Correlation of GIT2 and GIT2s expression with metabolic phenotype changes in aging rats.

<p>(A) Age-dependent changes in mean body weight for the multiple age ranges of rats. (B) Fasting blood glucose measurements for the multiple age ranges of rats. (C) Fasting plasma insulin measurements for the multiple age ranges of rats. (D) Fasting plasma leptin measurements for the multiple age ranges of rats. (E) Fasting plasma adiponectin measurements for the multiple age ranges of rats. (F) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat hypothalamic extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. (G) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat whole pancreatic extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. (H) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat whole liver extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. (I) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat hind-limb skeletal muscle extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. (J) GIT2 and GIT2s age-dependent expression in young (Y), middle-aged (M), and old rat somatic adipose tissue extracts. The associated histograms depict the mean ± SEM of the GIT2 and GIT2s expression in middle-aged (grey bars) and old (black bars) animals relative to the young (white bars) samples. Statistical significance is as follows: * = p<0.05, ** = p<0.01, *** = p<0.001.</p

KEGG signaling pathway analysis of aging-related hypothalamic proteins.

<p>Proteins significantly regulated in middle (M) or old (O) aged animals compared to young (Y) animals were used as input data for KEGG signaling pathway population analysis. (A) Venn diagram analysis of middle-aged (grey line) and old-aged (black line) significantly-regulated KEGG pathways demonstrated 27 common KEGG terms between old and middle-aged tissues. The common (27) significantly populated pathways for middle-aged (grey bars) and old aged (black bars) animals were then rationally clustered into subgroups focused upon disease pathology (B), neurophysiological activity (C), and intermediary cellular signaling activity (D). For each significantly-populated KEGG pathway a ‘hybrid’ score was generated which represents the −log₁₀ of the enrichment probability multiplied with the relative enrichment factor compared to the background proteomic expression.</p

Age-dependent expression profile of GIT2 in central nervous system tissues.

<p>(A) Expression profiles across three randomly chosen hypothalamic samples (Young, Y₁-Y₂-Y₃; Middle, M₁-M₂-M₃; Old, O₁-O₂-O₃) for ERK1/2, GRIT, GIT2, GIT2short (GIT2s), GIT1, β-PIX, and PAK1. The loading protein input control with coomassie staining of the gel is also indicated below. (B) Quantification (mean ± SEM) of age-dependent alterations in protein expression for middle aged (grey bars) or old (black bars) animals compared to the young controls (white bars). (C) Brain region-specific alterations of GIT2 and GIT2s in young (Y), middle (M), and old (O) age animals. Quantification of multi-brain region GIT2 (D) and GIT2s (E) expression across the experimental age-span (n = 10). Multiple brain region expression levels relative to hypothalamus of GIT2 in young (F), middle-aged (G), and old animals (H). Multiple brain region expression levels, relative to hypothalamus of GIT2s in young (I), middle-aged (J), and old animals (K). Statistical significance is as follows: * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Age-dependent protein expression changes in the rat hypothalamus.

<p>(A) Venn diagram analysis of significantly-regulated (p<0.05) proteins in the middle versus young (grey circle) and the old versus young (black circle) antibody array analyses. Proteins uniquely regulated in either middle age (22) or old age (64) animals are subsequently broken down into up-regulated (red numbers) or down-regulated (green numbers) groups. The 84 commonly-regulated proteins were further dissected into different regulatory-behavior groups represented in panels B–E. For each regulation-behavior group an exemplary protein is verified from the animal hypothalamic pools (Y–young, M–middle aged, O–old). (B) Proteins up-regulated in both M (grey bars) and O (black bars) hypothalami compared to Y animals, with O ratio >M ratio. The associated validation was performed using western blot for caspase 3 (Casp3). (C) Proteins up-regulated in both M (grey bars) and O (black bars) hypothalami compared to Y animals, with M ratio >O ratio. The associated validation was performed using western blot for Ran. (D) Proteins down-regulated in both M (grey bars) and O (black bars) hypothalami compared to Y animals, with M ratio >O ratio and O ratio >M ratio. The associated validation was performed using western blot for vinculin (Vcl). (E) Proteins differentially regulated between M or O timepoints relative to Y (up-regulated at M and down-regulated at O (verified using junction plakoglobin-Jup), or down-regulated at M and up-regulated at O (verified using nitric oxide synthase 1-Nos)). For each verification, data on each histogram is represented as mean ± SEM from the multiple animal pools. Statistical significance is as follows: * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Latent semantic indexing correlations of KEGG signaling pathways terms with proteins.

<p>(A) Latent semantic indexing (LSI) interrogation matrix between input significantly-regulated KEGG signaling pathway terms. Colored blocks represent the individual LSI implicit correlation of the specific protein (vertically organized on left of heatmap: 1–2524 – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036975#pone.0036975.s024" target="_blank">Table S20</a>) with the respective KEGG term (1-Regulation of actin cytoskeleton, 2-Chemokine signaling, 3-Alzheimer's disease, 4-Focal adhesion, 5-MAPK signaling, 6-Gap junction, 7-GnRH signaling, 8-Long term potentiation, 9-Notch signaling, 10-VEGF signaling, 11-p53 signaling, 12-Calcium signaling). The number of KEGG signaling pathway correlations for each protein is indicated by the color of the respective heatmap blocks (9 correlations-red; 8 correlations-orange; 7 correlations-yellow; 6 correlations-green; 5 correlations-light blue; 4 correlations-dark blue; 3 correlations-purple; 2 correlations-grey). (B) Mean ± SEM for the total implicitly-correlating proteins for each of the 12 input KEGG signaling pathways. (C) Box and whisker plot with 1–99% statistical cut-offs (GraphPad Prism) of the number of specific correlations to KEGG pathways each protein possessed. Twelve proteins demonstrated a statistically-significantly greater number of KEGG pathway correlations compared to the total protein mean number of correlations (*** = p<0.001). (D) Expanded heatmap identification of specific proteins possessing a significantly greater number of KEGG pathway correlations compared to the mean number of KEGG pathway correlations for all implicit proteins. (E) Mean ± SEM of LSI correlation scores (across all 9 correlations) for Grit and GIT2.</p

Age-related proteomic alterations in the hypothalamus.

<p>(A) Coomassie staining of pooled input hypothalami for Cy-dye labeling and hybridization with Cell Signaling antibody array: Y–young animal pool; M–middle-aged animal pool; O–old animal pool. (B) Prototypic single channel and merge images from scanned Panorama® Cell Signaling Antibody array chips. Specific antibody species are printed in duplicate across the chip. (C) Prototypic examples of protein results for factors up-regulated, down-regulated, or unchanged in either Cy3 or Cy5 channels are indicated. Positive and negative hybridization controls from the chips are also indicated. (D) Protein expression ratios (<0.5 or >1.5 ratio: middle versus young) for proteins in middle aged versus young hypothalami. Datapoints plotted represent the mean ± standard error of mean (SEM) from three separate experimental hybridizations which included Cys-Cy5 dye swaps for the samples. (E) Protein expression ratios (<0.5 or >1.5 ratio: old versus young) for proteins in old aged versus young hypothalami. Datapoints plotted represent the mean ± SEM from three separate experimental hybridizations which included Cys-Cy5 dye swaps for the samples. (F–K) Western blot validation of specifically identified proteins, from Panorama® array analysis, and their age-dependent expression trends (Y-young pool, M-middle aged pool, O-old pool). Proteins validated from pooled animal input were Myc (F), Akt-1 (G), Pyk2 (H), Map2 (I), FAK (J), and Cnp-1 (K). Data presented represents mean ± SEM from three separate blots. Statistical significance was assessed using a Student's t-test with GraphPad Prism: * = p<0.05; ** = p<0.01; *** = p<0.001. (L–Q) Expression patterns for specific proteins were also validated for each animal used as input for the Y (white circle), M (grey square), or O (black triangle) hypothalamic pools. Proteins validated from individual animal inputs were Myc (L), Akt-1 (M), Pyk2 (N), Map2 (O), FAK (P) and Cnp-1 (Q). Data on the histograms are represented as mean ± SEM from the multiple animals.</p

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