data are means ± SEM ( n = 4). * p < 0.05 vs. TPP (Tukey’s test). ( C ) Quantification of ROS production measured with the NBT reaction in microglial cells exposed or not (NSC) to the indicated treatments. Data are means ± SEM ( n = 4). * p < 0.05 vs. TPP (Tukey’s test).

Article Snippet: Mouse TNFα ELISA Kit (Thermo Fisher Scientific, BMS607-3), Mouse IL6 ELISA Kit (Thermo Fisher Scientific, BMS603-2TWO), Mouse IL1β ELISA Kit (Thermo Fisher Scientific, BMS6002), Mouse IL10 ELISA Kit (R&D Systems, M1000B), U-Plex (Meso Scale Discovery, K15069L-1), Mouse Pro-Inflammatory 7-Plex Tissue (Meso Scale Discovery, K15012B-1), Mouse IL1α ELISA Kit (Biolegend, #433404), Mouse CXCL5 ELISA Kit (R&D Systems, MX000), Hoechst 33342 (Tocris, #5117).

Techniques: Immunostaining, Staining