Journal:

Article Title: Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

doi: 10.1128/IAI.69.1.52-57.2001

Figure Lengend Snippet: Binding of purified and biotinylated TibA to HCT8 monolayers. Bound TibA was detected by peroxidase-coupled streptavidin. Color development in each well was measured at 405 nm. Symbols: ♦, saturation curve performed with 0.0, 1.0, 2.4, 4.8, 9.6, 19.2, and 48.0 pmol of TibA per well; ▴, competition curve performed with 4.8 pmol of labeled TibA plus 0.0, 1.0, 4.8, 9.6, or 48.0 pmol of unlabeled TibA. Every experiment was performed at least three times. The extent of biotin labeling varied from day to day, resulting in significant differences in color development. Therefore, both curves shown here represent the average of two experiments performed in duplicate.

Article Snippet: The human ileocecal epithelial cell line HCT8 (ATCC CCL 244) was maintained in RPMI 1640 medium containing 10% fetal bovine serum, 1 mM l -glutamine, and 1 mM sodium pyruvate (Life Technologies).

Techniques: Binding Assay, Purification, Labeling

Journal:

Article Title: Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

doi: 10.1128/IAI.69.1.52-57.2001

Figure Lengend Snippet: Inhibition of TibA-mediated invasion by anti-TibA antiserum. Invasion assays were performed in the absence of antibodies (▪) or in the presence of preabsorbed and affinity-purified IgG from rabbit preimmune serum or polyclonal anti-TibA antiserum. (A) Invasion of HCT8 cells relative to E. coli DH5α(pET109), representing 100% (actual invasion of this strain in the absence of antibody was 1.30% ± 0.05%). Statistically significant effects of antibody treatment on the invasion efficiency of DH5α(pET109) are indicated by ∗ (P < 0.01) or ∗∗ (P < 0.005) as determined by analysis of variance of three experiments, each performed in triplicate. (B) Invasion of HCT8 cells relative to ETEC strain H10407, representing 100% (actual invasion of this strain in the absence of antibody was 0.24% ± 0.02%). TIB3 is a tib locus deletion mutant of H10407. Data are shown as averages for three replicates. Statistically significant effects of antibody treatment on the invasion efficiency of H10407 are indicated by ∗ (P < 0.01) as determined by analysis of variance of three experiments, each performed in triplicate.

Article Snippet: The human ileocecal epithelial cell line HCT8 (ATCC CCL 244) was maintained in RPMI 1640 medium containing 10% fetal bovine serum, 1 mM l -glutamine, and 1 mM sodium pyruvate (Life Technologies).

Techniques: Inhibition, Affinity Purification, Mutagenesis

Journal: Scientific Reports

Article Title: hERG1 channels modulate integrin signaling to trigger angiogenesis and tumor progression in colorectal cancer

doi: 10.1038/srep03308

Figure Lengend Snippet: (A) p85 participates in the hERG1/β 1 /FAK complex. (C) p85 is phosphorylated in CRC cell lines, and its phosphorylation is significantly affected by hERG1 blocking. Parallel IP were done using rabbit IgG as negative control. (B–C) The p85 associated with the hERG1/β1 complex is phosphorylated (B), and its phosphorylation is modulated by hERG1 and β1 inhibition or integrin activation (C). (D–E) The effects of the E4031 on Akt activity (D) and on Akt phosphorylation (E) in HCT116, HCT8 and HT29 cell lines, as well as in HCT116 transfected with the pLKO.1 empty vector (HCT116-PLKO) or in hERG1-silenced HCT116 cells (HCT116-Sh-hERG1). Data were analyzed using ImageJ, and graphs, reported in Fig. 2S DA were plotted by Microcal Origin 6.0. (F) Immunofluorescence (IF) staining using an anti-Akt (Santa Cruz, SC-8312, dilution 1:500) on control, E4031 or anti-β1 (Bv7) treated HCT116 cells. Negative control is represented by Akt1/2-silenced cells (e.g. cells transfected with siRNA anti-Akt1 and 2 (see section Materials and methods)). IF protocol was performed as detailed in . Nuclear/cytoplasmic staining ratios measured by ImageJ (see for further details) were the following: Control = 1.8 ± 0.46, E4031 = 0.18 ± 0.028; anti-β1 Bv7 = 0.24 ± 0.025. ** p < 0.02. Full-length blots are reported in section titled “Full-length blots relative to the cropped images showed in the main Figures”.

Article Snippet: Colorectal cancer (CRC) cell lines HCT116, HCT8, were cultured in RPMI 1640 (Euroclone; Milan, Italy) with 10% Fetal Calf Serum (FCS) (Euroclone Defined; Euroclone; Milan, Italy).

Techniques: Phospho-proteomics, Blocking Assay, Negative Control, Inhibition, Activation Assay, Activity Assay, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Control

Journal: Scientific Reports

Article Title: hERG1 channels modulate integrin signaling to trigger angiogenesis and tumor progression in colorectal cancer

doi: 10.1038/srep03308

Figure Lengend Snippet: (A) Effect of β1 inhibition or activation on VEGF-A expression. (B) VEGF-A expression in HCT116 treated with E4031 or WAY (40 μM), in HCT116 and HCT8 transiently transfected with a mix of α- herg1 siRNAs 1 + 3 or in HCT116-Sh-hERG1. Controls are represented by cells cultured in standard conditions for the pharmacological treated samples, by HCT116-PLKO cells for HCT116-Sh-hERG1 and by HCT116 transfected with siRNA negative control for siRNAs 1 + 3, respectively. (C) Effect of hERG1 blocking on VEGF-A secretion in HCT116 and HCT8 cells. Cells were also used for RNA extraction and RT-qPCR assay shown in . Data are means ± SEM of two-four separate experiments, each carried out in duplicate. (D) VEGF-A secretion in HEK-Mock and HEK-hERG1 cells and in HT29-Mock and HT29-hERG1 cells. For quantitative analysis of hERG1 expression, see . (E) Effects of PI3K/Akt inhibitors LY294002 and perifosine on VEGF-A secretion (left panel) and of α-Akt1 and α-Akt2 siRNAs on VEGF-A expression (right panel). Data are reported as the percentage of control ± SEM of two experiments, each carried out in triplicate. (F) Effects of PI3K/Akt inhibitors LY294002 and perifosine, on HIF-1 transcriptional activity. Data are means ± SEM of three separate experiments. (G) Fold induction of HIF(s) target genes after hERG1, β1 inhibition and activation or after Akt1/2 silencing. Data are means ± SEM of three separate experiments, each carried out in duplicate. GLUT1 , glucose transporter 1; LDHA , lactate dehydrogenase A; ANGPTL-4 , angiopoietin-like 4.*: p < 0.05; **: p < 0.02; ***: p < 0.01 (Student's t test). Raw data are in .

Article Snippet: Colorectal cancer (CRC) cell lines HCT116, HCT8, were cultured in RPMI 1640 (Euroclone; Milan, Italy) with 10% Fetal Calf Serum (FCS) (Euroclone Defined; Euroclone; Milan, Italy).

Techniques: Inhibition, Activation Assay, Expressing, Transfection, Cell Culture, Negative Control, Blocking Assay, RNA Extraction, Quantitative RT-PCR, Control, Activity Assay

Journal: Scientific Reports

Article Title: hERG1 channels modulate integrin signaling to trigger angiogenesis and tumor progression in colorectal cancer

doi: 10.1038/srep03308

Figure Lengend Snippet: (A) Effect of hypoxia in HCT116 and HCT8 on HIF-1α protein. (B) Effect of hypoxia on HIF(s) target genes. (C–D) Effect of a cycle of an alternate hypoxic (5 h)/normoxic (4 h) condition, compared to a standard incubation in normoxia (5 h + 4 h) on HIF(s) angiogenic target genes (C) and HIF(s) transcripts (D). (E–F) Study of p53 involvement in the regulation of HIF-1α protein. In the left panel is reported a WB showing the effects of hERG1 and β1 inhibition, as well as β1 activation, on p53 protein. Densitometric analysis data were analyzed using ImageJ, and graphs reported in Fig. 5S DA , were plotted by Microcal Origin 6.0. (G) Percentage of E4031 inhibition on WT and p53-/- HCT116 cells cultured in FN. Raw data and Pfaffl analysis are reported in . Data are means ± SEM of three separate experiments. * p < 0.05; ** p < 0.02; *** p < 0.01 (Student's t test). Full-length blots are reported in section titled “Full-length blots relative to the cropped images showed in the main Figures”.

Article Snippet: Colorectal cancer (CRC) cell lines HCT116, HCT8, were cultured in RPMI 1640 (Euroclone; Milan, Italy) with 10% Fetal Calf Serum (FCS) (Euroclone Defined; Euroclone; Milan, Italy).

Techniques: Incubation, Inhibition, Activation Assay, Cell Culture