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:

Journal: PLOS One

Article Title: An in vitro tumor recurrence model based on platinum-resistant colon cancer cells as a research tool for studying cancer cell dormancy

doi: 10.1371/journal.pone.0333671

Figure Lengend Snippet: (A,B) LysoTracker-positive acidic lysosomal compartments in quiescent cells. Oxaliplatin-resistant HCT116 oxpl-R cells (A) and cisplatin-resistant HCT116 cspl-R cells (B) before (day 0) and after (day 8) platinum treatment (oxaliplatin or cisplatin, respectively), stained with LysoTracker Green™ and MitoTracker Orange™. Scale bars represent 50 µm. (C,D) Dynamics of autophagy marker expression in the in vitro cancer reсurrence model based on cisplatin-resistant colon cancer cells HCT116 cspl-R, analyzed by immunoblotting (C) and qPCR (D) . (C) Immunoblots probed with LC3, Beclin and Survivin antibodies. α-Tubulin was used as a loading control. Quantification values shown beneath each lane represent band intensity normalized to loading control and relative to day 0 control (set as 1.0). (D) Normalized expression of autophagy-related genes ( lc3, beclin ) after cisplatin exposure (days 0-33), relative to day 0. Expression of the GAPDH gene served as the endogenous control. Data represent biological triplicate experiments and are displayed as mean ± SEM.

Article Snippet: The primary antibodies used were against LC3 #4108 RRID: AB_2137703, Beclin #3738 RRID:AB_490837, Survivin #2808 RRID:AB_2063948 (Cell Signaling, USA), Cyclin A sc-751 RRID:AB_631329, and p27 (C-19) sc-528 RRID:AB_632129 (Santa Cruz Biotechnology, USA) with ɑ-Tubulin sc-32293 RRID:AB_628412 (Santa Cruz Biotechnology, USA) serving as a loading control.

Techniques: Staining, Marker, Expressing, In Vitro, Western Blot, Control