Journal: Oncotarget

Article Title: Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22–CCR4 axis

doi: 10.18632/oncotarget.14185

Figure Lengend Snippet: A . Prostate cancer cells are placed in transwell inserts and treated with CCL2 (0–30 ng/ml). After 24-h incubation, PC-3 and DU145 cells that had migrated through the membrane are stained. The mean OD value is read using a microreader at 595 nm. Migration of LNCaP cells is assessed with a wound-healing assay. Data are presented as mean ± SD. B . DU145 cells are co-cultured with THP-1 and U937 cells and treated with CCL2 (30 ng/ml) for 24 h, CM is collected, and CCL17 and CCL22 levels are analyzed using ELISA. The mean OD value is read using a microreader at 450 nm, and data are presented as mean ± SD. C, D . Total RNA and protein are extracted from prostate cancer cells, and CCR2 and CCR4 gene and protein expression levels are analyzed using PCR (C) and western blot (D). E . Prostate cancer cells (1.0 × 10 5 cells/well) are seeded into 6-well plates and cultured until they reach 60%–70% confluence. Cells are incubated with anti-CCR2 or anti-CCR4 antibody and detected using a second antibody conjugated with FITC (green). Cells are counterstained with 4',6-diamidino-2-phenylindole (blue). Adjustments of brightness, contrast, and size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. All experiments are performed in triplicate, and mean values are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Membrane, Staining, Migration, Wound Healing Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22–CCR4 axis

doi: 10.18632/oncotarget.14185

Figure Lengend Snippet: A . Prostate cancer cells are treated with CCL2 (30 ng/ml) for 24 h, and western blot for CCR2 is performed. B . Prostate cancer cells are treated with CCL2, CCL17, and CCL22 (30 ng/ml) for 24 h, and western blot for CCR4 is performed. C . Prostate cancer cells are co-cultured with U937 or U937-M cells for 24 h, and western blot for CCR4 is performed. D . Prostate cancer cells are placed in transwell inserts and treated with CCL17 and CCL22 (0–30 ng/ml). After 24-h incubation, PC-3 and DU145 cells that had migrated through the membrane are stained. The mean optical density (OD) value is read using a microreader at 595 nm. Migration of LNCaP cells is assessed with a wound-healing assay. Adjustments of brightness, contrast, and size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. Data are presented as mean ± SD. All experiments are performed in triplicate, and mean values are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Cell Culture, Incubation, Membrane, Staining, Migration, Wound Healing Assay

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22–CCR4 axis

doi: 10.18632/oncotarget.14185

Figure Lengend Snippet: Prostate cancer cells are incubated with a CCR2 antagonist (CCR2 ant; 20 μg/ml) or CCR4 antagonist (CCR4 ant; 10 μg/ml) for 30 min and then incubated with CCL2 (10–30 ng/ml), CCL17 (10–30 ng/ml), CCL22 (10–30 ng/ml), or CM of U937 or U937-M cells for 24 h for PC-3 and DU145 and 48 h for LNCaP. The mean optical density (OD) value is read using a microreader at 595 nm. Data are presented as mean ± SD. All experiments are performed in triplicate, and mean values are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22–CCR4 axis

doi: 10.18632/oncotarget.14185

Figure Lengend Snippet: A . Phosphorylation of Akt (Ser473) proteins in prostate cancer cells is assayed using western blot at 30 min after CCL2, CCL17, or CCL22 stimulation (left panels) and after CCR4 antagonist treatment with CCL2, CCL17, or CCL22 stimulation (right panels). B . U937 cell-induced phosphorylation of Akt (Ser473) proteins in prostate cancer cells with or without CCR2 and CCR4 antagonists is also assayed using western blot. Adjustments of brightness, contrast, and size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. C . PC-3 cells are incubated with the Akt inhibitor AZD5363 (10 μg/ml) with CCL17 (30 ng/ml) and CCL22 (30 ng/ml) for 24 h. The mean optical density (OD) value is read using a microreader at 595 nm. Data are presented as mean ± SD. All experiments are performed in triplicate, and mean values are shown. ** p < 0.01. All experiments are performed in triplicate, and representative data are shown.

Article Snippet: Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Western Blot, Incubation

Journal: Nature immunology

Article Title: STAT5 is critical in Dendritic Cells for development of T H 2- but not T H 1-dependent Immunity

doi: 10.1038/ni.2541

Figure Lengend Snippet: Representative of 2 experiments with 5 mice per group. STAT5 is required for TSLP-induced costimulatory molecule upregulation in FL-CD11b-DCs. ( a ) Phospho-Flow of indicated STAT proteins in three FL-DC populations, in triplicate, following 15 min incubation with 50ng/ml TSLP from WT (Cre − 5 fl/fl ) and Cre + 5 fl/fl BMDCs. Three independent experiments. ( b ) Immunoblot of total-STAT5 and –STAT3 in FL-DC populations. ( c ) pSTAT5 phospho-flow of WT and Cre + 5 fl/fl FL-CD11b-DC following 15 min incubation with TSLP. ( d ) Costimulatory molecule expression in TSLP-treated and –untreated FL-CD11b-DCs. Numbers represent MFI values. ( e ) Fold-change of TSLP-treated versus untreated FL-CD11b-DCs from WT, Cre + 5 fl/fl , STAT-1, -4, and -6 KO bone marrow in triplicate, 4 independent experiments. * = .01, ** = .0005, *** = .05( f ) Fold change of costimulatory molecule upregulation in WT (black bar) and Cre + 5 fl/fl (white bar) following LPS stimulation in triplicate. ( g ) Expression of CCL17 in FL-CD11b-DCs from e . * = .00003.

Article Snippet: The production of CCL17 was measured using a mouse CCL17 ELISA Kit (R&D Systems), according to the manufacturer’s protocol.

Techniques: Incubation, Western Blot, Expressing