Journal: Journal of Biological Chemistry

Article Title: Tumor Microenvironment Macrophage Inhibitory Factor Directs the Accumulation of Interleukin-17-producing Tumor-infiltrating Lymphocytes and Predicts Favorable Survival in Nasopharyngeal Carcinoma Patients

doi: 10.1074/jbc.m112.367532

Figure Lengend Snippet: FIGURE 3. Characterization and cytokine expression profiles of the Th17 cell subset among PBMCs and TILs from NPC patients. A, T cell surface markers were detected in Th17 cells from NPC patients. T cells were stimulated with phorbol 12-myristate 13-acetate/ionomycin for 4 h, then stained with fluorescence- conjugated antibodies against the markers shown, and analyzed by flow cytometry. B, graph of the percentage of cytokine-secreting Th17 cells among PBMCs and TILs from NPC patients (n 5) and PBMCs from healthy donors (n 5). Error bars represent the S.E. Significance was determined by the 2 test or paired t test. (*, p 0.001).

Article Snippet: Cytokines in the culture medium were measured using the Human Cytokine Array Panel A (Proteome Profiler; R&D Systems).

Techniques: Expressing, Staining, Fluorescence, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Tumor Microenvironment Macrophage Inhibitory Factor Directs the Accumulation of Interleukin-17-producing Tumor-infiltrating Lymphocytes and Predicts Favorable Survival in Nasopharyngeal Carcinoma Patients

doi: 10.1074/jbc.m112.367532

Figure Lengend Snippet: FIGURE 4. Generation and migration of Th17 cells mediated by NPC tumor cell lines. A, NPC tumor cell lines induce the differentiation of naive T cells into Th17cellsinvitro.PurifiedCD4naiveTcellsfromhealthydonorswerestimulatedwithOKT3andthenco-culturedwiththeirradiatedNPCcelllineC666(EBV) or CNE2 (EBV) in IL-2-containing medium for 7 days. The NP69 and LCL lines and the cytokines IL-1 and TGF were used as controls. All cultured cells were stained for Foxp3 and IL-17 for FACS analysis after stimulation of phorbol 12-myristate 13-acetate/ionomycin. The data represent one of three independent experiments. B, mean percentage of cytokine-secreting Th17 cells induced from CD4 naive T cells from three experiments. C, migration of Th17 cells was increased in response to cultured supernatants from the NPC cell lines C666 and CNE2, relative to that from the normal NP cell line NP69 or with medium alone. The data represent one of three independent experiments. D, mean percentage of cytokine-secreting Th17 cells in the inner well and outer well after migration induced by supernatants from NP69, C666, or CNE2 cells from three experiments. Error bars represent the S.E. Significance was determined by the 2 test or paired t test. (*, p 0.05).

Article Snippet: Cytokines in the culture medium were measured using the Human Cytokine Array Panel A (Proteome Profiler; R&D Systems).

Techniques: Migration, Cell Culture, Staining

Journal: Cell death and differentiation

Article Title: Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion.

doi: 10.1038/cdd.2009.133

Figure Lengend Snippet: Figure 4 Effects of calpain inhibitors, general caspase inhibitor or individual caspase inhibitors 3, 8 and 9 on a-fodrin fragmentation in BeWo cells and primary term trophoblasts. (a) Inhibitors of caspases 3, 8 and 9 (20 mM, DEVD, IETD and LEHD, respectively) attenuated the generation of the 120 kDa a-fodrin fragment in forskolin-treated (20 mM) BeWo cells after 48 h cultivation. The inhibitors of calpain – calpeptin and calpain inhibitor III (20 mM, 48 h) – did not affect forskolin-induced fragmentation of a-fodrin in BeWo cells. The general caspase inhibitor (20 mM, Q-VD-OPh) completely blocked a-fodrin fragmentation in forskolin-treated (20 mM, 48 h) BeWo cells. (b) Similar effects were observed in primary term trophoblasts after 48 h cultivation. (c) Efficiency of calpeptin and calpain inhibitor III was tested in ionomycin (10 mM, 2 h)-treated BeWo cells and HUVEC. Although a-fodrin fragmentation was weak and not effected by calpain inhibitors in ionomycin-treated BeWo cells, ionomycin-induced pronounced generation of the 150/145 kDa a-fodrin doublet in HUVEC, which was blocked in the presence of calpeptin and calpain inhibitor III. (d) Forskolin treatment (20 mM, 48 h) led to a slight increase of cleavage products of caspases 3, 8 and 9 (arrows) compared with vehicle control (DMSO, 0.2%). Staurosporine (2 mM, 2 h), but not ionomycin (10 mM, 1.5 h) strongly induced activation of caspases 3, 8 and 9 in BeWo cells. Nitrocellulose membranes were stained with Ponceau S solution to verify equal protein loading and blotting efficiency. Western blots are representative for three independent experiments

Article Snippet: On the next day, cells were preincubated for 30 min with calpeptin (Z-Leu-NLe-CHO, Calbiochem, Darmstadt, Germany), calpain inhibitor III (Z-Val-Phe-CHO, Calbiochem), caspase 3 inhibitor peptide (Z-DEVD-FMK, BD PharMingen, Franklin Lakes, NJ, USA), caspase 8 inhibitor peptide (Z-IETD-FMK, BD PharMingen), caspase 9 inhibitor peptide (Z-LEHD-FMK, R&D Systems, Minneapolis, MN, USA), general caspase inhibitor (Q-VD-OPh, R&D Systems) or negative control peptide (Z-FA-FMK, BD PharMingen) at a concentration of 20mM each.

Techniques: Control, Activation Assay, Staining, Western Blot

Journal: Cell death and differentiation

Article Title: Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion.

doi: 10.1038/cdd.2009.133

Figure Lengend Snippet: Figure 5 Effects of calpain and caspase inhibitors on bhCG secretion and syncytialization in BeWo cells and primary term trophoblasts. (a) Calpeptin and calpain inhibitor III (20 mM), as well as individual caspase inhibitors of caspase 8 (IETD, 20 mM), caspase 3 (DEVD, 20 mM), caspase 9 (LEHD, 20 mM) and the general caspase inhibitor (Q-VD-OPh, 20 mM) decreased forskolin (20 mM)-induced bhCG secretion in BeWo cells after 48 h. Secreted bhCG was normalized to total protein of treated cells. Secretion from BeWo cells treated with forskolin without inhibitor is given as 100%. (b) Inhibitors showed similar effects on bhCG secretion in primary term trophoblasts after 48 h. bhCG secretion from untreated primary term trophoblasts is given as 100%. (c) Syncytialization of forskolin (20 mM)-treated BeWo cells was not affected by calpain inhibitors (calpeptin and calpain inhibitor III, 20 mM) and inhibitors of caspases 3 (DEVD, 20 mM) and 9 (LEHD, 20 mM) after 48 h. Caspase 8 inhibitor (IETD, 20 mM) and the general caspase inhibitor Q-VD-OPh decreased syncytialization to 30.9 and 30.7%, respectively, compared with BeWo cells treated with forskolin alone (36.0 %). Data are presented as mean±S.D. from three independent experiments performed in triplicates. *Pp0.05, **Pp0.01, ***Pp0.001

Article Snippet: On the next day, cells were preincubated for 30 min with calpeptin (Z-Leu-NLe-CHO, Calbiochem, Darmstadt, Germany), calpain inhibitor III (Z-Val-Phe-CHO, Calbiochem), caspase 3 inhibitor peptide (Z-DEVD-FMK, BD PharMingen, Franklin Lakes, NJ, USA), caspase 8 inhibitor peptide (Z-IETD-FMK, BD PharMingen), caspase 9 inhibitor peptide (Z-LEHD-FMK, R&D Systems, Minneapolis, MN, USA), general caspase inhibitor (Q-VD-OPh, R&D Systems) or negative control peptide (Z-FA-FMK, BD PharMingen) at a concentration of 20mM each.

Techniques:

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: CCL3 correlates with bone resorption in vitro OCP ( n = 8 donors, four replicates/condition) differentiated into osteoclasts in medium containing anti-CCL3 or IgG1 antibody. ( A ) Anti-CCL3 exerted a concentration-dependent inhibition on osteoclastogenesis (i), reducing total tartrate-resistant acid phosphatase (TRAP)-positive cells (ii), multinucleated osteoclasts (iii) and resorption (iv). ( B ) Disks stained with TRAP and haematoxylin (day 14). M-CSF cultures lacked osteoclasts (top), M-CSF + RANKL cultures showed strong TRAP staining (bottom; scale bar = 50 µm). ( C ) Resorption pits visualized by toluidine blue (top, scale bar = 250 μm) or calcein (bottom) reduced in anti-CCL3 (8 ng/ml) cultures vs IgG1 (yellow arrows). Mean value per donor plotted. * P ≤ 0.05, ** P ≤ 0.01. OCP = osteoclast precursor cells.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: In Vitro, Concentration Assay, Inhibition, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: CCL3 inhibition had no effect on resorption pit parameters Calcein-stained ivory disks were imaged by fluorescence microscopy to quantify resorption pit parameters. ( A ) Representative topographical maps of M-CSF (i), M-CSF + RANKL + IgG1 (ii), M-CSF + RANKL + anti-CCL3 (iii) disks show their naturally undulating surface and resorption pits (spherical lacuna); scale bar = 40 μm. ( B ) Measured lacuna area (i), perimeter (ii), depth (iii) and volume (iv) for anti-CCL3 vs IgG1 were unchanged (8 ng/ml). ( C ) Levels of CCL2 (i) and sIL-6R (ii) were comparable in IgG1 and anti-CCL3 (8 ng/ml) cultures (day 14). Cells from healthy human volunteers ( n = 6) were cultured, n = ≤2 disks/condition, mean ( s . e . m .) for each donor plotted.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: Inhibition, Staining, Fluorescence, Microscopy, Cell Culture

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: Systemic inhibition of CCL3 reduced histological joint swelling Mice with CIA received IgG1 or anti-CCL3 on days 21, 23, 25, 27 and 28 (5 mg/kg, n = 6/group). ( A ) Arthritis progression monitored by clinical scores (i) and paw diameter (ii) saw no statistical differences (two-way analysis of variance). ( B ) Representative tartrate-resistant acid phosphatase (TRAP) and haematoxylin-stained elbow joints from IgG1 (i) and anti-CCL3 (ii). Assessment of inflammation (iii), erosion (iv) and arthritic index (v). ( C ) IgG1 wrist histology showed intense TRAP-staining (i), which reduced in anti-CCL3 (ii), with significant reductions in inflammation (iii), erosion (iv) and arthritic index (v). H = humerus, U = ulna, R = radius, C = carpal. * P ≤ 0.05, ** P ≤ 0.01, scale = 1 mm.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: Inhibition, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: Bone erosions decreased after anti-CCL3 treatment during CIA Front and hind paws from CIA mice, treated with IgG1 or anti-CCL3, were processed for histology. ( A ) tartrate-resistant acid phosphatase (TRAP)-positive cells in the elbow (i) and wrist (ii) joints significantly reduced with anti-CCL3. ( B ) Radiographs of hind paws and ( C ) front paws were acquired from mice treated with IgG1 (i) or anti-CCL3 (ii) ( n = 6 mice per group). Significant reductions in erosive radiographic score for in both hind [B (iii)] and front [C (iii)] paws were quantified in mice treated with anti-CCL3 compared with IgG1 controls. * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: