Journal: Molecular Cancer Research

Article Title: Norepinephrine Promotes the β1-Integrin–Mediated Adhesion of MDA-MB-231 Cells to Vascular Endothelium by the Induction of a GROα Release

doi: 10.1158/1541-7786.mcr-11-0130

Figure Lengend Snippet: Figure 6. Expression and function of the chemokine receptors CXCR1 and CXCR2 in tumor cell adhesion to HMVECs under flow conditions. A, the expression of the chemokine receptors CXCR1 and CXCR2 was investigated by PCR. b-Actin served as loading control. The standard is a 100-bp ladder. B, confirmatory experiments were carried out with PC-3 human prostate carcinoma cells. Left, adhesion of the cells to HMVECs under flow conditions; right, PCR of CXCR1 and CXCR2 expression. C and D, adhesion of MDA-MB-231 cells to HMVECs under flow conditions. C, the cells were pretreated with antibodies blocking the chemokine receptors (aCXCR1/2) or an isotypic control antibody (IgG2a). D, the endothelium was overlaid with GROa and IL-8 at a concentration of 1 mg/mL for 3 minutes before the experiment. Denaturated chemokines were used as control. Graphs in (C) and (D) show mean values and SDs of 3 independent experiments. , statistically significant changes (P < 0.05). Nor, norepinephrine.

Article Snippet: For blocking experiments, MDA-MB231 were preincubated for 10 minutes with monoclonal mouse anti-CXCR1 and anti-CXCR2 (each 5 mg/mL; R&D Systems), mouse IgG2a (10 mg/mL; R&D Systems), mouse anti-b1-integrin clone 4B4 (5 mg/mL; Beckman Coulter), or mouse IgG1 (5 mg/mL; Beckman Coulter).

Techniques: Expressing, Control, Blocking Assay, Concentration Assay

Journal: Molecular Cancer Research

Article Title: Norepinephrine Promotes the β1-Integrin–Mediated Adhesion of MDA-MB-231 Cells to Vascular Endothelium by the Induction of a GROα Release

doi: 10.1158/1541-7786.mcr-11-0130

Figure Lengend Snippet: Figure 7. Inhibition of b1-integrins and NF-kB abrogates the effect of norepinephrine on MDA-MB-231 cell adhesion. A, MDA-MB-231 cells were pretreated with either the b1-integrin–blocking antibody 4B4 or an isotypic control antibody (IgG1). B, HMVECs were preincubated with an NF-kB activation inhibitor (NF-kB Inh); norepinephrine (Nor) was used at 10 mmol/L. Both graphs show mean values and SDs of 3 independent experiments. , statistically significant changes (P < 0.05).

Article Snippet: For blocking experiments, MDA-MB231 were preincubated for 10 minutes with monoclonal mouse anti-CXCR1 and anti-CXCR2 (each 5 mg/mL; R&D Systems), mouse IgG2a (10 mg/mL; R&D Systems), mouse anti-b1-integrin clone 4B4 (5 mg/mL; Beckman Coulter), or mouse IgG1 (5 mg/mL; Beckman Coulter).

Techniques: Inhibition, Blocking Assay, Control, Activation Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: SAMSN1 Is a Tumor Suppressor Gene in Multiple Myeloma 1 2

doi: 10.1016/j.neo.2014.07.002

Figure Lengend Snippet: SAMSN1 expression is reduced in CD138 + PCs of patients with MM and HMCL. (A) SAMSN1 expression (as determined by real-time PCR) is significantly reduced in the BMs of patients with MM ( n = 34) compared with patients with MGUS ( n = 9) and healthy age-matched controls ( n = 5; * P < .05, ** P < .001, one-way ANOVA with Tukey’s multiple comparison test). (B) SAMSN1 expression in CD138 + MACS isolated PCs from patients with MM negatively correlates with BM PC burden ( n = 10, r 2 = 0.6147, P = .0043). (C) In silico analysis of published microarray data. CD138 + PCs were isolated by MACS from 414 patients with MM, 44 patients with MGUS, and 22 age-matched controls. RNA was extracted and analyzed using the Affymetrix U133Plus2.0 microarray platform (GEO Accession Nos GSE4581 and GSE5900). Expression of SAMSN1 is significantly reduced in PCs of patients with MM compared to those of patients with MGUS and normal controls. P < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. (D) Total RNA was extracted from six HMCLs and reverse transcribed. The levels of SAMSN1 expression were assessed by real-time PCR.

Article Snippet: PCs were isolated from flushed long bones and identified using rat anti-mouse CD138 (R&D Systems, Minneapolis, MN) followed by PE-conjugated goat anti-rat IgG.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison, Isolation, In Silico, Microarray, Reverse Transcription

Journal: The Journal of Clinical Investigation

Article Title: ADAMTS7 promotes smooth muscle foam cell expansion in atherosclerosis

doi: 10.1172/JCI187451

Figure Lengend Snippet: ( A ) Schematic outlining experimental design and the generation of the SMC transgenic ADAMTS7 mouse. Created with BioRender.com. ( B ) ORO staining of the en face aorta and quantification of lesion area. n = 9. ( C ) Representative images of H&E-stained aortic root sections, quantification of plaque areas ( n = 6). Scale bar: 500 μm. ( D ) Representative images of H&E-stained aortic root sections with necrotic core outlined in dotted line ( n = 6 mice). Scale bar: 300 μm. ( E ) Representative images of aortic root sections stained against α-SMA (Cy3; red), MAC2 (Alexa Fluor 488 green), and cell nuclei (DAPI blue) with their subsequent quantification relative to lesion area ( n = 6). Scale bar: 300 μm. ( F ) Representative Picrosirius red staining of aortic root lesions with bars indicating fibrous cap thickness and quantification of fibrous cap thickness ( n = 6). Scale bar: 300 μm. ( G ) Schematic outlining experimental design and the generation of the EC transgenic ADAMTS7 mouse. Created with BioRender.com. ( H ) ORO staining of the en face aorta and quantification of lesion area. n = 3–4. ( I ) Representative images of H&E-stained aortic root sections and quantification of plaque areas ( n = 5–6). Scale bar: 500 μm. **** P < 0.0001, * P < 0.05. Statistics were analyzed using a 2-tailed Student’s t test.

Article Snippet: The sections then were stained using the following Abs and dilutions: 1:1,000 α-SMA-Cy3 (MilliporeSigma, C6198) and 1:500 MAC2 (Cedarlane, CL8942AP).

Techniques: Transgenic Assay, Staining

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis.

doi: 10.1111/j.1538-7836.2006.02033.x

Figure Lengend Snippet: Fig. 4. Complement C3 deposition in joints. Healthy 5–8-week old female TMwt/wt (A) and TMLeD/LeD (B) mice were killed and knee joints were processed for immunohistochemical staining to detect complement factor C3 deposition. These sections are representative of three mice. No C3 was detectable in the joints of TMwt/wt mice (A). C3 was readily observed on the articular surface (small arrows) and in the bone marrow space of TMLeD/LeD mice (large arrows) (B). Higher power views of the articular surface are shown in the right upper corner of each panel.

Article Snippet: Specific immunostaining of histologic sections was achieved by overnight incubation with the following primary antibodies: CD45 (rat, 1:100; BecktonDickinson, Erembodegem, Belgium, no. 553076);Mac3 (rat, 1:300; BecktonDickinson, no. 553322); MPO (rabbit, 1:100, Dako A/S, Glostrup, Denmark, no. A0398); HMGB1 (rabbit; 1:250, Santa Cruz Biotechnology, Boechout, Belgium, no. sc12523); and complement factor C3 (rat, 1:200; Cedarlane,Uden, theNetherlands, no. CL7503AP), which recognizes intact C3, C3b, iC3b and C3d, but not C3a.

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia

doi: 10.3389/fcvm.2021.745810

Figure Lengend Snippet: Myeloid-specific Pfkfb3 deficiency attenuates LPS-induced inflammatory responses. (A) Representative images (left) and quantification (right) for immunohistochemical staining of the neutrophil marker Ly6G in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (B) Representative images (left) and quantification (right) for immunohistochemical staining of the macrophage marker Mac2 in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (C–E) qPCR analysis of the mRNA levels of Il1b (C) , Il6 (D) and Nos2 (E) in the lung of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 6). (F,G) ELISA analysis of Il1b (F) and Il6 (G) in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 4). (H) NO levels in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 3–4). All data are represented as mean ± SEM, ** P < 0.01 and *** P < 0.001 for Pfkfb3 WT vs. Pfkfb3 ΔMϕ (unpaired two-tailed Student's t test).

Article Snippet: After antigen retrieval with Antigen Unmasking Solution (H-3301, Vector Laboratories, Burlingame, CA, USA) at 98°C for 10 min, sections were blocked with avidin solution with 10% normal rabbit serum for 1 h at room temperature, and incubated in biotin blocking solution with primary antibodies against Mac2 (3 μg/mL, CL8942F, Cedarlane, Burlington, NC, USA), or Ly6G (3 μg/mL, 551459, BD biosciences, San Jose, CA, USA) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Marker, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: BMC Molecular and Cell Biology

Article Title: Transcriptome and proteome profiling reveal complementary scavenger and immune features of rat liver sinusoidal endothelial cells and liver macrophages

doi: 10.1186/s12860-020-00331-9

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: CD11b/c Biotin (OX-42) , CD11b/c, CR3 , Cedarlane , CL042B , 2 μg/ml.

Techniques: Concentration Assay, Flow Cytometry, Control, Staining, FACS

Journal: Journal of immunotoxicology

Article Title: The early growth response factor-1 contributes to interleukin-13 production by mast cells in response to stem cell factor stimulation.

doi: 10.1080/15476910802129612

Figure Lengend Snippet: FIG. 4. Egr-1 deficiency has no effects on c-Kit and IgE receptor expression or mast-cell viability. (A) Bone marrow cells from wild-type mice or Egr-1– deficient mice were cultured in conditioned media in vitro for 4 wk and exam- ined by flow cytometry for c-Kit and IgE receptor expression. For c-Kit anal- ysis, BMMCs were stained with FITC conjugated rat anti-mouse c-Kit mAb or FITC conjugated rat IgG2a isotypic control. For analysis of IgE receptor expression, BMMC were sensitized with IgE overnight and then stained with FITC-conjugated anti-IgE antibody (mouse IgG1). No difference in c-Kit or IgE receptor expression was observed between Egr-1+/+ and Egr-1−/−BMMCs. (B) BMMC from Egr-1-deficient mice or wild-type mice cultured in complete media containing WEHI-3B supernatants (a source of IL-3) were healthy and showed ≥96% viability. To induce mast cell death, WEHI-3B supernatant was removed from culture media for various days. Mast cell viability was examined by trypan blue exclusion assay. Data were expressed as mean ± SD (n = 3 independent experiments).

Article Snippet: Fluorescein isothiocyanate (FITC) anti-mouse CD117 monoclonal antibody (mAb) (CL8936F), FITC rat IgG2a (CLCR2A01) were purchased from Cedarlane Laboratories Limited (Ontario, Canada).

Techniques: Expressing, Cell Culture, In Vitro, Cytometry, Staining, Control, Trypan Blue Exclusion Assay