Chronic minimal peroxide-mediated alteration of protein expression and ligand responses.

<p>(A) Representative western immunoblot (IB) analysis of control (PBS-vehicle treated) or CMP treatment (7 days, 10 nM hydrogen peroxide treatment) effects upon cellular expression of lamin A (LMNA), G protein-coupled receptor kinase interacting ArfGAP 2 (GIT2), calreticulin (CALR) and calmodulin (CALM1). Quantification of CMP-mediated expression changes of LMNA (B), GIT2 (C), CALM1 (D) and CALR (E). Quantification was achieved as follows: western blot relative absorbance units (x1000) minus background absorbance subtraction per square pixel: ((AU-B)/px²). Quantification was performed using ImageQuant 5.2 and statistical analysis was performed on three independent experiments using GraphPad Prism version 5.02 with a Student’s t-test (p<0.05, *; p<0.01, **; p<0.001, ***). Data is represented as mean ± standard error of mean (SEM). (F) Log dose-response curves for acetyl-β-methycholine (MeCh)-mediated ERK1/2 activation in control (black circles) or CMP-treated (white square) cells. (G) Response-normalized MeCh ERK1/2 log dose-response curves. The maximal response for MeCh in each cellular context (control versus CMP) was considered to be 100% in each circumstance. Each experimental point on the curves (fitted using sigmoidal dose-response functions in GraphPad Prism) represents the mean ± SEM of three independent experiments.</p

VENNTURE data input and output scenarios.

<p>Simplistic data (two set analysis) input into VENNTURE is achieved using an Excel™ input file. The data representation type can be selected using a drop-down menu for numerical set analysis, set order demonstration for eventual output and also textual set content. The resultant set data output can be directly generated for PowerPoint™ (Venn diagram) and Excel™ (dataset descriptions).</p

Cellular context modification of downstream receptor signaling activity in SH-SY5Y cells.

<p>(A) Comparison of phosphoprotein/GO term group/canonical signaling pathway activation in the non-stimulated (blue circle) and multiple MeCh-stimulated conditions (red circles) in either control-state (solid line) or CMP-state (dashed lines) SH-SY5Y cells. (B) Summary of potential non-stimulated and MeCh-induced signaling functionality (aggregate of highest-scoring GO-term group and canonical signaling pathway enrichments) in a dose-dependent manner compared between control-state or CMP-state cells.</p

VENNTURE analysis of log dose-response ligand data in diverse cellular contexts.

<p>VENNTURE Venn diagram set distribution analysis (1–63 set intersections) of extracted phosphoproteins in the stimulated or non-stimulated SH-SY5Y cells in the control state (‘Control’ - A) or peroxide-treated state (‘CMP’ - B). Specific column color coding is as follows: blue - phosphoproteins unique to non-stimulated state only, red – phosphoproteins unique to a specific MeCh stimulation dose only; grey – phosphoproteins common to multiple MeCh doses but not present in the non-stimulated set; black – phosphoproteins common to multiple MeCh doses also present in the non-stimulated set; green – phosphoproteins common to all doses and the non-stimulated set. GO term enrichment analysis was performed using the initial phosphoprotein sets from cells in the control or CMP-treated states. The significantly enriched (p≤0.05) GO term groups for each non-stimulated or MeCh-stimulated set were then separated using VENNTURE in a similar manner to the actual phosphoprotein identifications. VENNTURE Venn diagram set distribution analysis of GO term groups significantly populated by the extracted phosphoproteins in the control state (C) or CMP-state (D) cells is depicted. Color-coding of the histogram is as described previously. Canonical signaling pathway enrichment analysis was also performed using the initial phosphoprotein sets from cells in the control or CMP-treated states. The significantly enriched (p≤0.05) canonical signaling pathways for each non-stimulated or MeCh-stimulated set were then separated using VENNTURE in a similar manner to the actual phosphoprotein identifications. VENNTURE Venn diagram set distribution analysis of the canonical signaling pathways significantly populated by the extracted phosphoproteins in the control state (E) or CMP-state (F) cells is depicted.</p