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

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) Schematic illustration of the 1D11 administration paradigm for histological analysis. (B) Representative images of H&E stained sections (left) showing whole eyes (top panels) and corneas (lower panels) from E18.5 mice and quantification graph (right) showing increased corneal stromal thickness in Col4a1+/G1344D mice treated with 1D11 compared to those that received the control IgG1 antibody. Scale bars = 200 μm (top) and 50 μm (bottom). n = 20 and 24 corneas from IgG1- and 1D11-treated Col4a1+/+ mice, and 24 and 30 corneas from IgG1- and 1D11-treated Col4a1+/G1344D mice, respectively. (C) Schematic illustration of the 1D11 administration paradigm for qPCR analyses. (D) qPCR analyses revealed that 1D11 treatment partially prevented the increased expression of TGFβ target genes in anterior segments from P0 Col4a1+/G1344D mice compared to their IgG1-treated counterparts. n = 5–6 samples per genotype. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, two-way ANOVA and Tukey’s multiple comparison test.

Article Snippet: Timed-pregnant B6 females crossed with Col4a1 +/ G1344D males were injected intraperitoneally with the 1D11 pan-TGFβ neutralizing antibody (clone 1D11.16.8, BioXCell, West Lebanon, NH) or IgG1 isotype control antibody (clone MOPC-21, BioXCell) diluted in inVivoPure Dilution Buffer (pH 7.0, BioXCell) (20 mg/kg) every other day from E8.5 to E16.5 and animals were harvested at E18.5 or P0 for histological and molecular analyses, respectively.

Techniques: Staining, Control, Expressing, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Tumor suppression of novel anti–PD-1 antibodies mediated through CD28 costimulatory pathway

doi: 10.1084/jem.20182359

Figure Lengend Snippet: Anti–PD-1 Abs significantly enhance proliferation of Ag-specific exhausted CD8 T cells. (A) Recovery of proliferation in HIV-specific CD8 T cells following stimulation with an HIV-derived peptide. Results from a representative experiment are shown and expressed as the percentage of CFSE-low CD8 T cells. 8–10 replicates were performed for each experimental condition. (B) Cumulative results from multiple CFSE experiments ( n = 2–6) are shown for the 10 anti–PD-1 Abs identified with antagonistic properties similar to pembrolizumab. Results have been generated assessing the proliferation of CD8 T cells specific to three HIV-derived peptides (FLGKIWPSYK restricted by A*0201 and RLRPGGKKK or RMRGAHTNDVK restricted by A*0301) in patients B08 and B09. For comparative purposes across multiple assays and the different anti–PD-1 Abs, the level of proliferation in the pembrolizumab-treated samples was set as a 100% reference. Pembrolizumab was used as a positive control, while untreated (Neg) or mouse IgG1 isotype control Ab was used as a negative control in each experiment. Graphs show the mean ± SD. ****, P < 0.0001 for all anti–PD-1 Abs relative to the IgG1 control (unpaired t test with Welch’s correction).

Article Snippet: The PD-1–PDL-1 biochemical protein–protein interaction assay was performed by preincubating PD-1 Fc coated Bio-Plex beads in the presence of 20 nM of anti–PD-1 Ab (clones 137F2, 135C12, and 136B4) or a mouse IgG1 isotype control for 1 h. Duplicate samples of biotinylated PD-L1 Fc protein (R&D Systems) were added in a concentration range from 0.08 to 10 nM to the preformed Ab–PD-1 complex and incubated for a further 4 h before removing unbound PDL-1 and staining beads with PE-labeled streptavidin.

Techniques: Derivative Assay, Generated, Positive Control, Control, Negative Control

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: