Fig. 1. Regulatory networks for PACAP-dependent transcripts in bovine chromaffin cells (BCC). Transcripts with an abundance of two-fold or greater in PACAP-treated (6 h) compared to vehicle-treated bovine chromaffin cells by two-color microarray analysis (187 transcripts; see Supplementary Table S2) were used as the input for analysis of potential networks using the signal transduction knowledge environment of Ingenuity (http://www.ingenuity.com). (a) Network solely involving 35 PACAP-regulated transcripts. (b) Network involving 16 PACAP-regulated transcripts and 12 additional components with Ingenuity-confirmed direct or indirect links to them. (c) Network involving 12 PACAP-regulated transcripts and 8 additional components with Ingenuity-confirmed direct or indirect links to them. PACAP-regulated transcripts are depicted in gray and linked components in white. Circular lines above symbols indicate auto-regulation; connecting lines without arrows indicate direct protein interaction deduced from either yeast two-hybrid, enzymatic, or immunoprecipitation assays; dashed and solid arrows indicate indirect (e.g. regulation of mRNA levels) and direct (e.g. enzymatic) activation, and lines ending in orthogonal dash indicate inhibition. The node shapes denote enzymes ( ), phosphatases ( ), kinases ( ), peptidases ( ), G-protein coupled receptor ( ), transmembrane receptor ( ), cytokines ( ), growth factor ( ), ion channel ( ), transporter ( ), translation factor ( ), nuclear receptor ( ), transcription factor ( ) and other ( ). All gene products are defined in the glossary in Supplementary Table S5, which includes active links to Entrez Gene for each component. In this and all following figures, nodes and relationships are hyperlinked to the Ingenuity-curated references validating the relationship or describing the nodal protein (see supplementary Figures for hyperlinks). 73.8% of the relationships were validated by a single literature reference in Ingenuity. For the relationships with multiple references, we chose the reference we judged to be the single most comprehensive one. Four relationships without references are under revision.

Fig. 2. Regulatory networks for PACAP-dependent transcripts in PC12 cells. Transcripts with an abundance two-fold or greater in PACAP-treated PC12 cells at 0–6 h (deduced from cycloheximide superinduction at 6 h), 6 h or 48 h (168 transcripts; see Supplementary Table S3 and references [28], [45] and [57], respectively) were used as the input for analysis of regulatory networks using Ingenuity as described in Fig. 1. (a) Network involving 15 PACAP-regulated transcripts and 5 additional components with Ingenuity-confirmed direct or indirect links. (b) Network involving 14 PACAP-regulated transcripts and 11 additional components with Ingenuity-confirmed direct or indirect links. Symbols and abbreviations are as described in Fig. 1 and Supplementary Table S5.

Fig. 3. Regulatory networks for PACAP-dependent transcripts in middle cerebral artery occlusion (MCAO). Up-regulated transcripts in wild-type compared to PACAP-deficient mice (a) 1 h and (b) 24 h after MCAO. Sixty-four and 74 transcripts, respectively (see Supplementary Table S4 and reference [15]) were used as the input for analysis of regulatory networks using Ingenuity as described in Fig. 1. (a) Network involving 15 PACAP-regulated transcripts and 17 additional components with Ingenuity-confirmed direct or indirect links. (b) Network involving 15 PACAP-regulated transcripts and 20 additional components with Ingenuity-confirmed direct or indirect links. Symbols and abbreviations are as described in Fig. 1 and Supplementary Table S5.

Fig. 4. Regulatory networks for PACAP-dependent transcripts in PC12 cells and MAG, BCC or MCAO. PACAP-regulated transcripts common to PC12 cells and either MAG, BCC or cerebral cortex in MCAO as listed in Table 2 were used as input for analysis of regulatory networks using Ingenuity as described in Fig. 1. (a) Network involving 13 PACAP-regulated transcripts and 5 additional components with Ingenuity-confirmed direct or indirect links. (b) Network involving 8 PACAP-regulated transcripts and 17 additional components with Ingenuity-confirmed direct or indirect links. Symbols and abbreviations are as described in Fig. 1 and Supplementary Table S5.

Mouse adrenal gland and bovine chromaffin cell microarray analysis

cDNA from mouse adrenal gland (MAG) of wild-type vs. PACAP-deficient animals was hybridized and represented as wild-type:PACAP-deficient ratio for each gene (i.e. up-regulation refers to greater transcript abundance in wild-type than in PACAP-deficient MAG). cDNA from bovine chromaffin cells (BCC) of 6-h PACAP-treated (100 nM PACAP-27) vs. untreated cells was hybridized and represented as PACAP-treated:untreated ratio for each gene (i.e. up-regulation refers to greater transcript abundance in PACAP-treated than in untreated cells). Each array consisted in a separate paired experiment. The number of clones per array shown above represents the gene targets passing the filter for signal quality in each set (see Section 2). Significantly up-regulated transcripts were those determined using SAM (statistical analysis of microarrays) at the false discovery rates (FDR) shown, after filtration for named genes (named up-regulated) and after further filtration to remove values less than two-fold up-regulated (named up-regulated genes >two-fold).

PACAP-dependent transcripts in PC12 cells and MAG, BCC or MCAO

PACAP-responsive transcripts identified in the six data sets described in Table 1 and Tables S1-S4 and regulated in both the anchor set (PC12 cells; Table S4) and at least one other (mouse adrenal gland, MAG; bovine chromaffin cells, BCC; mouse middle cerebral artery occlusion model, MCAO) are depicted. Filled square, transcript elevated at least two-fold; empty square, transcript not significantly elevated.