Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Induction of apoptosis in 70Z/3 cells treated with 3CPA at 250 μ M and 500 μ M . ( A ) Cytochrome c release into the cytosol after exposure to 3CPA for indicated time periods measured by Western blot analysis. ( B ) Pro-caspase-9 processing determined in cytosolic fractions after exposure to 3CPA. Actin was used as an internal standard of the protein load and the results shown are representatives of two separate experiments. ( C ) Caspase inhibitors reduce 3CPA-induced apoptosis in 70Z/3 cells. Apoptosis, measured as AnnexinV + 7AAD − cells, was determined after exposure to 3CPA at 250 μ M (left) and 500 μ M (right) for 24 h. The pan-caspase inhibitor ZVAD-fmk (50 μ M , n =4), the caspase-9 inhibitor LEHD-fmk (50 μ M , n =3), or the caspase-8 inhibitor IETD-fmk (50 μ M , n =3) were added 1 h before treatment with 3CPA. The level of apoptosis is expressed relative to the level in cell cultures treated with the inhibitor only. Results show mean±s.d., and (*) indicates a statistical difference compared to 3CPA only (paired t -test, P <0.05).

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Over-expression of Bcl-2 in 70Z/3 cells inhibits 3CPA-induced apoptosis. ( A ) FACS-analysis of 70Z/3 (upper panel) and 70Z/3 Bcl-2+ (lower panel) cells after exposure to 3CPA (500 μ M ) for 18 h. 7AAD negative (viable) cells were gated (gate A) and analysed for Annexin V + expression (% of gated cells). ( B ) Levels of Bcl-2 and cytochrome c expression were determined by Western blot analysis in mitochondrial enriched fractions after 18 h incubation with 3CPA at the indicated concentrations (30 μg protein per lane). ( C ) Pro-caspase-9 processing into active caspase-9 was determined in the cytosolic fraction after 18 h incubation with 3CPA at the indicated concentrations. Actin was used as an internal standard to control the amount of cytosolic protein loaded in each lane. Results shown are representatives of two independent experiments.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Over Expression, Expressing, Western Blot, Incubation

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: N-substituted benzamides induce cytochrome c release and activation of caspase-9 in HL60 cells. ( A ) Cytochrome c release induced by MCA (500 μ M ) and 3CPA (250 μ M ) in HL60 cells after 24 and 48 h, measured by Western blot analysis of mitochondria or cytosol fractions (30 μg protein/lane). Cells were incubated with etoposide for 6 and 18 h at 100 μ M as a positive control. ( B ) Activation of caspase-9 after treatment of HL60 cells with 3CPA at 250 and 500 μ M for 12 and 18 h (48 μg protein per lane). One representative experiment out of two is shown.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Activation Assay, Western Blot, Incubation, Positive Control

Journal:

Article Title: Upregulation of CD40 Expression on Endothelial Cells Infected with Human Cytomegalovirus

doi: 10.1128/JVI.76.24.12803-12812.2002

Figure Lengend Snippet: Immunoblot analysis of CD40 in whole-cell lysates. Cell monolayers were infected with HCMV TB40E at an MOI of 1 (inf.) or mock infected (noninfected [n.i.]). Cell lysates were prepared at the indicated times and analyzed by immunoblotting. (A) CD40 protein levels were detected with a polyclonal rabbit serum directed against human CD40. (B) Blots from panel A were stripped and reprobed with a rabbit serum directed against calreticulin. (C) Signal intensities from panels A and B were densitometrically quantified; data represent the ratio of CD40 to calreticulin (y axis) versus time p.i. (x axis). (D) Protein lysates from infected or noninfected cells were treated with N-glycosidase F (+) or left untreated (−) prior to immunoblot analysis with anti-CD40 antibodies. Molecular masses are given in kilodaltons.

Article Snippet: The following monoclonal antibodies or polyclonal sera were used: mouse IgG1-fluorescein isothiocyanate (FITC)-conjugated anti-human CD54 (Calbiochem, La Jolla, Calif.), anti-human major histocompatibility complex class I (MHC-I) (BD PharMingen, San Diego, Calif.), mouse IgG1-FITC anti-human CD40 (Dianova, Hamburg, Germany), mouse IgG1-FITC anti-human CD62E (Calbiochem), antibodies G28.5 and Ro1 (anti-human CD40) (gifts from H. Engelmann, Institute for Immunology, University of Munich, Munich, Germany) ( 39 ), polyclonal rabbit anti-CD40 serum (Santa Cruz Biotechnology), rabbit antiserum against human calreticulin (Dianova), biotin-conjugated rabbit anti-mouse antibodies, and biotinylated swine anti-rabbit antibodies (Dako, Hamburg, Germany).

Techniques: Western Blot, Infection

Journal: PLoS ONE

Article Title: The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue In Vivo and Give Rise to Distinct NETs In Vitro

doi: 10.1371/journal.pone.0069575

Figure Lengend Snippet: A ) Neutrophils were immunofluorescently stained for OLFM4 (green) together with specific granule marker NGAL or azurophil granule marker CD63 (red), and DNA was labeled with DAPI (blue). Colocalization was analyzed by imaging flow cytometry and images show representative cells from the double positive populations (BF = brightfield). The diagram shows the mean colocalization score for the two fluorophores +SD from at least three experiments. The technical positive control was FITC-conjugated mouse anti-human CD16 antibody followed by Alexa Fluor 647-coupled goat anti-mouse secondary antibody and the technical negative control was FITC-conjugated mouse anti-human CD16 antibody together with DAPI, showing the minimum and maximum values that can be obtained by this analysis. The biological positive control was specific granule marker lactoferrin (LF) together with NGAL, and the biological negative control was lactoferrin together with CD63, showing the resolution of the analysis. B ) Pooled neutrophils from three donors were subjected to subcellular fractionation, and the fractions containing peak content of azurophil granule marker (MPO; fraction 1), specific granule marker (lactoferrin; fractions 10-12), gelatinase granule marker (gelatinase; fractions 13-15) and secretory vesicle marker (latent alkaline phosphatase; fraction 22) were subjected to Western blot with anti-OLFM4 antibody using rOLFM4 as a positive control. One representative blot out of three is shown. Lactoferrin (specific granule marker) and gelatinase (gelatinase granule marker) blots are also shown for fractions 10-15.

Article Snippet: The contents of lactoferrin and gelatinase were analyzed by Western blotting, using rabbit anti-human lactoferrin (DAKO) and rabbit anti-human gelatinase (Merck, Darmstadt, Germany), both at 2 μg/ml.

Techniques: Staining, Marker, Labeling, Imaging, Flow Cytometry, Positive Control, Negative Control, Fractionation, Western Blot

Journal: Cancer Science

Article Title: Quantitative proteomic analysis of mitochondria from human ovarian cancer cells and their paclitaxel-resistant sublines

doi: 10.1111/cas.12710

Figure Lengend Snippet: (a) Electron microscopy image of mitochondria morphology (10 000×). The purity of the mitochondria was confirmed using electron microscopy, including intactness and contamination. (b) Western blot analyses of total cell proteins (Cell) and mitochondrial preparations isolated from paclitaxel-sensitive SKOV3 cells (Smt), paclitaxel-resistant SKOV3 cells (TSmt), paclitaxel-sensitive A2780 cells (Amt), and paclitaxel-resistant A2780 cells (TAmt) for COX4 (mitochondrial marker), lamin-B (nuclear marker), flotillin-1 (cell membrane marker) and β-actin (cytoskeletal protein). There was little contamination in the mitochondria-enriched fractions.

Article Snippet: Mouse anti-human flotillin-1 and rabbit anti-human β-actin antibodies were obtained from eBioscience (San Diego, CA, USA).

Techniques: Electron Microscopy, Western Blot, Isolation, Marker, Membrane

Journal: Journal of Thoracic Disease

Article Title: Bioinformatics and integrated analyses of prognosis-associated key genes in lung adenocarcinoma

doi: 10.21037/jtd-21-49

Figure Lengend Snippet: Kaplan-Meier survival curves. Survival analysis indicating that dynein light chain roadblock-type 2 (DYNLRB2) (A) and mouse homolog of ß1 spectrin (SPTBN1) (B) are good prognostic factors in lung adenocarcinoma by Kaplan-Meier Plotter (Logrank P both <0.05).

Article Snippet: The membranes were further incubated with primary antibodies targeting rabbit anti-human DYNLRB2 (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) and rabbit anti-human GAPDH (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) at 4 °C overnight, then washed 5 times with TBST.

Techniques:

Journal: Journal of Thoracic Disease

Article Title: Bioinformatics and integrated analyses of prognosis-associated key genes in lung adenocarcinoma

doi: 10.21037/jtd-21-49

Figure Lengend Snippet: Verification of from dynein light chain roadblock-type 2 (DYNLRB2) and mouse homolog of ß1 spectrin (SPTBN1) expression level in The Cancer Genome Atlas database. Wilcoxon rank test showed that the expression of DYNLRB2 expression was lower in lung adenocarcinoma (LUAD) tissues than in normal tissues (A). SPRBN1 was lower in LUAD tissues than in normal tissues (B). *P<0.05.

Article Snippet: The membranes were further incubated with primary antibodies targeting rabbit anti-human DYNLRB2 (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) and rabbit anti-human GAPDH (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) at 4 °C overnight, then washed 5 times with TBST.

Techniques: Expressing

Journal: Journal of Thoracic Disease

Article Title: Bioinformatics and integrated analyses of prognosis-associated key genes in lung adenocarcinoma

doi: 10.21037/jtd-21-49

Figure Lengend Snippet: Dynein light chain roadblock-type 2 (DYNLRB2) and mouse homolog of ß1 spectrin (SPTBN1) correlated enrichment gene analysis with gene set enrichment analysis (GSEA). GSEA results showed that high DYNLRB2 and SPTBN1 co-expression was enriched in drug metabolism cytochrome P450 (A), cardiac muscle contraction (B), and retinol metabolism (C). Downregulated DYNLRB2 and SPTBN1 expressions were associated with homologous recombination (D), progesterone-mediated oocyte maturation (E), and base excision repair (F).

Article Snippet: The membranes were further incubated with primary antibodies targeting rabbit anti-human DYNLRB2 (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) and rabbit anti-human GAPDH (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) at 4 °C overnight, then washed 5 times with TBST.

Techniques: Expressing, Homologous Recombination

Journal: Journal of Thoracic Disease

Article Title: Bioinformatics and integrated analyses of prognosis-associated key genes in lung adenocarcinoma

doi: 10.21037/jtd-21-49

Figure Lengend Snippet: Quantitative reverse transcription polymerase chain reaction validation of the expression level of dynein light chain roadblock-type 2 (DYNLRB2) in empty vector transfected cells and DYNLRB2 transfected cells. ***P<0.001.

Article Snippet: The membranes were further incubated with primary antibodies targeting rabbit anti-human DYNLRB2 (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) and rabbit anti-human GAPDH (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) at 4 °C overnight, then washed 5 times with TBST.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Transfection

Journal: Journal of Thoracic Disease

Article Title: Bioinformatics and integrated analyses of prognosis-associated key genes in lung adenocarcinoma

doi: 10.21037/jtd-21-49

Figure Lengend Snippet: Dynein light chain roadblock-type 2 (DYNLRB2) Protein expression levels were determined by western blot. (A) Western blot bands representing DYNLRB2 protein level in control, empty vector transfected cells and DYNLRB2 transfected cells. GAPDH was used as an inner control. (B) Relative protein level of DYNLRB2 in control, empty vector transfected cells and DYNLRB2 transfected cells. **P<0.01.

Article Snippet: The membranes were further incubated with primary antibodies targeting rabbit anti-human DYNLRB2 (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) and rabbit anti-human GAPDH (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) at 4 °C overnight, then washed 5 times with TBST.

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection

Journal: Journal of Thoracic Disease

Article Title: Bioinformatics and integrated analyses of prognosis-associated key genes in lung adenocarcinoma

doi: 10.21037/jtd-21-49

Figure Lengend Snippet: Cell viability assay results by cell counting kit-8 in empty vector transfected cells and dynein light chain roadblock-type 2 (DYNLRB2) transfected cells. **P<0.01.

Article Snippet: The membranes were further incubated with primary antibodies targeting rabbit anti-human DYNLRB2 (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) and rabbit anti-human GAPDH (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) at 4 °C overnight, then washed 5 times with TBST.

Techniques: Viability Assay, Cell Counting, Plasmid Preparation, Transfection

Journal: Journal of Thoracic Disease

Article Title: Bioinformatics and integrated analyses of prognosis-associated key genes in lung adenocarcinoma

doi: 10.21037/jtd-21-49

Figure Lengend Snippet: Apoptosis assay in empty vector transfected cells, normal cells and dynein light chain roadblock-type 2 (DYNLRB2) transfected cells. ***P<0.001.

Article Snippet: The membranes were further incubated with primary antibodies targeting rabbit anti-human DYNLRB2 (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) and rabbit anti-human GAPDH (Sigma-Aldrich, St. Louis, MO, USA) (1:1,000) at 4 °C overnight, then washed 5 times with TBST.

Techniques: Apoptosis Assay, Plasmid Preparation, Transfection