Journal: International Journal of Molecular Sciences

Article Title: Distinct Plasma Immune Profile in ALS Implicates sTNFR-II in pAMPK/Leptin Homeostasis

doi: 10.3390/ijms24065065

Figure Lengend Snippet: Plasma levels of leptin, CCL16 and sTNF-RII measured by ELISA. ( a ) concentration (ng/mL) of leptin measured by ELISA in healthy controls (38) and sALS (51). ( b , d ) Concentration (ng/mL) of leptin ( b ), CCL16 ( c ) and sTNF-RII ( d ) measured by ELISA in slow (40) and fast (11) progressing ALS patients. ( e , f ) Concentration (ng/mL) of leptin in sALS and controls sub-grouped by sex ( e ) and by sex and rate of progression ( f ) (Data are mean ± SEM * p ≤ 0.05, ** p < 0.01, **** p < 0.0001, NS, not significant).

Article Snippet: CCL16 plasma levels were measured using a CCL16 ELISA kit (Invitrogen, Waltham, MA, #EHCCL16) with 1:10 dilution. sTNF-RII plasma levels were measured using human sTNF-RII quantikine ELISA kit (R&D system, Minneapolis, MN, USA, #DRT200) with 1:10 dilution.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Distinct Plasma Immune Profile in ALS Implicates sTNFR-II in pAMPK/Leptin Homeostasis

doi: 10.3390/ijms24065065

Figure Lengend Snippet: Exposure to plasma from sALS reduces leptin production in human adipocytes. ( a ) Oil red O coloration demonstrating lipid droplet accumulation (red) in mature adipocytes. ( b ) Human adipocytes were treated for 12 h with 1:100 diluted mixed plasma samples from healthy controls (12), slow progressors (12) and fast progressors (6). The supernatant was collected, and leptin levels were measured by ELISA (pg/mL) and normalized with plasma sample levels of leptin. ( c ) Leptin levels (pg/mL) secreted by mature adipocytes after plasma samples treatment. The adipocytes were pretreated with 1 μM of mTOR inhibitor (PP242) or 10 μM of AMPK inhibitor (compound C) for 1h. ( d ) Proteins from treated adipocytes ( c ) were extracted using RIPA buffer. Graph exhibits pAMPK/GAPDH fold change observed by immunoblotting with or without 10 μM AMPK inhibitor pretreatment. ( e , f ) Leptin levels (pg/mL) secreted by mature adipocytes after insulin and sTNF-RII treatment ( e ) and insulin and CCL16 treatment ( f ). ( g ) Immunoblotting showing pAMPK levels in adipocytes treated with increasing concentration of sTNF-RII. ( h ) Quantification of immunoblotting in ( g ) showing levels of pAMPK/GAPDH fold change in adipocytes treated with sTNF-RII ((Data are mean ± SEM * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) n = 3, two-way ANOVA). NS, not significant.

Article Snippet: CCL16 plasma levels were measured using a CCL16 ELISA kit (Invitrogen, Waltham, MA, #EHCCL16) with 1:10 dilution. sTNF-RII plasma levels were measured using human sTNF-RII quantikine ELISA kit (R&D system, Minneapolis, MN, USA, #DRT200) with 1:10 dilution.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Reagent information used in this study.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Enzyme-linked Immunosorbent Assay, Magnetic Beads, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Primer sequences for qPCR.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques:

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Tumor cell-derived CCL16 mediates the recruitment and M2 polarization of macrophages in the liver cancer microenvironment. (A) Expression of CCL16 and CCR1 in different cell types at the single-cell level. (B) Expression levels of CCL16 in different liver cancer cell lines from the CCLE database. (C) mRNA expression of CCL16 in different liver cancer cell lines detected by qPCR. (D) Validation of CCL16 knockdown in HEPG2 cell line. (E) Validation of CCL16 overexpression in SNU761 cell line. (F) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 knockdown HEPG2 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of HEPG2 cells. (G) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 overexpressing SNU761 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of SNU761 cells. (H) Schematic diagram illustrating the cell co-culture system and the macrophage migration assay. (I) Flow cytometry analysis to detect the proportion of M2-polarized cells after co-culture of THP1 cells with CCL16 knockdown or overexpressing tumor cells. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test. ns, not significant; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Derivative Assay, Expressing, Over Expression, Transwell Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Co-Culture Assay, Migration, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: The recruitment of tumor-associated macrophages mediated by CCL16 depends on the macrophage receptor CCR1. (A) Immunoprecipitation assay to detect the interaction between Flag-CCL16 and CCR1, CCR2, CCR5, CCR8 in THP1 cell culture medium. (B) Immunofluorescence assay to detect the co-localization of Flag-CCL16 and CCR1 in THP1 cells after the addition of Flag-CCL16. Scale bar = 20 μm. (C) qPCR validation of CCR1 knockdown in THP1 cells. (D) Transwell assay to evaluate the cell migration ability of CCR1 knockdown THP1 cells co-cultured with HEPG2 cells. (E) Recruitment of THP1 cells by control or 5μM BX471-treated CCL16 overexpressing SNU761 cells after 24 hours. (F) Recruitment of THP1 cells by CCL16 overexpressing SNU761 cells after CCR1 knockdown. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test or two-way ANOVA was used for statistical analysis. ns, not significant; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Immunoprecipitation, Cell Culture, Immunofluorescence, Transwell Assay, Migration

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Clinical characteristics of patients with high and low expression of CCL16.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: High expression of CCL16 is positively correlated with the infiltration of M2 macrophages and the expression of CCR1 in clinical tissues. (A) Immunohistochemistry representative images and Pearson correlation analysis of CCL16 with CCR1, CD68, and CD206 in liver cancer patient samples, as well as the Mean Optical Density (MOD) values. Scale Bar = 100μm. (B) Immunofluorescence detection of CCR1+ macrophage infiltration. Scale Bar = 20μm. Statistical analysis of the difference in CD68+CCR1+ cell numbers between high and low CCL16 expression groups using unpaired Student’s t-test. **: P < 0.01. Pearson correlation analysis of CCL16 expression and CCR1+ macrophage infiltration, r = 0.5743, P < 0.001.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Reagent information used in this study.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, Magnetic Beads, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Primer sequences for qPCR.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques:

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Tumor cell-derived CCL16 mediates the recruitment and M2 polarization of macrophages in the liver cancer microenvironment. (A) Expression of CCL16 and CCR1 in different cell types at the single-cell level. (B) Expression levels of CCL16 in different liver cancer cell lines from the CCLE database. (C) mRNA expression of CCL16 in different liver cancer cell lines detected by qPCR. (D) Validation of CCL16 knockdown in HEPG2 cell line. (E) Validation of CCL16 overexpression in SNU761 cell line. (F) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 knockdown HEPG2 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of HEPG2 cells. (G) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 overexpressing SNU761 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of SNU761 cells. (H) Schematic diagram illustrating the cell co-culture system and the macrophage migration assay. (I) Flow cytometry analysis to detect the proportion of M2-polarized cells after co-culture of THP1 cells with CCL16 knockdown or overexpressing tumor cells. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test. ns, not significant; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Derivative Assay, Expressing, Over Expression, Transwell Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Co-Culture Assay, Migration, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: The recruitment of tumor-associated macrophages mediated by CCL16 depends on the macrophage receptor CCR1. (A) Immunoprecipitation assay to detect the interaction between Flag-CCL16 and CCR1, CCR2, CCR5, CCR8 in THP1 cell culture medium. (B) Immunofluorescence assay to detect the co-localization of Flag-CCL16 and CCR1 in THP1 cells after the addition of Flag-CCL16. Scale bar = 20 μm. (C) qPCR validation of CCR1 knockdown in THP1 cells. (D) Transwell assay to evaluate the cell migration ability of CCR1 knockdown THP1 cells co-cultured with HEPG2 cells. (E) Recruitment of THP1 cells by control or 5μM BX471-treated CCL16 overexpressing SNU761 cells after 24 hours. (F) Recruitment of THP1 cells by CCL16 overexpressing SNU761 cells after CCR1 knockdown. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test or two-way ANOVA was used for statistical analysis. ns, not significant; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Immunoprecipitation, Cell Culture, Immunofluorescence, Transwell Assay, Migration

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Clinical characteristics of patients with high and low expression of CCL16.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: High expression of CCL16 is positively correlated with the infiltration of M2 macrophages and the expression of CCR1 in clinical tissues. (A) Immunohistochemistry representative images and Pearson correlation analysis of CCL16 with CCR1, CD68, and CD206 in liver cancer patient samples, as well as the Mean Optical Density (MOD) values. Scale Bar = 100μm. (B) Immunofluorescence detection of CCR1+ macrophage infiltration. Scale Bar = 20μm. Statistical analysis of the difference in CD68+CCR1+ cell numbers between high and low CCL16 expression groups using unpaired Student’s t-test. **: P < 0.01. Pearson correlation analysis of CCL16 expression and CCR1+ macrophage infiltration, r = 0.5743, P < 0.001.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence