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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Distinct roles for CCR4 and CXCR3 in the recruitment and positioning of regulatory T cells in the inflamed human liver.

doi: 10.4049/jimmunol.0901216

Figure Lengend Snippet: FIGURE 6. CCR4 ligands are detected in diseased liver tissue and secreted by LIDCs. A, LITregs migrate toward CCR4 ligands in vitro. Migration of LITregs to CCR4 ligands CCL22 and CCL17 was assessed using fibronectin-coated 5-mm-pore transwell migration chambers. The lower chambers were filled with 600 ml assay media (RPMI 1640 medium with 0.1% BSA) alone or supplemented with chemokines (100 ng/ml recombinant human CCL22 and CCL17). BSA was used as control. A total of 5 3 105 Tregs isolated from inflamed livers (n = 4) were loaded per well in the upper chamber; Tregs were collected from the top and bottom chambers after 4 h of incubation at 37˚C in 5% CO2. The proportion of transmigrated cells was calculated by fixed- volume counting and phenotyped for Treg markers and chemokine receptor expression by flow cytometry. To inhibit chemokine-mediated signaling, Tregs were incubated with PTx (100 ng/ml) prior to the assay. Results are expressed as mean 6 SEM. LITregs showed significant migration responses to CCL22 and CCL17 compared with migration to BSA, and these were inhibited by PTx. pp , 0.05, Student t test. B, To determine whether liver DCs secrete CCL17 and CCL22, we isolated and cultured myeloid DCs from human liver tissue and stimulated them with LPS (1 mg/ml) for 24 h. CCL17 and CCL22, measured by ELISA, were detected in cell culture supernatants of LPS-stimulated liver-derived DCs isolated from patients with AIH (n = 2) or PBC (n = 1) or from NL (n = 3). All DCs secreted detectable CCL17 and CCL22, with a .2-fold increase in secretion by DCs derived from inflamed liver tissue compared with NL. Data represent mean 6 SEM of three experiments using supernatants from different DCs. Values represent the chemokine concentration (pg/ml) of three replicate wells relevant to a standard curve of known concentration. pp # 0.05; ppp # 0.01. C, Expression of CCL22 in human liver tissue samples by immunohistochemistry. CCL22 staining (i) was detected on LIDCs within inflammatory infiltrates (positive staining cells brown) in a patient with AIH. No detectable CCL22 was found in NL or when control Abs were used (ii). (Original magnification 3200.) D, Dual staining of FoxP3-expressing cells (brown) and CCL22 (red) in liver tissue from a patient with AIH reveals CCL22+ cells in close association with FoxP3+ Tregs within an inflammatory infiltrate (original magnification 3200). E, Confocal image of dual staining of FoxP3-expressing cells (red/TRITC) and CD11c (green/FITC) in liver tissue from a patient with AIH reveals CD11c+ cells in close association with FoxP3+ Tregs within an inflammatory infiltrate (original magnification 340). F, Colocalization of DCs and CCL22 in inflamed liver tissue. Serial liver sections from a patient with PBC were stained with CD11c (top panels) and CCL22 (bottom panels). Cells that coexpress CD11c and CCL22 can be determined by comparing staining of CD11c (top right panel) and CCL22 (bottom right panel) at higher power (original magnification 3200). Staining with isotype-matched control Abs was negative (left panels, original magnification 3100.) G, Colocalization of DCs and CCL22 in inflamed liver tissue. Staining of FITC-labeled CCL22 (green) with Texas Red-labeled CD11c (red). Areas of coexpression (top left panel) appear yellow. Nuclei were stained with DAPI (blue). Control Abs demonstrated minimal background tissue fluorescence. The findings are representative of the pattern of immunofluorescence staining seen in tissue sections from four donors each for PBC, ALD, AIH, and HCV

Article Snippet: The following primary Abs were used for immunohistochemistry, immunofluorescence, and confocal microscopy: mouse anti-human CCL22 mAb (MAB 336, R&D Systems, Minneapolis, MN), goat anti-human CCL17 mAb (AF 364, R&D Systems), mouse anti-human FoxP3 (Abcam, 236A/ E7, Cambridge, U.K.), rabbit anti-human polyclonal to CCL22 amino terminal end (Ab 53002-100; Abcam), rabbit anti-human monoclonal to CD11c (Abcam, EP13474), rabbit monoclonal to phosphorylated STAT5 (phosphoSTAT5)(Abcam/E208), mouse anti-human CD4 IgG 2b (eBioscience, Hatfield, U.K.), anti-human CD3 IgG2a (eBioscience), and mouse anti-human CD8 IgG2a (eBioscience).

Techniques: In Vitro, Migration, Recombinant, Control, Isolation, Incubation, Expressing, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Immunohistochemistry, Staining, Labeling