Journal: Science Advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: ( A ) Percentage of CD25 hi Foxp3 + T reg cells [fluorescence-activated cell sorting (FACS)] in human PBMC cultures treated with IL-12a C96S (20 ng/ml) and EBI3 (20 ng/ml) or the combination of both IL-12a C96S and EBI3 ( n = 8). Data are presented as means + SEM. Statistical significance was determined by Friedman test. * P < 0.05 and ** P < 0.01. ( B ) Concentrations of IL-4 (ELISA) in culture supernatants from SEA-stimulated human PBMCs alone or in combination with IL-12 C96S or EBI3 ( n = 8 donors). The dotted line indicates mean secretion of IL-4 from PBS-treated PBMCs. ( C ) Concentrations of CCL17 (ELISA) in culture supernatants from IL-4–stimulated human PBMCs alone or in combination with IL-12 C96S or EBI3 ( n = 3 donors). (B and C) Data are presented as individual values. Donor-dependent effect is shown by the connecting line. Statistical significance was determined by Wilcoxon test. * P < 0.05 and ** P < 0.01.

Article Snippet: PBMC supernatants were analyzed for IL-4 using the IL-4 Human ELISA Kit from Thermo Fisher Scientific (KHC0041) and for CCL17 using the human CCL17/TARC DuoSet ELISA Kit from R&D systems (DY364).

Techniques: Fluorescence, FACS, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: CCL17/TARC and CCR4 expression in Merkel cell carcinoma

doi: 10.18632/oncotarget.25836

Figure Lengend Snippet: Cells were transfected with a luciferase reporter vector driven by CCL17/TARC promoter fragments spanning nucleotides -2535/+ 40, -1084/+ 40 or -375/+40, respectively. Expression plasmid for full-length LT (FLTA), MKL-1, MKL-2 or MS-1 LT, or pcDNA3 control, were co-transfected. Luciferase activity was assessed after an overnight cultivation of transfected cells. The promoter activity in presence of pcDNA3 was arbitrary set as 1.0 and the promoter activity measured in the presence of LT was related to this. Each bar represents the average of three independent parallels +SD. The experiment was repeated two more times, and similar results were obtained. Luciferase values were normalized with a total protein in each sample. P * ≤ 0.05, P ** ≤ 0.01, P *** ≤ 0.001 and P **** ≤ 0.0001.

Article Snippet: The empty expression plasmid pcDNA3.1(+) was purchased from Invitrogen. pcDNA3-full large T-antigen (pcDNA6.MCV.cLT206.V5_CM2B4) was purchased from Addgene (Cambridge, MA, USA), while pcDNA3-MKL-1 large T-antigen, pcDNA3-MKL-2 large T-antigen and pcDNA3-MS-1 large T-antigen were constructed by site-directed mutagenesis using the original pcDNA6.MCV.cLT206.V5_CM2B4 plasmid. pCMV2-CCL17-flag (cat.# HG10233-M-F) expression plasmid was purchased from SinoBiological (Beijing, China).

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Activity Assay

Journal: Oncotarget

Article Title: CCL17/TARC and CCR4 expression in Merkel cell carcinoma

doi: 10.18632/oncotarget.25836

Figure Lengend Snippet: (A) MCC13 cells were seeded, and after 24 hrs the cells were serum starved. After serum starvation for 24 hrs, the cells were co-transfected with either CCL17/TARC expression plasmid or empty expression vector pcDNA3.1, and luciferase reporter plasmid containing either the MCPyV early or the late promoter. Luciferase activity was measured 24 hrs after transfection. The activity of the MCPyV early (respectively late) promoter in the presence of pcDNA3 was arbitrary set as 1.0 and the activity in the presence of CCL17 was related to this. (B) Cells were transfected as in (A), and were then exposed to rhCCL17/TARC (12ng/ml) or vehicle (PBS) for 4 hrs with a subsequent analysis of luciferase activity. Each bar represents the average of three independent parallels +SD * P < 0.05, *** P < 0.001.

Article Snippet: The empty expression plasmid pcDNA3.1(+) was purchased from Invitrogen. pcDNA3-full large T-antigen (pcDNA6.MCV.cLT206.V5_CM2B4) was purchased from Addgene (Cambridge, MA, USA), while pcDNA3-MKL-1 large T-antigen, pcDNA3-MKL-2 large T-antigen and pcDNA3-MS-1 large T-antigen were constructed by site-directed mutagenesis using the original pcDNA6.MCV.cLT206.V5_CM2B4 plasmid. pCMV2-CCL17-flag (cat.# HG10233-M-F) expression plasmid was purchased from SinoBiological (Beijing, China).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: CCL17/TARC and CCR4 expression in Merkel cell carcinoma

doi: 10.18632/oncotarget.25836

Figure Lengend Snippet: MCC13 cells were transfected with an empty vector or expression plasmid for MCPyV full-length LT (FLTA), or truncated MKL-1, MKL-2, or MS-1 LT. (A) qRT-PCR analysis shows CCL17/TARC mRNA levels normalized with eukaryotic 18S rRNA levels. (B) CCL17/TARC protein levels analyzed by Western blotting. The uttermost right lane represents baseline expression of CCL17/TARC by MCC13 cells. (C) CCR4 protein levels analyzed by Western blotting. A Western blot with ERK2 antibodies was used as a loading control. (D) Representative figures of CCL17/TARC (B) and CCR4 (C) Western blots. The lane most to the left in the lower part contains the protein molecular mass marker (in kDa). Bars in (B) and (C) shows a densitometric scanning of the Western blot signals. P * ≤ 0.05 and P *** ≤ 0.001.

Article Snippet: The empty expression plasmid pcDNA3.1(+) was purchased from Invitrogen. pcDNA3-full large T-antigen (pcDNA6.MCV.cLT206.V5_CM2B4) was purchased from Addgene (Cambridge, MA, USA), while pcDNA3-MKL-1 large T-antigen, pcDNA3-MKL-2 large T-antigen and pcDNA3-MS-1 large T-antigen were constructed by site-directed mutagenesis using the original pcDNA6.MCV.cLT206.V5_CM2B4 plasmid. pCMV2-CCL17-flag (cat.# HG10233-M-F) expression plasmid was purchased from SinoBiological (Beijing, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Marker

Journal: Oncotarget

Article Title: CCL17/TARC and CCR4 expression in Merkel cell carcinoma

doi: 10.18632/oncotarget.25836

Figure Lengend Snippet: (A) PhosphoERK1/2 (pERK1/2) expression levels were determined by Western blot analysis using a phospho-specific antibody detecting Thr202/Tyr204 phosphorylation and compared to total ERK1/2 (tERK1/2) levels using an anti-pan-ERK1/2 antibody. MCC13 (1x10 5 ) cells were seeded in a 6-well plate. Cells were then serum-starved for 24 hrs, and thereafter stimulated with either PBS (control or C) or with 2.5, 5, 7.5, 10 and 15 ng/ml of rhCCL17/TARC for 45 min. (B) Densitometry scanning represent the expression of pERK1 (respectively pERK2) protein relative to GAPDH.

Article Snippet: The empty expression plasmid pcDNA3.1(+) was purchased from Invitrogen. pcDNA3-full large T-antigen (pcDNA6.MCV.cLT206.V5_CM2B4) was purchased from Addgene (Cambridge, MA, USA), while pcDNA3-MKL-1 large T-antigen, pcDNA3-MKL-2 large T-antigen and pcDNA3-MS-1 large T-antigen were constructed by site-directed mutagenesis using the original pcDNA6.MCV.cLT206.V5_CM2B4 plasmid. pCMV2-CCL17-flag (cat.# HG10233-M-F) expression plasmid was purchased from SinoBiological (Beijing, China).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: CCL17/TARC and CCR4 expression in Merkel cell carcinoma

doi: 10.18632/oncotarget.25836

Figure Lengend Snippet: (A) MCC13 (1x10 5 ) cells were seeded in a 6-well plate and were serum starved for 24 hrs. The cells were either pre-exposed to PBS, C021 dihydrochloride (+), a specific CCR4 receptor antagonist, or left untreated (-) for 15 min (15’) or 30 min (30’) as indicated. The cells were subsequently incubated with rhCCL17/TARC (15ng/ml) or left untreated (-) for another 45 min. Cell lysates were prepared and phosphoERK1/2 and total ERK1/2 levels were monitored. GAPDH levels were used as loading control. Densitometry represents the expression of pERK1 (respectively pERK2) protein relative to (B) total ERK (tERK1/2) and (C) GAPDH.

Article Snippet: The empty expression plasmid pcDNA3.1(+) was purchased from Invitrogen. pcDNA3-full large T-antigen (pcDNA6.MCV.cLT206.V5_CM2B4) was purchased from Addgene (Cambridge, MA, USA), while pcDNA3-MKL-1 large T-antigen, pcDNA3-MKL-2 large T-antigen and pcDNA3-MS-1 large T-antigen were constructed by site-directed mutagenesis using the original pcDNA6.MCV.cLT206.V5_CM2B4 plasmid. pCMV2-CCL17-flag (cat.# HG10233-M-F) expression plasmid was purchased from SinoBiological (Beijing, China).

Techniques: Incubation, Expressing

Journal: Oncotarget

Article Title: CCL17/TARC and CCR4 expression in Merkel cell carcinoma

doi: 10.18632/oncotarget.25836

Figure Lengend Snippet: (A) NFκB-p65 activation was determined by monitoring phosphorylation of p65 and p105. MCC13 cells (1x10 5 ) were seeded in a 6-well plate. Cells were serum starved for 24 hrs, and then stimulated with 2.5, 5, 7.5, 10 and 15 ng/ml of rhCCL17/TARC for 45 min. PBS was used as a control. Relative phospho p65 and phospho p105 were determined by western blot with phosphospecific antibodies. (B) Shows densitometry bars of western blot. (C) NFκB activity was measured by using a luciferase reporter plasmid containing a NFκB-responsive promoter. Cells were stimulated with 12ng/ml or 15ng/ml of rhCCL17/TARC for 4 hrs. Luciferase values were normalized with total protein. (D) The CCR4 receptor antagonist interferes with CCL17/TARC-induced activation of NFκB. Phospop65 and phosphor p105 levels were determined by western blot. MCC13 (1x10 5 ) cells were seeded in a 6-well plate and were serum starved for 24 hrs. The cells were pre-incubated with C021 dihydrochloride (0.3μM) for 30 min. The cells were then stimulated with rhCCL17/TARC (15ng/ml) in the presence or without CCR4 receptor antagonist for 45 min. DMSO was used as a control. (E) C021 ablates CCL17/TARC-induced activation of an NFκB-responsive promoter. Cells were transfected with the luciferase reporter plasmid with an NFκB responsive promoter and exposed to rhCCL17/TARC (15ng/ml) in the presence or without CCR4 receptor antagonist C012 (0.1 or 1 μM) for 45 min. DMSO was used as a control. Each bar represent the average of three independent parallels. Luciferase values were corrected for protein concentration of the samples. P * ≤ 0.05 and P ** ≤ 0.01. (F) CCL17/TARC stimulates proliferation of MCC13 cells. Cell were exposed to PBS or increasing concentrations of rhCCL17/TARC /12-100 ng/ml) and cell proliferation was measured after 24 and 48 hrs. Each bar represents the average of three independent parallels. (G) Cells were incubated for 45 min with 0.1 or 1 0μM C021 and rhCCL17/TARC (12 or 25 ng/ml) was subsequently added. Proliferation was monitored 24 hrs later. P * ≤ 0.05 and P ** ≤ 0.01.

Article Snippet: The empty expression plasmid pcDNA3.1(+) was purchased from Invitrogen. pcDNA3-full large T-antigen (pcDNA6.MCV.cLT206.V5_CM2B4) was purchased from Addgene (Cambridge, MA, USA), while pcDNA3-MKL-1 large T-antigen, pcDNA3-MKL-2 large T-antigen and pcDNA3-MS-1 large T-antigen were constructed by site-directed mutagenesis using the original pcDNA6.MCV.cLT206.V5_CM2B4 plasmid. pCMV2-CCL17-flag (cat.# HG10233-M-F) expression plasmid was purchased from SinoBiological (Beijing, China).

Techniques: Activation Assay, Western Blot, Activity Assay, Luciferase, Plasmid Preparation, Incubation, Transfection, Protein Concentration

Journal: Oncotarget

Article Title: CCL17/TARC and CCR4 expression in Merkel cell carcinoma

doi: 10.18632/oncotarget.25836

Figure Lengend Snippet: (A) HE, (B) CCL17/TARC, (C) CCR4, (D) CK20, (E) LTA and (F) Isotype control. The displayed images are representative stainings from a panel of MCC primary tumors (Scale bar= 500 μm).

Article Snippet: The empty expression plasmid pcDNA3.1(+) was purchased from Invitrogen. pcDNA3-full large T-antigen (pcDNA6.MCV.cLT206.V5_CM2B4) was purchased from Addgene (Cambridge, MA, USA), while pcDNA3-MKL-1 large T-antigen, pcDNA3-MKL-2 large T-antigen and pcDNA3-MS-1 large T-antigen were constructed by site-directed mutagenesis using the original pcDNA6.MCV.cLT206.V5_CM2B4 plasmid. pCMV2-CCL17-flag (cat.# HG10233-M-F) expression plasmid was purchased from SinoBiological (Beijing, China).

Techniques: