Journal: Nature Communications

Article Title: PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

doi: 10.1038/ncomms2879

Figure Lengend Snippet: ( a , b ) The effect of Akt silencing on Plk1 phosphorylation, Plk1 interaction with 14-3-3γ ( a ) and Plk1 catalytic activity ( b ). ( c, f ) Using Tet-On HeLa cells expressing Flag-Akt1 WT ( c ) or Myc–Plk1 T210D ( f ), we performed the combination of DTB synchronization and the treatment with Akt1- and Akt2-specific siRNAs (siAkt1/2) as described in Hirota et al. Just after release from second thymidine block, the cells were cultured with (+) or without (−) doxycycline in the medium. Each protein level was checked by the immunoblotting with anti-Akt and anti-Plk1 ( c ) or anti-Myc ( f ). ( d ) Live-cell imaging analyses using H2B-GFP-expressing HeLa cells ( n ≥130 from two independent experiments). ( e ) The effect of Akt silencing on bipolar spindle formation. ( g ) KD or CA Flag-Akt1 was introduced in Tet-ON HeLa cells, and then mitotic phosphorylation of Plk1 was evaluated. ( h , i ) In vitro Akt kinase assays using depleted/reconstituted HeLa cell extracts ( h ) or anti-Plk1 immunoprecipitates ( i ) were performed as described in ‘Methods’.

Article Snippet: Antibodies from commercial sources were as follows: mouse anti-Plk1 (F-8), anti-α-tubulin (DM1A), anti-Bad (C-7) and rabbit anti-14-3-3γ (C-16) from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-Cdk1 pY15 (10A11), anti-pan Akt (40D4), anti-Akt1 (C73H10), anti-Akt2 (D6G4), anti-Bad (D24A9), anti-Bad pSer136 (D25H8), anti-Wee1 and HRP-conjugated anti-GAPDH from Cell Signaling Technology (Beverly, MA); mouse anti-Plk1 pThr210 (2A3) from Abcam (Cambridge, UK); mouse anti-14-3-3γ (CG31-2B6) and anti-Myc (4A6) from Millipore (Bedford, MA); mouse anti-γ-tubulin (GTU-88), anti-Vimentin (V9), anti-Flag (M2) and rabbit anti-INCENP from Sigma (St Louis, MO); mouse anti-Cdk1, anti-Aurora A and anti-Aurora B from BD Transduction Laboratories (San Diego, CA); rabbit anti-Mad2 from Covance (NJ, USA); mouse anti-BubR1 (8G1) and rabbit anti-histone H3 pSer10 from Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan); and CREST antiserum from Fitzgerald Industries International (North Acton, MA).

Techniques: Activity Assay, Expressing, Blocking Assay, Cell Culture, Western Blot, Live Cell Imaging, In Vitro

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Chemokine induction in Hct-8 cells infected with LEE-negative and LEE-positive STEC strains. Hct-8 cells were infected with the indicated STEC strains (Table ​(Table1).1). (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, culture supernatants were collected and assayed for IL-8 protein by ELISA as described in Materials and Methods. Data are shown as the means ± standard errors of the means (SEM) from two experiments. The significance of differences between IL-8 secretion by infected versus uninfected cells is indicated as follows: **, P < 0.001; *, P < 0.05.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Stimulation of Hct-8 cells by strain EDL933 derivatives. Cells were infected with strains 98NK2, EDL933, EDL933 expressing wild-type or defective copies of the ler gene carried on a multicopy plasmid (designated EDL933 Ler and 95SF2 Ler, respectively), and EDL933 with a deletion mutation in eae (Δeae) (all described previously [23]). (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. *, significant difference relative to control cells (P < 0.001).

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Expressing, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with purified Stx1 and Stx2. Hct-8 cells were treated with purified Stx1 or Stx2 at the indicated concentrations or with STEC strain 98NK2 or EDL933. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Purification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with H21 flagellin. Hct-8 cells were stimulated with H21 flagellin at the indicated concentrations or with STEC strains 98NK2 and EDL933. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells infected with strain 98NK2 stx2 and fliC deletion mutants. Hct-8 cells were stimulated with strain 98NK2, 98NK2Δstx2, or 98NK2ΔfliC. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. *, significant difference relative to strain 98NK2-treated cells (P < 0.01).

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with flagellin from strains 95HE4 and 95ZG1. Hct-8 cells were stimulated with strain 95HE4 (H7) or 95ZG1 flagellin at the indicated concentrations or with STEC strain 95HE4, 95ZG1, or 98NK2. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay