Journal: Frontiers in Oncology

Article Title: AXL Overexpression in Tumor-Derived Endothelial Cells Promotes Vessel Metastasis in Patients With Hepatocellular Carcinoma

doi: 10.3389/fonc.2021.650963

Figure Lengend Snippet: AXL/SOX2/DKK-1 axis in HUVECs promotes HCC metastasis. (A) DKK-1 and CCL14 secretion was significantly downregulated in CM from HUVEC-AXL-KD compared with that from HUVEC-AXL-NC, as detected with a human cytokine antibody array. (B) DKK-1 and CCL14 expression was markedly upregulated in the CM of HUVECs overexpressing AXL (CCL14: p < 0.001; DKK-1: p = 0.003) compared with the CM of HUVEC-AXL-NC, as detected by ELISA assay. (C) AXL siRNA downregulated DKK-1 and CCL14 secretion in the CM of HUVEC-AXL-NC and HUVEC-AXL-OE cells (CCL14: p < 0.001 and p < 0.001; DKK-1: p < 0.001 and p < 0.001). (D) DKK1 siRNA (MHCC-97L: p < 0.001 and p < 0.001; HCC-LM3: p <0.001 and p < 0.001), but not CCL14 siRNA (MHCC-97L: p = 0.126 and p = 0.711; HCC-LM3: p = 0.901 and p = 0.694) could attenuate the effect of the CM from HUVEC-AXL-NC and HUVEC-AXL-OE cells on the migration of HCC-LM3 cells and MHCC-97L cells. (E) SOX2 mRNA expression was significantly increased in HUVEC-AXL-OE cells and decreased in HUVEC-AXL-KD cells compared with HUVEC-AXL-NC cells (HUVEC-AXL-KD: p < 0.001, HUVEC-AXL-OE: p < 0.001). (F) AXL overexpression could significantly increase SOX2 and DKK-1 protein expression in HUVEC-AXL-OE cells compared with HUVEC-AXL-NC cells, and SOX2 siRNA inhibited SOX2 and DKK-1 protein expression in HUVEC-AXL-OE and HUVEC-AXL-NC cells.

Article Snippet: The protein concentrations of CCL14 and DKK-1 in the supernatants were also measured using an enzyme-linked immunosorbent assay (ELISA) kit (CCL14: EK1123 Boster, Wuhan, China; DKK-1: EK0867 Boster, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Ab Array, Expressing, Enzyme-linked Immunosorbent Assay, Migration, Over Expression

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: Cells were also treated with different concentrations of recombinant CCL16 (R&D systems, Minneapolis, MN, USA, # 802-HC-025) or recombinant sTNF-RII (MyBioSource, MBS343136, London, UK).

Techniques: Clinical Proteomics, 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: Cells were also treated with different concentrations of recombinant CCL16 (R&D systems, Minneapolis, MN, USA, # 802-HC-025) or recombinant sTNF-RII (MyBioSource, MBS343136, London, UK).

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

Journal: Oncotarget

Article Title: Chemokines CCL2, 3, 14 stimulate macrophage bone marrow homing, proliferation, and polarization in multiple myeloma

doi:

Figure Lengend Snippet: A. qPCR analysis of expression of different MO chemokines in MM patient BM cells. The value indicates the relative expression to GAPDH. One representative sample of 4 patient samples analyzed is shown. B. Levels of CCL3, CCL14, and CCL2, measured by ELISA, in BM plasma of healthy donors (CTR) and patients with MM or MGUS. The numbers of CTR, MGUS and MM patients used for measuring CCL2 are 4, 10, and 23, respectively; for CCL14 are 7, 10, and 11, respectively; and for CCL3 are 7, 10, and 13, respectively. C. Immunohistochemistry analysis of CCL3, CCL14, and CCL2 expression in BM biopsies of 2 healthy donors (CTR1 and CTR2) and 2 representatives (MM1 and MM2) out of five MM patients. D. Linear regression analysis of the relationship between the percentage of BM MΦs and concentration of chemokines CCL14 ( n = 20) and CCL3 ( n = 18) in BM plasma in MM patients. * p < 0.05.

Article Snippet: Neutralizing antibodies to human CCL3 (MAB270), CCL14 (MAB3241) and CCL2 (MAB679), and mouse CCL3 (AB-450-NA), CCL2 (AB-479-NA) were purchased from R&D Systems.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Immunohistochemistry, Concentration Assay

Journal: Oncotarget

Article Title: Chemokines CCL2, 3, 14 stimulate macrophage bone marrow homing, proliferation, and polarization in multiple myeloma

doi:

Figure Lengend Snippet: A. qPCR analysis of CCL3, CCL14, and CCL2 expression in CD138 + primary MM cells and CD138 − non-malignant cells in BM aspirates from 3 different MM patients (MM1 to MM3). The y-axis indicates the fold change relative to GAPDH value. B. Concentration of chemokines secreted by 4 human MM cell lines. MM cells (2 ×10 5 cell per well) were cultured in vitro for 24 hours; then the chemokine expression in culture supernatant was analyzed by ELISA. C. BMSCs were cocultured with ARP-1 or MM.1S MM cells for 48 hours. CCL3, CCL14, and CCL2 expression in BMSCs was analyzed by qPCR. D. BMSCs were cultured in transwells with MM cells ARP-1 for 48 hours, followed by removal of MM cells and cultured alone in fresh medium for 24 hours. BMSCs from the same donors were cultured alone in parallel as controls. Chemokine concentrations were determined by ELISA using culture supernatants collected at the end of culture. * p < 0.05, ** p < 0.01.

Article Snippet: Neutralizing antibodies to human CCL3 (MAB270), CCL14 (MAB3241) and CCL2 (MAB679), and mouse CCL3 (AB-450-NA), CCL2 (AB-479-NA) were purchased from R&D Systems.

Techniques: Expressing, Concentration Assay, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Chemokines CCL2, 3, 14 stimulate macrophage bone marrow homing, proliferation, and polarization in multiple myeloma

doi:

Figure Lengend Snippet: A. nMΦ or mMΦ were generated as described in the Methods. The cells were then cultured in vitro in the presence of each of the chemokine neutralizing antibodies (αCCL3, αCCL14 and αCCL2, at a final concentration of 10 μg/ml each), and their combination (MIX). An equal amount of IgG was used as control. Cell proliferation at different time points was examined by MTS. B. nMΦs were cultured for 3 days in the presence of each of the recombinant chemokines (final concentration of 1 μg/ml) or their combination (MIX). mMΦs cultured in medium alone were used as a positive control. Cell proliferation at different time points was determined by MTS assay. C. Western blot showing the levels of p27Kip1 and cyclin D1 in nMΦs and mMΦs from 2 healthy donors (Do1 and Do2). D. Western blot showing the levels of p27Kip1 and cyclin D1 in nMΦs and mMΦs. mMΦs were generated in coculture with MM cells in transwells in the absence (CTR) or presence of control IgG or a combination of the chemokine neutralizing antibodies (MIX). E. Western blot showing the levels of different kinases, IL-6, c-myc, cyclin D1, and p27Kip1 in nMΦs in overnight cultures in medium or with addition of each of the chemokines CCL2, CCL14, or CCL3 individually or all three combined (MIX). mMΦs were used as a positive control. F. Proliferative response of mMΦs in medium alone (CTR) or in the presence of the PI3K-Akt inhibitor LY294002 (LY; 50 μM) or Erk1/2 inhibitor U0126 (UO; 2 μM). * p < 0.05, ** p < 0.01.

Article Snippet: Neutralizing antibodies to human CCL3 (MAB270), CCL14 (MAB3241) and CCL2 (MAB679), and mouse CCL3 (AB-450-NA), CCL2 (AB-479-NA) were purchased from R&D Systems.

Techniques: Generated, Cell Culture, In Vitro, Concentration Assay, Control, Recombinant, Positive Control, MTS Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Comparison of mRNA levels for selected chemokines in the endometrium of pregnant vs. non-pregnant cows as determined by microarray analysis (Fold > 2.0; p < 0.05).

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Comparison, Microarray

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Changes in relative amounts of mRNA for ( a ) CCL2, ( b ) CCL8, ( c ) CCL11, ( d ) CCL14, ( e ) CCL16, and ( f ) CXCL10 in the endometrium at days 15 and 18 of non-pregnant cows (NP) and pregnant cows (P). Data are means ± SEM of four cows per stage and are expressed as relative ratios of the mRNAs to SUZ12 polycomb repressive complex 2 subunit (SUZ12). p -Values show significant differences between NP and P.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Localization of CCR1 (binds to CCL8, CCL14, and CCL16), CCR2 (binds to CCL2, CCL8, and CCL16), CCR3 (binds to CCL11), and CXCR3 (binds to CXCL10) in the bovine endometrium and fetal trophoblast obtained from cows in their 18th day of pregnancy. Intensive immunoreactivity was observed in endometrial epithelial cells, glandular epithelial cells, or fetal trophoblast. No positive immunoreactivity was observed in the negative control (Control). Scale bar = 50 µm.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Negative Control, Control

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Effects of the supernatant derived from homogenized fetal trophoblast (FMP; 200 ng/mL) and interferon-τ (IFNT; 100 ng/mL) on the mRNA expression of ( a ) CCL2, ( b ) CCL8, ( c ) CCL11, ( d ) CCL14, ( e ) CCL16, and ( f ) CXCL10 in cultured bovine endometrial tissues. Homogenization buffer was added at the control group. Data are means ± SEM of five cows and are expressed as relative ratios of the mRNAs to SUZ12. p -Values show significant differences between treated group and control group.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Derivative Assay, Expressing, Cell Culture, Homogenization, Control

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Effects of CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10 (50 ng/mL each) on the mRNA expression of ( a ) interferon-stimulated gene 15 (ISG15), ( b ) myxovirus-resistance gene 1 (MX1), ( c ) cyclooxygenase 2 (COX2), ( d ) oxytocin receptor (OTR), and ( e ) estrogen receptor α (ESR1) in cultured bovine endometrial tissues. Data are means ± SEM of five cows and are expressed as relative ratios of the mRNAs to SUZ12. p -Values show significant differences between treated group and control group.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Primers used in real-time PCR.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Hypothetical model for inhibition of luteolysis by IFNT and chemokines. Although this model is not concerned with the effects of steroids or growth factors, IFNT, CCL2, CCL8, CCL16, CXCL10, and LIF may block TNF-stimulated-COX2 expression in bovine endometrial cells, leading to the reduction of TNF-induced PGF2α output from the cells. Furthermore, IFNT and CCL16 may stimulate anti-viral activity by up-regulating ISG15 and MX1 expression at the time of maternal recognition in cows. Red and blue arrows show stimulatory and inhibitory actions of each substance, respectively. IFNT may stimulate both CCL8 and CXCL10 production and inhibit CCL14 production from bovine endometrium. Effects of CCL11 on bovine endometrial function are still unclear, although its receptor (CCR3) is expressed in the endometrial epithelial cells.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Inhibition, Blocking Assay, Expressing, Activity Assay

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: CCL16 expression in the liver occurs homeostatically. CCL16 mRNA was quantified in normal human liver tissue ( n = 4) in comparison to livers with cirrhosis ( n = 7), hepatitis B virus (HBV) and hepatitis C virus (HCV) infection ( n = 4–5), as well as cholangiocarcinoma (CCC; n = 7) and hepatocellular carcinoma (HCC; n = 7). Values are expressed as relative units (RU) normalised to 18S. Measurements of individual samples and the mean of each group are shown. A Mann–Whitney U test was performed to assess statistical significance (* p < 0.05).

Article Snippet: Normal human liver RNA samples ( n = 4) were commercially available (Clontech; AMS Biotechnology, Abingdon, United Kingdom); diseased liver tissues with liver cirrhosis, hepatitis B virus (HBV) infection with and without tumour, hepatitis C virus (HCV) infection with and without tumour, cholangiocarcinoma (CCC), and hepatocellular carcinoma (HCC) were obtained from the Universitätstumorzentrum (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany).

Techniques: Expressing, Infection, MANN-WHITNEY