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: Experimental and molecular pathology

Article Title: Structural Changes in Hair Follicles and Sebaceous Glands of Hairless Mice Following Exposure to Sulfur Mustard

doi: 10.1016/j.yexmp.2014.03.002

Figure Lengend Snippet: Histological sections prepared 1 and 14 days after air exposure (CTL), and 1, 3, 7 and 14 days after SM exposure were stained with an antibody to FGFR2. Antibody binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown.

Article Snippet: Tissue sections were incubated at overnight at 4°C with rabbit affinity purified polyclonal antibodies against cleaved caspase-3 (1:200, Cell Signaling, Danvers, MA), fatty acid synthase (1:100, Cell Signaling), fibroblast growth factor receptor 2 (FGFR2) (1:2000, Abcam, Cambridge, MA), phospho-histone 2A.X (Ser139) (phospho-H2A.X) (1:100, Cell Signaling), proliferating cell nuclear antigen (PCNA) (1:250, Abcam), goat affinity purified polyclonal antibody galectin-3 (1:2000, R&D Systems, Minneapolis, MN) or appropriate controls.

Techniques: Staining, Binding Assay

Journal: Nucleic Acids Research

Article Title: DFF-ChIP: a method to detect and quantify complex interactions between RNA polymerase II, transcription factors, and chromatin

doi: 10.1093/nar/gkae760

Figure Lengend Snippet: DFF-ChIP allows targeting of non-DNA bound factors. ( A ) UCSC genome browser tracks showing HeLa PRO-Seq (GSE100742) and DFF-ChIP targeng MED1, DSIF, NELF, and H3K4me3 (GSE185763) on two promoters. ( B ) MED1, DSIF, and NELF DFF-ChIP heatmaps of 11235 HeLa expressed genes sorted by DSIF signal from -50 to + 250. ( C ) MED1 DFF-ChIP and TBP DFFChIP (GSE185763) heatmaps of 11,235 HeLa expressed genes sorted by MED1 signal from -50 to + 250. ( D ) FragMaps of Non-TATA (10380) and TATA (500) genes from the MED1, DSIF, and NELF datasets. ( E ) Narrow range fragMaps of TBP from HFF cells and MED1 over Non-TATA and TATA genes. ( F ) Fragment center distribution of either paused (40–60 bp) or abutted (170–210 bp) sized fragments from DSIF (blue) or NELF (red) DFF-ChIP datasets relative to 11235 HeLa TSSs (see ).

Article Snippet: Afterwards, the supernatants were removed from beads and incubated with ∼5 μg of antibodies for CTCF (D31H2 XP ® Rabbit mAb #3418), NELF-A (A-20 sc-23599), DSIF (H-300 sc-28678), MED1 (A300-793A Bethyl Labs), or 5 μg antibody for GR (IA-1), a polyclonal rabbit antibody raised against human GR amino acids 84–112 , overnight at 4°C with rotation.

Techniques:

Journal: Nucleic Acids Research

Article Title: DFF-ChIP: a method to detect and quantify complex interactions between RNA polymerase II, transcription factors, and chromatin

doi: 10.1093/nar/gkae760

Figure Lengend Snippet: GR associates with engaged Pol II. ( A ) UCSC genome browser tracks showing DFF-ChIP targeting GR under both native and crosslinked conditions (SUP-B15 cells) with Pol II and H3K4me3 DFF-ChIP (HFFs). ( B ) FragMaps comparing DFF-ChIP targeting Pol II (Ser5P in HFFs) to native and crosslinked DFF-ChIP targeng GR (SUP-B15 cells). FragMaps are centered on the TSSs of 11,229 genes expressed in HFF cells discovered utilizing the truQuant program on a PRO-Cap dataset. ( C ) Heatmaps utilizing Pol II (Ser5P) DFF-ChIP and both crosslinked and native GR DFF-ChIP datasets sorted by abutted Pol II signal (+1 to +250) from the Ser5P dataset at the 12,229 genes expressed in HFF cells. ( D ) UCSC genome browser tracks showing four enhancers (E1–4) surrounding DDIT4. Tracks shown are PRO-Seq and DSIF (HeLa), native and crosslinked GR DFF-ChIP (SUP-B15 cells), as well as the GR ChIP-Seq peaks containing GR motifs.

Article Snippet: Afterwards, the supernatants were removed from beads and incubated with ∼5 μg of antibodies for CTCF (D31H2 XP ® Rabbit mAb #3418), NELF-A (A-20 sc-23599), DSIF (H-300 sc-28678), MED1 (A300-793A Bethyl Labs), or 5 μg antibody for GR (IA-1), a polyclonal rabbit antibody raised against human GR amino acids 84–112 , overnight at 4°C with rotation.

Techniques: ChIP-sequencing

Journal: Nanomaterials

Article Title: Few Layer Graphene Does Not Affect Cellular Homeostasis of Mouse Macrophages

doi: 10.3390/nano10020228

Figure Lengend Snippet: Cytokine secretion by BMDMs. ( A ) IL-6, ( B ) TNF-α, and ( C ) IL-1β levels in cell supernatant after treatment with or without FLG (3, 10, 30, 100 µg/mL), and LPS (1 µg/mL) for 24 h. A One-way ANOVA followed by Bonferroni’s post-test was performed to determine the statistical differences for control untreated cells versus FLG-treated samples and LPS-treated cells (* p < 0.05).

Article Snippet: In short, polyvinyl microtiter 96-well plates (Falcon) were coated overnight at 4 °C with 50 µL/well of purified rat anti-mouse IL-6 (0.5 mg/mL, BD Pharmingen #554400), rabbit anti-mouse TNF-α (0.5 mg/mL, BD Pharmingen #557516) and IL-1β (1 mg/mL, BD Biosciences, Le Pont de Claix, France), #51_26661E), diluted in coating buffer (Carbonate/bicarbonate buffer 0.05 M, pH 9.6).

Techniques: