Journal: Journal of Neuroscience

Article Title: Vascular Remodeling versus Amyloid -Induced Oxidative Stress in the Cerebrovascular Dysfunctions Associated with Alzheimer's Disease

doi: 10.1523/jneurosci.4031-05.2005

Figure Lengend Snippet: Figure6. NitrosativestressinthecerebralcortexofAPP andTGF mice.Nitrotyrosineimmunoreactivityisupregulatedin the vessel walls of young APP mice, whereas it is distributed in the neuropil and around intraparenchymal blood vessels in 12-month-old APP mice. As shown in the adjacent panels, nitrotyrosine immunoreactivity (red) is mainly located in CD11b- immunopositive microglial processes (green; the merge with nitrotyrosine is yellow) and cells (open arrows), but some neurons are also nitrotyrosine immunopositive (small arrows). In TGF mice, nitrotyrosine immunoreactivity is primarily distributed to glial elements in both wild-type (WT) and transgenic mice. Scale bar, 75 m. mo, Month.

Article Snippet: Changes in pial and/or intracortical microvessels in transgenic mice (perfusion-fixed brains) and AD patients (50- m-thick sections) were assessed by immunostaining for the following markers: SOD2 (rabbit anti-MnSOD; 1:400; Stressgen), nitrosative stress (mouse anti-nitrotyrosine; 1:1000; Upstate Biotechnology, Lake Placid, NY), basement membrane proteins collagen-I (goat anti- collagen type I; 1:400; Southern Biotechnology, Birmingham, AL) and collagen-IV (goat anti-collagen type IV; 1: 400; Chemicon), and astroglial (GFAP, rabbit anti-GFAP; 1:300; Dako, Glostrup, Denmark) or microglial (rat anti-CD11b; 1:100; Serotec, Raleigh, NC) activation.

Techniques: Transgenic Assay

Journal: Journal of the American Heart Association

Article Title: Transplantation of Allogeneic Pericytes Improves Myocardial Vascularization and Reduces Interstitial Fibrosis in a Swine Model of Reperfused Acute Myocardial Infarction

doi: 10.1161/jaha.117.006727

Figure Lengend Snippet: Figure 6. Flow cytometry analyses of APCs isolated from human and swine saphenous veins. A and B, Representative flow cytometry gating procedure of hAPC line #1 and sAPC line #1 at P5. Total cell populations and the single cells (singlets) were gated according to FSC-A vs SSC-A and FSC-A vs FSC-H parameters (i and ii). Viable cells were distinguished from dead cells using Fixable Viability Dye eFluor780 (iii) and further gated for selected antigens (iv through xi and iv through xiii). Pericyte, mesenchymal, endothelial, and hematopoietic markers were studied. The FMO control was used in the assessment and gating of CD146+ and PDGFRb+ cells, because of the use of multiple fluorochromes (vii through ix and viii through x). The same approach was used when studying the expression of CD45 and CD11b on sAPCs to exclude hematopoietic cell contamination in the cell culture system (xi through xiii). Data were acquired using FACSCantoII (BD Biosciences) or Novocyte 3000 flow cytometer (ACEA Biosciences, San Diego, CA, USA) and analyzed using the FlowJo v10.3 software. C and D, Flow cytometry histograms for each surface marker in representative hAPC (C) and sAPC lines (D). Negative control staining profiles are shown by the red histograms, whereas specific antibody staining profiles are shown by light blue histograms. Bar graphs show the meanSEM values of 3 hAPC and sAPC lines. E and F, Gating and histograms of fresh isolated swine PB-MNCs and swine PAEC line #1 at P5 used as positive control for the staining of hematopoietic and endothelial markers, respectively. In both cell lines, the negative control staining profile is shown by full red histogram, while the positive staining profile is shown by full light-blue histogram. FSC-A indicates Forward Scatter Area; FSC-H, Forward Scatter Height; FMO, fluorescence minus 1; hAPCs, human adventitial pericytes; PB-MNCs, Peripheral blood mononuclear cells; PDGFRb, platelet-derived growth factor receptor-b; sAPCs, swine adventitial pericytes.

Article Snippet: Antibodies used in flow cytometry studies on sAPCs Marker Permeabilization Reactivity Primary antibody source dilution Fluorophores CD90 No Swine eBioscience, 1:20 Pe-Cy7 CD44 No Swine eBioscience, 1:20 APC CD105 No Swine LifeSpan, 1:5 PE CD45 No Swine Serotec, 1:25 FITC CD11b No Human/Swine eBioscience, 1:20 Pe-Cy7 CD31 No Swine Serotec, 1:80 PE CD146 No Swine Serotec, 1:60 FITC PDGFR No Human Biolegend, 1:25 PE Dye eFluor 780 No eBioscience, 1:1000 APC-Cy7 Cells (1x106, N=3 biological replicates) were washed in 1X DPBS (Life Technologies) and treated with 10X trypsin/EDTA (Life Technologies).

Techniques: Flow Cytometry, Isolation, Cytometry, Control, Expressing, Cell Culture, Software, Marker, Negative Control, Staining, Positive Control, Derivative Assay

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

Article Title: The IL-6/JAK/Stat3 Feed-Forward Loop Drives Tumorigenesis and Metastasis 1 2

doi:

Figure Lengend Snippet: IL-6R blockade inhibits growth of tumors and metastases and prevents metastatic progression. (A) 1833-IL-6 cells (1 x 105) were injected into the tail vein (n = 10/group) and were randomized to receive weekly intraperitoneal injections of control IgG (C) or IL-6RAb (IL-6R) given at the time of injection. Tumor growth was determined weekly by normalized BLI. Representative lung images are shown. (B) qRT-PCR for GFP relative to β-actin (tumor cells: GFP+) was performed to quantify tumor lung colonization described in A (n = 5). (C) 1833-IL-6 cells (1 x 106) were injected into the MFP of athymic nude mice and randomized to receive weekly intraperitoneal injections of control IgG (C) or IL-6RAb (IL-6R). Growth was determined weekly by normalized BLI. (D) qRT-PCR for GFP was performed on the lungs of mice (n = 5) in C. Quantification of CD11b+ and Gr1+ cells/hpf in the lungs (E) and MFP tumors (F) of mice described in A and C, respectively (n = 10 sections/group; **P < .01, *P < .05).

Article Snippet: Flow Cytometry Cells were isolated from tumor, spleen, or lung and stained with CD45 (Pe-Cy7 rat anti-mouse, 30-F11; BD), CD11b (APC rat anti-mouse, M1/70; BD), Gr1 (APC-Cy7 rat anti-mouse, RB6-8C5; BD), F4/80 (Brilliant Violet 421, anti-mouse, BM8; Biolegend, San Diego, CA), CD3 (PE rat anti-mouse, 17A2; BD), and CD4 (PerCP rat antimouse, RM4-5; BD).

Techniques: Injection, Quantitative RT-PCR

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

Article Title: The IL-6/JAK/Stat3 Feed-Forward Loop Drives Tumorigenesis and Metastasis 1 2

doi:

Figure Lengend Snippet: Stat3 deficiency abrogates tumorigenesis. (A) Representative IHC images examining pStat3 expression within the central portion, edge, and invasive (lymphovascular invasion) regions of human and MMTV-PyMT breast cancers. (B) Tumor pieces (1 mm) from Stat3+/+ (PyMT S3+) and Stat3-/- (PyMT S3-) PyMT-bearing mice were implanted orthotopically in the MFP of syngeneic C57/BL6 hosts, and tumor growth (volume and weight) was determined over 3 weeks (n = 8/group). (C) Representative histologic images of PyMT S3+ and PyMT S3- tumors stained for hematoxylin and eosin (H&E), pStat3, SMA, and Meca-32. (D) Flow cytometric analysis of PyMT S3+ and PyMT S3- tumors for CD11b+/Gr1+ cells (n = 5/group). (E) qRT-PCR analysis to determine the fold change (2-ddCT) in the expression of Stat3-regulated genes in CD11b+/Gr1+ cells isolated from PyMT S3+ and PyMT S3- tumors. (F) Representative co-IF staining for pStat3 and CD33 in primary invasive ductal carcinomas (n = 20). Thirty-three percent of the CD33+ cells were also pStat3+ (**P < .01).

Article Snippet: Flow Cytometry Cells were isolated from tumor, spleen, or lung and stained with CD45 (Pe-Cy7 rat anti-mouse, 30-F11; BD), CD11b (APC rat anti-mouse, M1/70; BD), Gr1 (APC-Cy7 rat anti-mouse, RB6-8C5; BD), F4/80 (Brilliant Violet 421, anti-mouse, BM8; Biolegend, San Diego, CA), CD3 (PE rat anti-mouse, 17A2; BD), and CD4 (PerCP rat antimouse, RM4-5; BD).

Techniques: Expressing, Staining, Quantitative RT-PCR, Isolation

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

Article Title: The IL-6/JAK/Stat3 Feed-Forward Loop Drives Tumorigenesis and Metastasis 1 2

doi:

Figure Lengend Snippet: Tumor-extrinsic effects of Stat3 deficiency. (A) Representative IHC images of the lungs of tumor-bearing (PyMT S3+ and PyMT S3-) mice stained for pStat3. The proportion (%) of CD11b+/Gr1+ cells in the lungs of these mice was determined by flow cytometry. (B) IL-6 levels in the serum were determined by ELISA and tumors were determined by qRT-PCR in PyMT S3+ and PyMT S3- mice, respectively (n = 5/group). (C) Tumor pieces (1mm) from mice bearing Stat3+/+ (PyMT S3+) cancerswere implanted orthotopically in theMFP of syngeneic (C57/BL6) IL6+/+ and IL-6-/- hosts, and tumor growth (weight) was determined after 3 weeks (n = 4/group). **P < .01, *P < .05.

Article Snippet: Flow Cytometry Cells were isolated from tumor, spleen, or lung and stained with CD45 (Pe-Cy7 rat anti-mouse, 30-F11; BD), CD11b (APC rat anti-mouse, M1/70; BD), Gr1 (APC-Cy7 rat anti-mouse, RB6-8C5; BD), F4/80 (Brilliant Violet 421, anti-mouse, BM8; Biolegend, San Diego, CA), CD3 (PE rat anti-mouse, 17A2; BD), and CD4 (PerCP rat antimouse, RM4-5; BD).

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

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

Article Title: The IL-6/JAK/Stat3 Feed-Forward Loop Drives Tumorigenesis and Metastasis 1 2

doi:

Figure Lengend Snippet: JAK inhibition alters the cellular immune phenotype of the tumor microenvironment. (A) The proportion (%) of CD3+, CD4+, and CD8+/CD45+ cells (n = 5/group) found in the tumors of 4T1 tumor-bearing mice treated with vehicle control (C) or JAKi (30 mg/kg twice daily) was determined by flow cytometry. (B) Flow cytometric analysis for CD11b+/Gr1+ cells in tumors and lungs of mice bearing MMTV-PyMT, Met-1, and 4T1 MFP tumors treated with vehicle control or JAKi (30 mg/kg twice daily). (C) qRT-PCR analysis to determine the fold change (2-ddCT) in the expression of Stat3-regulated genes in CD11b+/Gr1+ cells isolated from 4T1 tumors treated with vehicle (C) or JAKi (30 mg/kg twice daily). (D) The urea and NO2- concentrations observed in the CD11b+/Gr1+ cells isolated from 4T1 tumor-bearing mice treated with vehicle control (C) or JAKi (30 mg/kg) were determined by arginase and NO assays, respectively (n = 6/group). (E) The proportion (%) of CD11b+ and F480+ cells (n = 5/group) found in the tumors of MMTV-PyMT transgenic mice and in Met-1 and 4T1 xenografts from mice treated with vehicle control (C) or JAKi (30 mg/kg) was determined by flow cytometry. (F) qRT-PCR analysis to determine the fold change (2-ddCT) in the expression of immunosuppressive genes (Arg1, Il-10, Ccl2, Fizz1, Ym1, Ifnγ, S100a8, and S100a9) from CD11b+/F480+ cells isolated from 4T1 xenografts from tumor-bearing mice treated with vehicle control or JAKi (30 mg/kg twice daily; **P < .01, *P < .05).

Article Snippet: Flow Cytometry Cells were isolated from tumor, spleen, or lung and stained with CD45 (Pe-Cy7 rat anti-mouse, 30-F11; BD), CD11b (APC rat anti-mouse, M1/70; BD), Gr1 (APC-Cy7 rat anti-mouse, RB6-8C5; BD), F4/80 (Brilliant Violet 421, anti-mouse, BM8; Biolegend, San Diego, CA), CD3 (PE rat anti-mouse, 17A2; BD), and CD4 (PerCP rat antimouse, RM4-5; BD).

Techniques: Inhibition, Flow Cytometry, Quantitative RT-PCR, Expressing, Isolation, Transgenic Assay

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

Article Title: The IL-6/JAK/Stat3 Feed-Forward Loop Drives Tumorigenesis and Metastasis 1 2

doi:

Figure Lengend Snippet: The IL-6/JAK/Stat3 feed-forward loop drives mammary tumorigenesis. A schematic diagram demonstrating that the selective targeting of the IL-6/JAK/Stat3 signaling pathway (IL-6mAb, JAKi, Stat3-/-) has tumor-extrinsic effects on angiogenesis (Meca-32+), fibroblasts (SMA+), and the immune microenvironment [specifically, a decrease in the levels of pro-inflammatory CD11b+/F480+ and CD11b+/Gr1+ cells and an increase in T (CD4+, CD8+) cell responses]. These effects are IL-6-dependent and result in a decrease in tumor size and metastatic potential.

Article Snippet: Flow Cytometry Cells were isolated from tumor, spleen, or lung and stained with CD45 (Pe-Cy7 rat anti-mouse, 30-F11; BD), CD11b (APC rat anti-mouse, M1/70; BD), Gr1 (APC-Cy7 rat anti-mouse, RB6-8C5; BD), F4/80 (Brilliant Violet 421, anti-mouse, BM8; Biolegend, San Diego, CA), CD3 (PE rat anti-mouse, 17A2; BD), and CD4 (PerCP rat antimouse, RM4-5; BD).

Techniques: