Journal: Cellular signalling

Article Title: The gep proto-oncogene Gα 12 mediates LPA-stimulated activation of CREB in ovarian cancer cells

doi: 10.1016/j.cellsig.2013.08.012

Figure Lengend Snippet: A. Silencing of Gα12 in HeyA8 cells using Gα12-specific shRNA was monitored by immunoblot analysis using lysates of 25 µg protein derived from three distinct clones of Gα12-silenced cells along with cells from vector control clone. B. Gα12-shRNA-HeyA8 clones were analyzed by quantitative RT-PCR for Gα12 expression. The expression levels of Gα12 for each clone in relation to vector control cells are presented in the bar graph. C. Hey cells stably expressing shRNA against Gα12 or the vector alone (non-specific scrambled shRNA vector) were serumstarved overnight. The stably silenced Gα12 cells were treated with 20 µM of LPA for 16 hours along with one group of HeyA8 cells stably-expressing the vector alone. Additionally, one group of the vector control cells was left in serum-free media for the 16-hour treatment period. After the 16-hour treatment, nuclear lysate was obtained from each cell group and analyzed by a Protein/DNA array according to manufacturer’s protocol. Representative array data from two independent experiments are presented. Each spot on the array that corresponds to a specific transcription factor was identified according to manufacturer’s protocol. Transcription factors stimulated by LPA but absent or down-regulated in Gα12-silenced cells are scored. The arrows indicate the spots corresponding to CREB. The profiles of activated transcription factors as indicated by the binding of the respective transcription factors to the DNA-elements printed in the array were analyzed in serum-starved HeyA8 cells (Upper Panel), HeyA8 cells stimulated with LPA (Middle Panel), and LPA-stimulated HeyA8 cells in which the expression of Gα12 was silenced (Lower Panel).

Article Snippet: The nuclear lysate was then analyzed for transcription factor activation using an Affymetrix Combo Protein/DNA Array (MA1215; Santa Clara, CA) according to the manufacture’s instructions.

Techniques: shRNA, Western Blot, Derivative Assay, Clone Assay, Plasmid Preparation, Quantitative RT-PCR, Expressing, Stable Transfection, DNA Array, Binding Assay

Journal: Cellular signalling

Article Title: The gep proto-oncogene Gα 12 mediates LPA-stimulated activation of CREB in ovarian cancer cells

doi: 10.1016/j.cellsig.2013.08.012

Figure Lengend Snippet: LPA-stimulated and Gα 12 -dependent Transcription Factors in HeyA8 Cells Control HeyA8 cell expressing non-specific sh-vector or HeyA8 cells in which Gα 12 were stimulated with 20 µM LPA for 16 hrs. Nuclear extracts from these cells along with unstimulated controls were analyzed for the activation of different transcription factors using “Affymetrix Combo Protein/DNA Array” as described under Methods section. Representative array data from two independent experiments are presented here. Each spot on the array, which corresponds to a specific transcription factor, was identified using the template from the user manual. The intensities of the spots were quantified using Carestream Molecular Imaging Software version 5. Transcription factors stimulated by LPA but absent or down-regulated in Gα 12 -silenced cells were scored, quantified, and tabulated.

Article Snippet: The nuclear lysate was then analyzed for transcription factor activation using an Affymetrix Combo Protein/DNA Array (MA1215; Santa Clara, CA) according to the manufacture’s instructions.

Techniques: Expressing, Activation Assay, Imaging, Software, Inhibition, Binding Assay, Methylation

Journal: Molecular Plant Pathology

Article Title: Distinguishing bacterial pathogens of potato using a genome‐wide microarray approach

doi: 10.1111/j.1364-3703.2008.00482.x

Figure Lengend Snippet: Percentage of probes that recognized four bacterial species tested by microarray analysis.

Article Snippet: Image and data analysis Microarray slides were scanned with a GenePix 4200 AL scanner (Axon Instruments, Foster City, CA) using a pixel resolution of 5 μm.

Techniques: Microarray

Journal: Molecular Plant Pathology

Article Title: Distinguishing bacterial pathogens of potato using a genome‐wide microarray approach

doi: 10.1111/j.1364-3703.2008.00482.x

Figure Lengend Snippet: Scanned images of the signals detected on the microarray. A view of the whole microarray with eight subarrays is shown in (A), whereas areas covered by c. 800 probes (of the total of 9676 probes of one subarray) are shown at higher magnification in B and C. Total DNA extracted from pure cultures of bacteria and pooled from several strains of each species was used for hybridization. (A) Two samples were hybridized on each of the eight subarrays. The sample labelled with Cy5 (red) in all eight subarrays was Pectobacterium atrosepticum. The other samples labelled with Cy3 (illustrated as green) were (1) Streptomyces scabies, (2) Dickeya sp., (3) P. carotovorum, (4) Clavibacter michiganensis, (5) P. atrosepticum and (6–8) S. turgidiscabies. The amount of DNA per sample was 500 ng in subarrays 1–6. Signals were clear also with 50 ng of sample DNA (dilution 1 : 10, subarray 7). The image shown here was scanned using constant laser power and detector gain, and signals in subarray 8 (5 ng of DNA; dilution 1 : 100) cannot be seen. However, using increased detector gain, the most species‐specific signals (highest signal intensity) could be detected on subarray 8. (B) Magnification of a part of subarray 5: two samples of P. atrosepticum labelled each with a different dye. Intensive yellow spots (equal hybridization) correspond to probes specific to P. atrosepticum, whereas the spots with faint signal indicate non‐specific hybridization. (C) Magnification of part of the subarray 1: P. atrosepticum labelled with Cy5 and S. scabies labelled with Cy3. A ‘black spot’ (no signal) indicates no hybridization with the probe. The probes were designed to be gene‐specific, taking the whole‐genome sequence information of the species into consideration. Results indicate that most probes detect only the respective species based on which the probes were designed.

Article Snippet: Image and data analysis Microarray slides were scanned with a GenePix 4200 AL scanner (Axon Instruments, Foster City, CA) using a pixel resolution of 5 μm.

Techniques: Microarray, Bacteria, Hybridization, Sequencing

Journal: Molecular Plant Pathology

Article Title: Distinguishing bacterial pathogens of potato using a genome‐wide microarray approach

doi: 10.1111/j.1364-3703.2008.00482.x

Figure Lengend Snippet: Pooled DNA of the strains of Clavibacter michiganensis ssp. sepedonicus (Cms) (labelled with Cy3) and Pectobacterium atrosepticum (Pat) (labelled with Cy5) analysed on the microarray. (A) Scatterplot shows signal intensities from each probe on the array. Signals for Cms are given on the x‐axis and those for Pat on the y‐axis. Data reveal that the samples are not detected with common probes giving high signal intensities. (B) The scatterplot presented in a logarithmic domain places the probes within four groups: (1) high signal intensities for both samples (very few probes); (2) non‐specific probes detecting both samples (relatively low signal intensities); (3) probes giving high signal intensities only for Pat; and (4) probes giving high signal intensities only for Cms. In (C) (Cms) and (D) (Pat), the histograms of the logarithmic signal intensities show three peaks (histograms smoothened by the kernel density method). A threshold value of ~10 separates the two right‐most peaks (II and III) corresponding to the non‐specific and specific probes, respectively, as shown in B. The threshold value corresponds to the raw (non‐logarithmic) intensity value of c. 1000. In (E) (Cms) and (F) (Pat) the hybridization signal intensities are indicated per groups of probes. In the boxplot, the horizontal line in the middle of the box indicates the median value of the data. The box itself shows the first and third quartile of data. Whiskers outside the box indicate the range of data up to 1.5× the box height from both ends. Data beyond these limits are shown as circles. The intensity values of all probes are shown; however, in the final classification, the probes with intensities below the threshold obtained from the intensity histogram would be eliminated. Abbreviations used in the probe group names: Pat, P. atrosepticum; Sca, S. scabies; Cms, C. michiganensis spp. sepedonicus; IGS, 16S–23S intergenic spacer; Pca, P. carotovorum; IGS Dic, probes to the IGS of Dickeya spp.; Stu, S. turgidiscabies; Rso, R. solanacearum; nip, gene for necrosis‐inducing protein; Dic Nip30‐Nip50, probes of different lengths (30–50 nt) designed for the nip gene of D. dadantii; PAI, pathogenicity island.

Article Snippet: Image and data analysis Microarray slides were scanned with a GenePix 4200 AL scanner (Axon Instruments, Foster City, CA) using a pixel resolution of 5 μm.

Techniques: Microarray, Hybridization

Journal: Molecular Plant Pathology

Article Title: Distinguishing bacterial pathogens of potato using a genome‐wide microarray approach

doi: 10.1111/j.1364-3703.2008.00482.x

Figure Lengend Snippet: Pooled DNA of the strains of Streptomyces scabies (Sca) and S. turgidiscabies (Stu) analysed on the microarray. (A) Scatterplot showing signal intensities from each probe on the array. Signals for Sca are given on the x‐axis and those for Stu on the y‐axis. (B) The scatterplot presented on a logarithmic scale places the probes within four groups: (1) high signal intensities for both samples [of the total of 3894 probes designed to target genes of Sca, 1462 probes (c. 40%) show high signal intensities also for Stu]; (2) non‐specific probes giving relatively weak signals for both samples; (3) probes giving high signals only for Stu; and (4) probes giving high signals only for Sc. In (C) (Sca) and (D) (Stu), the histograms of the logarithmic signal intensities show three peaks corresponding to the groups of probes in B, as explained in Fig. 2. In (E) (Sca) and (F) (Stu) the hybridization signal intensities are indicated per three groups of probes. Interpretation of the boxplots is as in Fig. 1. The data indicate that the probes targeting the 16S–23S intergenic spacer (IGS) can be used to distinguish the two species.

Article Snippet: Image and data analysis Microarray slides were scanned with a GenePix 4200 AL scanner (Axon Instruments, Foster City, CA) using a pixel resolution of 5 μm.

Techniques: Microarray, Hybridization