Journal: PLoS ONE

Article Title: LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway

doi: 10.1371/journal.pone.0263997

Figure Lengend Snippet: A, B: the expression of HOTAIR and CCL22 mRNA were detected by RT-qPCR. C: CCL22 protein analyzed by ELISA assay. ****, P<0.001.

Article Snippet: The concentration of CCL22 in the supernatant was tested according to the instructions of ELISA kit operation (CUSABIO, CSB-E04660h).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway

doi: 10.1371/journal.pone.0263997

Figure Lengend Snippet: A: The HOTAIR and CCL22 mRNA expression examined by RT-qPCR B: Cell apoptosis detected by Flow cytometry. C: The cell proliferation tested by MTS assay. D: The CCL22 protein expression tested by ELISA. E: The cell migration and invasion detected by Transwell assay. **, P<0.05; ***, P<0.01; ****, P<0.001;ns, no significance.

Article Snippet: The concentration of CCL22 in the supernatant was tested according to the instructions of ELISA kit operation (CUSABIO, CSB-E04660h).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, MTS Assay, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay

Journal: Scientific Reports

Article Title: Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer

doi: 10.1038/srep35855

Figure Lengend Snippet: ( a ) Fucoidan modulates CC chemokines transcription of THP-1-derived macrophages during the polarization process. The expression of selected CC chemokines in non-treated and fucoidan-treated M0/M1/M2-like macrophages were assessed by quantitative real-time PCR and expressed as a fold change compared with M0 macrophages. ( b ) Fucoidan modulates CCL22 secretion level of THP-1-derived macrophages during the polarization process. After 48 h polarization, the conditioned media created by non-treated and fucoidan-treated M0/M1/M2-like macrophages were collected. ELISA was then used on the various conditioned media to measure the levels of secreted CCL22. ( c , d ) CCL22 transcription ( c ) and concentration in culture media ( d ) of peripheral blood monocyte-derived macrophages. ( e , f ) CCL22 transcription ( e ) and concentration ( f ) in non-treated and fucoidan-treated polarized M2 macrophages exposure to IL-4 and/or IL-13. The quantitative real-time PCR data expressed as relative fold change compared with control (polarized M2 macrophages). The value represent means ± SD, n = 3: *P < 0.05, **P < 0.01.

Article Snippet: Recombinant human CCL22, M-CSF, IFN-γ, IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer

doi: 10.1038/srep35855

Figure Lengend Snippet: ( a ) Diagram showing the protocol for MHCC-97 migration assay and the PBMC/CD4 + lymphocytes recruitment assay. Upper: Supernatants of THP-1-derived M2 macrophages under non- or fucoidan pretreatment were collected and add into lower chamber of transwell with or without neutralizing anti-CCL22 antibody or recombine human CCL22, respectively. Down left: MHCC-97H were seeded in upper chamber. Transwelled cells were stained after 24 h and counted. Down right: PBMC (I) from healthy human peripheral blood separated by lymphocyte separation liquid or microbeads selected CD4 + lymphocytes (II) were used as upper chamber components of transwell. Transwelled cells were collected after 6h, and flow cytometry was used to detect the cellular components. FCM: flow cytometry. ( b ) Migration assays were carried out in MHCC-97H cells by using transwell chamber assay. Migrating cells located on the lower surface were fixed in methanol and stained with eosin. The scale bar represents 50 μm. The graph represents fold change. ( c ) Flow cytometry settings used to detect human CD3 + CD4 −/+ cells among PBMC recruited to lower chamber contained non-treated and fucoidan-treated THP-1-derived M2 macrophages supernatants from donor human PBMC seeded in the upper chambers (left). %CD3 + CD4 + and %CD3 + CD4 − cells are the percentage of CD3 + CD4 + or CD3 + CD4 − cells among recruited PBMC (right). ( d , e ) Flow cytometry settings used to detect human CD25 + FoxP3 + (Treg) cells among CD4 + lymphocytes recruited to the bottom chamber filled with conditioned M2 supernatants as tumor cell migration assay. Graphs showed the total numbers of CD4 + lymphocytes ( d ) and the flow cytometry (left) and percentage (right) of CD25 + FoxP3 + cells among recruited CD4 + lymphocytes ( e ). Control group: the bottom chamber filled with M2 supernatants. The value represent means ± SD, n = 3: *P < 0.05, **P < 0.01 versus the control.

Article Snippet: Recombinant human CCL22, M-CSF, IFN-γ, IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Derivative Assay, Staining, Flow Cytometry, Transwell Chamber Assay, Cell Migration Assay, Control

Journal: Scientific Reports

Article Title: Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer

doi: 10.1038/srep35855

Figure Lengend Snippet: ( a , b ) THP-1-derived M2 macrophages were treated with the NF-κB inhibitor BAY11-7082, the PI3K inhibitor Wortmannin or the p38-MAPK inhibitor SB203580 for 24 h. The cells and culture supernatants were collected. CCL22 transcription ( a ) and concentration ( b ) were measured by quantitative real-time PCR and ELISA, respectively. ( c ) M2 macrophages derived from THP-1 cells were pretreated with the Wortmannin or the SB203580 for 30 min, followed by incubation with or without Fucoidan for 1 h. Whole-cell lysates were analyzed by western blot using the appropriate antibodies. The original blots are presented in . Densitometry ratios of phospho-p65-NF-κB, AKT and p38-MAPK were normalized to β-actin levels. The cells treated with DMSO were used as control. The value represent means ± SD, n = 3. *P < 0.05, **P < 0.01 versus the control. ( d ) p65-NF-κB immunofluorescence of THP-1-derived M0 and M2 polarized macrophages, which were treated as described in ( c ). Green (anti-p65-NF-κB) indicates p65-NF-κB distribution, and blue indicates the location of nucleus. The scale bar represents 10 μm.

Article Snippet: Recombinant human CCL22, M-CSF, IFN-γ, IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Control, Immunofluorescence

Journal: Scientific Reports

Article Title: Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer

doi: 10.1038/srep35855

Figure Lengend Snippet: Green arrow represents activation; truncated red line, inhibition. The dotted lines represent the non-critical effect of AKT and p38-MAPK on fucoidan-mediated downregulation of CCL22.

Article Snippet: Recombinant human CCL22, M-CSF, IFN-γ, IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Inhibition