Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased Oxidative Stress Induced by Rubus Bioactive Compounds Induce Apoptotic Cell Death in Human Breast Cancer Cells

doi: 10.1155/2019/6797921

Figure Lengend Snippet: Immunofluorescence: caspase 9, p53, and Bax. (a) Control cells showing very few caspase 9-positive cells; (b) C1 4.69 μ g/mL treated showing higher caspase 9-positive cells; (c) C2 8.36 μ g/mL treated showing medium caspase 9-positive cells. (d) Control showing very few nuclear staining for active p53; (e) C1 4.69 μ g/mL treated showing strong nuclear staining for active p53; (f) C2 8.36 μ g/mL treated showing moderate nuclear staining for active p53. (g) Control showing weak staining for active Bax; (h) C1 4.69 μ g/mL treated showing strong cytoplasmic staining for active Bax; (i) C2 8.36 μ g/mL treated showing moderate cytoplasmic staining for active Bax. C1-treated cells showed increased expression of caspase 9, p53, and Bax proteins than C2-treated cells (×20, original magnification; arrow indicates the presence of proteins; nuclear stain: DAPI (blue color); p53 and Bax protein stain: FITC (green color)).

Article Snippet: Cells were blocked in PBS containing 3% bovine serum albumin for 1 h, and primary antibody recognizing p53 (Santa Cruz Biotechnology, SC393031), Bax (Santa Cruz Biotechnology, SC493), and caspase 9 (Santa Cruz Biotechnology, SC56076) were added at 1 : 100 dilution for 2 h at room temperature.

Techniques: Immunofluorescence, Control, Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased Oxidative Stress Induced by Rubus Bioactive Compounds Induce Apoptotic Cell Death in Human Breast Cancer Cells

doi: 10.1155/2019/6797921

Figure Lengend Snippet: Expression of apoptotic proteins. (a) The effect of C1 and C2 on the expression of caspase 9 by ELISA. (b, c) The effect of C1 and C2 on the expression of p53, Bax, cleaved PARP, and cytochrome c by western blotting (L1: control; L2: C1 treated; L3: C2 treated). (d) The relative expression levels of p53, Bax, cytochrome c, and cleaved PARP. The results showing that apoptotic proteins such as caspase 9, p53, Bax, cytochrome c, and cleaved PARP levels were significantly increased in C1-treated groups than C2 and control cells.

Article Snippet: Cells were blocked in PBS containing 3% bovine serum albumin for 1 h, and primary antibody recognizing p53 (Santa Cruz Biotechnology, SC393031), Bax (Santa Cruz Biotechnology, SC493), and caspase 9 (Santa Cruz Biotechnology, SC56076) were added at 1 : 100 dilution for 2 h at room temperature.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased Oxidative Stress Induced by Rubus Bioactive Compounds Induce Apoptotic Cell Death in Human Breast Cancer Cells

doi: 10.1155/2019/6797921

Figure Lengend Snippet: Proposed cell death mechanism. An illustration of proposed cell death induced by C1 and C2 in MCF-7 cells. Mitochondria mediated caspase-dependent intrinsic apoptotic pathway is upregulated. Increased expression of p53, caspase 9, cleaved PARP, and Bax proteins mediated the cell death.

Article Snippet: Cells were blocked in PBS containing 3% bovine serum albumin for 1 h, and primary antibody recognizing p53 (Santa Cruz Biotechnology, SC393031), Bax (Santa Cruz Biotechnology, SC493), and caspase 9 (Santa Cruz Biotechnology, SC56076) were added at 1 : 100 dilution for 2 h at room temperature.

Techniques: Expressing

Journal: Retrovirology

Article Title: HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

doi: 10.1186/s12977-018-0422-5

Figure Lengend Snippet: CDK2 phosphorylates Tat Ser-16 and PKR phosphorylates Tat Ser-46 in vitro. a Sequence of Flag-tagged HIV-1 Tat indicating peptides that were used for the analysis of Tat phosphorylation in vitro. Potential phosphorylation sites are underscored. Tat Ser-16 and Ser-46 residues are further indicated with superscript numbering. Ser-16 (peptide 12–29); Thr-39 and Thr-40 (peptide 29–45); Ser-46 (peptide 41–57); and Ser-62, Thr-64, Ser-68 and Ser-70 (peptide 57–71). b Phosphorylation of Tat peptides in vitro. Upper panels, Tat peptides (4 µg) were phosphorylated in vitro with recombinant enzymes CDK2/cyclin E (lanes 1–4), CDK9/cyclin T1 (lanes 5–8), and PKR (lanes 9–12) with γ( 32 P)ATP as described in Methods. Phosphorylated peptides were resolved on 12% SDS Tris-Tricine gel containing 6 M urea, stained with SimpleBlue SafeStain (Coomassie), dried and analyzed by Phospho Imager. Phosphorylated Tat peptides position indicated with arrows on the right. Lower panel, Coomassie stained gel of Tat peptides showing 20 μg of Tat peptides 12–29, 29–45 and 57–71 and 2 μg of Tat peptide 41–57 resolved on 12% SDS Tris-Tricine gel with urea and stained with SimpleBlue SafeStain (Coomassie). c Relative intensities of the peptides phosphorylated by CDK2/cyclin E, CDK9/cyclin T1 or PKR were quantified with OptiQuant software (Packard)

Article Snippet: Cell suspensions were fixed and permeabilized using BD Biosciences kit (554714) followed by staining with primary antibodies against CDK2 (Santa Cruz, sc6248) or PKR (Santa Cruz, sc393038) and secondary anti-rabbit IgG antibody (FITC) from Invitrogen.

Techniques: In Vitro, Sequencing, Phospho-proteomics, Recombinant, Staining, Software

Journal: Retrovirology

Article Title: HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

doi: 10.1186/s12977-018-0422-5

Figure Lengend Snippet: Hunter peptide mapping analysis of Tat phosphorylation by CDK2 and PKR. Tat-derived peptides were phosphorylated in vitro by CDK2/cyclin E or PKR, as indicated. The reactions were loaded on nitrocellulose plates and peptides were resolved by thin layer electrophoresis as described in Methods. Plates were dried and stained with ninhydrin (left panels) or exposed to Phospho Imager screen (right panels). Origin and peptide positions are indicated on figure. The results are representative from 2 experiments

Article Snippet: Cell suspensions were fixed and permeabilized using BD Biosciences kit (554714) followed by staining with primary antibodies against CDK2 (Santa Cruz, sc6248) or PKR (Santa Cruz, sc393038) and secondary anti-rabbit IgG antibody (FITC) from Invitrogen.

Techniques: Phospho-proteomics, Derivative Assay, In Vitro, Electrophoresis, Staining

Journal: Retrovirology

Article Title: HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

doi: 10.1186/s12977-018-0422-5

Figure Lengend Snippet: Tat phosphorylation and the effect of CDK2 and PKR in cultured cells. a Mutation of Ser-16 or Ser-46 residue reduced HIV-1 Tat phosphorylation in cultured cells. Flag-tagged Tat, WT and S16A and S46A mutants were expressed in 293T cells and metabolically labeled with ( 32 P) orthophosphate. Tat protein was immunoprecipitated from cell lysates, resolved on 10% SDS-PAGE and exposed to Phosphor Imager screen. Tat expression was verified by Western blotting with anti-Flag antibodies. Lane 1, mock-transfected cells. Lane 2, WT Tat. Lane 3, Tat S16A mutant. Lane 4, Tat S46A mutant. The figure represents one of the three independent experiments. b Relative intensities of Tat and the mutants phosphorylation from three independent experiments. The mean ± SD are shown. * p < 0.01. c Label-free quantitative analysis of the high resolution MS spectra produced by Orbitrap MS scans for Tat by SIEVE 2.1 software. Average intensities of the indicated Tat peptide are shown with mean and standard deviations. d Quantification of non-phosphorylated and Ser-16 phosphorylated LEPWEHPGSQPK + Phospho(9) peptides derived from the data on c . Data are further adjusted to indicate the ratio of non-phosphorylated versus phosphorylated peptides. * p < 0.05

Article Snippet: Cell suspensions were fixed and permeabilized using BD Biosciences kit (554714) followed by staining with primary antibodies against CDK2 (Santa Cruz, sc6248) or PKR (Santa Cruz, sc393038) and secondary anti-rabbit IgG antibody (FITC) from Invitrogen.

Techniques: Phospho-proteomics, Cell Culture, Mutagenesis, Residue, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Expressing, Western Blot, Transfection, Produced, Software, Derivative Assay

Journal: Retrovirology

Article Title: HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

doi: 10.1186/s12977-018-0422-5

Figure Lengend Snippet: Effect of CDK2 and PKR Knock downs on HIV-1 replication. a – d CDK2 and PKR knockdown were generated in CEM T cells stably transduced with lentiviruses expressing CDK2 or PKR-targeting shRNA, or control non-targeting shRNA. a , c Expression of PKR and CDK2 in CEM T cells respectively determined by FACS analysis. Representative histogram shows isotype antibody staining (black), shRNA control (green or purple respectively) and CDK2 or PKR-targeting shRNA (orange or blue respectively). Bar graph of mean fluorescent intensity (MFI, y axis starts at mean fluorescence intensity of the isotype control; n = 2 per group).Mean ± SD. b , d Protein expression levels of CDK2 and PKR in stable knock down cell lines. Actin was used as normalization control. Bar graphs show the extent of the corresponding protein knock outs normalized to actin. e CEM T cells were infected with HIV-1-LUC-G virus and luciferase activity was measured at 48 h post infection. The mean ± SD are shown. * p ≤ 0.01

Article Snippet: Cell suspensions were fixed and permeabilized using BD Biosciences kit (554714) followed by staining with primary antibodies against CDK2 (Santa Cruz, sc6248) or PKR (Santa Cruz, sc393038) and secondary anti-rabbit IgG antibody (FITC) from Invitrogen.

Techniques: Knockdown, Generated, Stable Transfection, Transduction, Expressing, shRNA, Control, Staining, Fluorescence, Infection, Virus, Luciferase, Activity Assay

Journal: Retrovirology

Article Title: HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

doi: 10.1186/s12977-018-0422-5

Figure Lengend Snippet: Schematic representation of the effect of Tat phosphorylation on HIV-1 transcription regulation. CDK2/cyclin E or DNA-PK phosphorylates Tat Ser-16 which facilitates binding to TAR RNA and reduces the interaction with CDK9/cyclin T1. PKR phosphorylates Tat Ser-46 which may affect Tat nuclear localization and prevents Tat binding to cyclin T1

Article Snippet: Cell suspensions were fixed and permeabilized using BD Biosciences kit (554714) followed by staining with primary antibodies against CDK2 (Santa Cruz, sc6248) or PKR (Santa Cruz, sc393038) and secondary anti-rabbit IgG antibody (FITC) from Invitrogen.

Techniques: Phospho-proteomics, Binding Assay