Journal: Rheumatology (Oxford, England)

Article Title: Cyclophilin A up-regulates MMP-9 expression and adhesion of monocytes/macrophages via CD147 signalling pathway in rheumatoid arthritis

doi: 10.1093/rheumatology/ken225

Figure Lengend Snippet: IF of NF-κB activity induced by CypA in the THP-1 cell. ( a ) The u and d THP-1 cells were precubated with control (a, b, i, j) and AP-9 (c, d, k, l), then stimulated with CypA (200 ng/mL, b, d, j, l), CypA/CSA (f, n), LPS (1 µg/mL, g, o) or LPS/CSA (h, p) respectively for 1h. The p50 protein of NF-κB activity was analysed by IF with FITC-conjugated anti-p50 antibody (green fluorescence) for the uTHP-1 and Cy3-conjugated anti-p50 antibody (red fluorescence) for the dTHP-1, respectively. CypA-induced nuclear translocation of NF-κB 2 h after treatment (b and h). Nuclear translocation of NF-κB induced by CypA was blockaded by AP-9 (d and l) or CSA (f and n) respectively. ( b ) Merged images of Cy3 and DAPI in the dTHP-1 were obtained using computer software. After CypA stimulating THP-1, the red color for NF-κB overlayed with the blue color for nucleus in cell nucleus under immunofluorescence microscopy, which showed plenty of p50 transducted into nucleus. The scale bars indicate 25 μm ( a ) and 10 μm ( b ). The data shown are representative of similar results from three independent experiments.

Article Snippet: The cells were then treated with anti-p50 monoclonal antibody (SC-8414, Santa Cruz, CA, USA), followed by incubation, respectively, with FITC-conjugated rabbit anti-mouse IgG antibody (Serotec) or Cy3-conjugated sheep anti-mouse IgG antibody (C2181, Sigma-Aldrich; FITC for the uTHP-1 and Cy3 for the dTHP-1) according to manufacturer's instructions.

Techniques: Activity Assay, Control, Fluorescence, Translocation Assay, Software, Immunofluorescence, Microscopy

Journal: Cytometry

Article Title: Three-color versus four-color multiparameter cell cycle analyses of primary acute myeloid leukemia samples

doi: 10.1002/(sici)1097-0320(20000415)42:2<83::aid-cyto1>3.0.co;2-g

Figure Lengend Snippet: FIG. 1. Four-color flow cytometry assays allow reproducible and accurate immunodetection of CD34 in hematopoietic cell lines, as well as reproducible BCL-2 and PCNA immunodetection. Cumulative data from eight independent 3C versus 4C flow assays of each cell line are presented, with means and standard deviations denoted. Panel A: CD34 immunopositive cell fractions (% CD341) were consistently high in KG1a cells and low in K562 cells, whereas SSC-gated fractions were high in 3C and 4C analyses of both cell lines, as expected. Panel B: BCL-2 immunopositivity was reproducible in KG1a (high) and K562 (low) cells. Four-color K562 data represents SSC-gated events. Panel C: PCNA immunopositivity was high in both cell lines, as expected for highly proliferative cells. Four-color K562 data represents SSC-gated events. BCL-2 and PCNA determinations are consistent with data collected earlier with 3C flow cytometry assays of the same cell lines (1–3). Panels D and E: Cell-cycle distributions were reproducible for KG1a and K562 cells and were concordant for 3C SSC-gated (D) versus 4C CD34-gated (E) KG1a cells and for 3C SSC-gated (D) versus 4C SSC-gated (E) K562 cells.

Article Snippet: Cells were subsequently incubated in the dark for 1 h with FITC-labeled mouse anti-BCL-2 clone 124 antibody (Dako, Carpinteria, CA) at 200 ng/2–5 3 105 cells in PBS/SER and with R-phycoerythrin (R-PE)-labeled mouse anti-human PCNA clone PC10 (Dako) at 375 ng/ sample, or with isotype control antibodies (Dako), in the dark for 1 h. After further washing, the DNA-specific dye 7AAD (Sigma) was added at 25 mg/ml and cells were incubated for 30 min before analysis.

Techniques: Cytometry, Immunodetection

Journal: Cytometry

Article Title: Three-color versus four-color multiparameter cell cycle analyses of primary acute myeloid leukemia samples

doi: 10.1002/(sici)1097-0320(20000415)42:2<83::aid-cyto1>3.0.co;2-g

Figure Lengend Snippet: FIG. 2. Three-color flow cytometry assays of primary AML cell samples allow determinations of BCL-2 and PCNA immunopositive cell fractions and cell-cycle distributions in PCNA-gated 7AAD-stained DNA histograms. Data are presented for one primary AML cell sample, NCr, as an example. Cells were fixed with ethanol and permeabilized with Triton-X. FITC-labeled mouse anti-BCL-2 antibody 124 (Dako) or isotype control antibody (Dako) and R-PE-labeled mouse anti-human PCNA antibody PC10 (Dako) or isotype control antibody (Dako) were used. DNA was stained with 7AAD (Sigma). Representative histograms are shown. FL3A/FL3H single parameter ratio gates were used to exclude doublets (data not shown). Panel A: DNA (FL3-H) 3 SSC gates were used to exclude lymphocytes and small debris from the myeloid-enriched cell population. Panels B and C: BCL-2 (FL1-H) immunopositive cell fractions were determined (C) relative to the 98th percentile of isotype control antibody staining (B). Panel D: PCNA (FL2-H) immunopositive cell fractions were determined in single parameter gates relative to the 98th percentile of isotype control staining. Panels E and F: Cell-cycle fractions of PCNA-gated, 7AAD-stained DNA 3 SSC histograms (E) were determined (F) using the MultiCycle AV curve fitting software (Phoenix Flow Systems).

Article Snippet: Cells were subsequently incubated in the dark for 1 h with FITC-labeled mouse anti-BCL-2 clone 124 antibody (Dako, Carpinteria, CA) at 200 ng/2–5 3 105 cells in PBS/SER and with R-phycoerythrin (R-PE)-labeled mouse anti-human PCNA clone PC10 (Dako) at 375 ng/ sample, or with isotype control antibodies (Dako), in the dark for 1 h. After further washing, the DNA-specific dye 7AAD (Sigma) was added at 25 mg/ml and cells were incubated for 30 min before analysis.

Techniques: Cytometry, Staining, Labeling, Control, Software

Journal: Cytometry

Article Title: Three-color versus four-color multiparameter cell cycle analyses of primary acute myeloid leukemia samples

doi: 10.1002/(sici)1097-0320(20000415)42:2<83::aid-cyto1>3.0.co;2-g

Figure Lengend Snippet: FIG. 3. Four-color flow cytometry assays allow determinations of CD34, BCL-2, and PCNA immunopositivity, and cell-cycle distributions for primary AML cell samples. Data are presented for the NCr sample, as in Fig. 2. Cells were stained with IgM anti-CD34 antibody (I. Bernstein, FHCRC) followed by biotin-conjugated secondary anti-mouse IGM antibody (Cappel) and streptavidin RED 670 reagent (GIBCO-BRL). Cells were then fixed with paraformaldehyde followed by ethanol. A FITC-labeled mouse anti-BCL-2 antibody 124 (Dako) or isotype control was used for BCL-2 staining and a R-PE-labeled mouse anti-human PCNA antibody PC10 (Dako) or isotype control was used for PCNA staining. DNA was stained with H33342 dye. FL3A/FL3H single parameter ratio gates were used to exclude doublets (data not shown). Panel A: A DNA (FL4-H) 3 CD341 (FL3-H) gate was set to enrich for leukemia cell subsets, relative to isotype control antibody staining and negative cell controls (data not shown). Panels B and C: BCL-2 (FL1-H) immunopositive cell fractions (C) were determined relative to the 98th percentile of isotype control antibody staining (B). Panel D: Single parameter PCNA (FL2-H) immunopositive gates were determined relative to the 98th percentile of isotype controls. Panels E and F: Cell-cycle fractions of PCNA-gated, H33342-stained DNA 3 SSC histograms (E) were determined (F) using the MultiCycle AV curve fitting software (Phoenix Flow Systems).

Article Snippet: Cells were subsequently incubated in the dark for 1 h with FITC-labeled mouse anti-BCL-2 clone 124 antibody (Dako, Carpinteria, CA) at 200 ng/2–5 3 105 cells in PBS/SER and with R-phycoerythrin (R-PE)-labeled mouse anti-human PCNA clone PC10 (Dako) at 375 ng/ sample, or with isotype control antibodies (Dako), in the dark for 1 h. After further washing, the DNA-specific dye 7AAD (Sigma) was added at 25 mg/ml and cells were incubated for 30 min before analysis.

Techniques: Cytometry, Staining, Labeling, Control, Software

Journal: Cytometry

Article Title: Three-color versus four-color multiparameter cell cycle analyses of primary acute myeloid leukemia samples

doi: 10.1002/(sici)1097-0320(20000415)42:2<83::aid-cyto1>3.0.co;2-g

Figure Lengend Snippet: FIG. 4. SSC gating versus CD34 gating enumerates different leukemia blast fractions and different levels of BCL-2 in some primary AML cell samples, whereas PCNA immunopositivity is largely concordant. Panel A: Leukemia blast fractions were measured by 3C DNA 3 SSC-gating (x-axis) versus 4C DNA 3 CD34 gating (y-axis) in analyses of 13 primary AML BM samples. Panel B: Leukemia blast fractions were determined by 4C DNA 3 SSC gating (x-axis) versus 4C DNA 3 CD34 gating (y-axis) in analyses of primary AMLs. Panel C: BCL-2 immunopositive fractions were determined after 3C DNA 3 SSC gating (x-axis) versus 4C DNA 3 CD34 gating (y-axis) in analyses of 13 primary AML BM samples. Panel D: PCNA immunopositive fractions were determined after 3C DNA 3 SSC gating (x-axis) versus 4C DNA 3 CD34 gating (y-axis) in analyses of 13 primary AML BM samples.

Article Snippet: Cells were subsequently incubated in the dark for 1 h with FITC-labeled mouse anti-BCL-2 clone 124 antibody (Dako, Carpinteria, CA) at 200 ng/2–5 3 105 cells in PBS/SER and with R-phycoerythrin (R-PE)-labeled mouse anti-human PCNA clone PC10 (Dako) at 375 ng/ sample, or with isotype control antibodies (Dako), in the dark for 1 h. After further washing, the DNA-specific dye 7AAD (Sigma) was added at 25 mg/ml and cells were incubated for 30 min before analysis.

Techniques:

Journal: Cell reports

Article Title: Mutations in an Innate Immunity Pathway Are Associated with Poor Overall Survival Outcomes and Hypoxic Signaling in Cancer

doi: 10.1016/j.celrep.2018.11.093

Figure Lengend Snippet: See also . (A) mRNA expression fold changes (FC) and p values for complement regulators CD35, CD46, CD55, and CD59 in different colorectal cancer/adenocarcinoma versus normal tissue pairs. Data were acquired from https://www.oncomine.org/resource/login.html (GSE20916) . (B) Relative expression of CD55 (log 10 conversion) in COADREAD patients is shown against hypoxia signature expression (log 10 conversion) . Two-tailed p value is shown for the Pearson r (correlation coefficient). (C) KM curve for colorectal cancer patients with high (red) or low (blue) CD55 mRNA expression levels is shown. This analysis was based on the PrognoScan database ( http://dna00.bio.kyutech.ac.jp/PrognoScan/ ) using the publicly available Gene Expression Omnibus data ( https://www.ncbi.nlm.nih.gov/geo ) with the accession number GSE14333 ( ; ). p = 0.011327. (D) mRNA expression of CD55/18S is shown. qPCR was carried out following exposure of HCT116 cells to 0, 6, or 24 hr of hypoxia (1% O 2 ) or 24 hr of hypoxia followed by 1 hr reoxygenation (21% O 2 ). ** = p value < 0.01, 1-way ANOVA with Tukey’s multiple comparison test. Error bars represent the SEM for a representative experiment. n = 3. (E) HCT116 cells were treated with 0, 6, or 24 hr of hypoxia (1% O 2 ) or 24 hr of hypoxia followed by 1 hr reoxygenation (21% O 2 ). Western blotting (WB) was carried with the antibodies indicated. β-actin, loading control. n = 3. (F) CT26 cells were treated with 0 or 24 hr of hypoxia (1% O 2 ). WB was carried with the antibodies indicated. β-actin, loading control. n = 3. (G) Graph shows the % CMC/total lysis in HCT116 cells treated in 21% O 2 (normoxia) and either CD55 blocking antibody or IgG control (for the last hour of treatment). % CMC/total lysis was assessed by calculating calcein release/total lysis (and total cell number) following treatment with either normal human serum or heat-inactivated normal human serum. **** = p value < 0.0001, unpaired t test, two-tailed. Error bars represent the SEM for a representative experiment. n = 3. (H) Graph shows the % CMC/total lysis in HCT116 cells treated with 24 hr of 1% O 2 (hypoxia) and either CD55 blocking antibody or IgG control (for the last hour of treatment). % CMC/total lysis was assessed by calculating calcein release/total lysis (and total cell number) following treatment with either normal human serum or heat-inactivated normal human serum. **** = p value < 0.0001, unpaired t test, two-tailed. Error bars represent the SEM for a representative experiment. n = 3 (I) Graph shows the % CMC/total lysis in CT26 cells treated in 21% O 2 (normoxia) and either CD55 blocking antibody or IgG control (for the last hour of treatment). % CMC/total lysis was assessed by calculating calcein release/total lysis following treatment with either normal human serum or heat-inactivated normal human serum. * = p value < 0.05, unpaired t test. Error bars represent the SEM for a representative experiment. n = 3. (J) Graph shows the % CMC/total lysis in CT26 cells treated with 24 hr of 1% O 2 (hypoxia) and either CD55 blocking antibody or IgG control (for the last hour of treatment). % CMC/total lysis was assessed by calculating calcein release/total lysis following treatment with either normal human serum or heat-inactivated normal human serum. **** = p value < 0.0001, unpaired t test, two-tailed. Error bars represent the SEM for a representative experiment. n = 3.

Article Snippet: Antibodies used were anti-human mouse CD55 (BioRad, concentration # MCA914: 1:500), anti-mouse rat CD55 (R&D # MAB5376, concentration: 1:500) β-actin (Sigma # A5441, concentration: 1:5000), H3 (Abcam, #ab1791, concentration: 1:1000), HIF1α (BD-Biosciences, #610959, concentration: 1:500), HIF2α (Novus, # NB100–122, concentration: 1:500), ARNT (Novus, # NB100–124, concentration: 1:500), CD59 (Santa Cruz, concentration # sc-133171, 1:500).

Techniques: Expressing, Two Tailed Test, Western Blot, Lysis, Blocking Assay

Journal: Cell reports

Article Title: Mutations in an Innate Immunity Pathway Are Associated with Poor Overall Survival Outcomes and Hypoxic Signaling in Cancer

doi: 10.1016/j.celrep.2018.11.093

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used were anti-human mouse CD55 (BioRad, concentration # MCA914: 1:500), anti-mouse rat CD55 (R&D # MAB5376, concentration: 1:500) β-actin (Sigma # A5441, concentration: 1:5000), H3 (Abcam, #ab1791, concentration: 1:1000), HIF1α (BD-Biosciences, #610959, concentration: 1:500), HIF2α (Novus, # NB100–122, concentration: 1:500), ARNT (Novus, # NB100–124, concentration: 1:500), CD59 (Santa Cruz, concentration # sc-133171, 1:500).

Techniques: Blocking Assay, Viability Assay, Recombinant, shRNA, Software