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1.
Figure 6

Figure 6. Clustering compression for primary oxygen tension transitional k-means analysis.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

(A) Specific description of compression of k-means clusters into morphometric clusters (A–K) and eventually four group clusters (ONE-FOUR). (B) Numerical z ratio comparisons of individual morphometric clusters that comprise the larger group clustering (ONE-FOUR).

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
2.
Figure 2

Figure 2. Venn diagram analysis of oxygen tension-dependent transcriptional responses in primary astrocytes.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

(A) Three-way proportional Venn diagram for transcriptional responses (compared to ambient 20% O2) for 24 hour exposure to 1% (red circle), 4% (blue circle) and 9% (green circle) ambient oxygen tension. The intersection subsets (A–G) are specified in the associated boxed key. (B) Oxygen tension-dependent variations in transcriptional regulation of multi-tension responsive genes from intersection G (1% O2, red; 4% O2, blue; 9% O2, green).

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
3.
Figure 4

Figure 4. Venn diagram analysis of oxygen tension-dependent transcriptional PAGE gene collection population in primary astrocytes.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

(A) Three-way proportional Venn diagram for transcriptional responses (compared to ambient 20% O2) for 24 hour exposure to 1% (red circle), 4% (blue circle) and 9% (green circle) ambient oxygen tension. The intersection subsets (A–G) are specified in the associated boxed key. (B) Oxygen tension-dependent variations in transcriptional regulation of multi-tension responsive genes from intersection G (1% O2, red; 4% O2, blue; 9% O2, green).

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
4.
Figure 3

Figure 3. Focused MSigDB PAGE pathway collection analysis of oxygen tension-dependent gene transcription.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

(A) Oxygen-tension related PAGE gene pathway collections generated by the multiple oxygen tension environments. (B) Receptor signaling-related PAGE gene pathway collections generated by the multiple oxygen tension environments. (C) Energy regulation-related PAGE gene pathway collections generated by the multiple oxygen tension environments. (D) Aging- related PAGE gene pathway collections generated by the multiple oxygen tension environments. (E) Cytokine-related PAGE gene pathway collections generated by the multiple oxygen tension environments. (F) Translation and stress-response- related PAGE gene pathway collections generated by the multiple oxygen tension environments. The magnitude of the specific pathway collection cumulative z-scores are indicated in the scale at the bottom of each histogram (AF). Filled color bars indicate PAGE collections populated with genes with a positive cumulated z-score, striped bars indicate PAGE collections populated with genes with a negative cumulated z-score. Each PAGE pathway collection group was significantly populated by at least two separate genes with a co-probability of p≤0.05.

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
5.
Figure 8

Figure 8. Canonical signaling pathway analysis of group cluster composition.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

(A) Four way Venn diagram analysis of significantly populated canonical signaling pathways populated by group clusters ONE, TWO, THREE and FOUR. (BE) Proportional pie charts created from multiple associated canonical signaling pathway hybrid scores generated by the group cluster-unique (B-ONE unique; C-TWO unique; D-THREE unique; E-FOUR unique) signaling pathways populated by at least two genes with a co-probability of at least ≤0.05. Associated percentage values indicate the relative distribution of the respective pathway scores for each unique group cluster.

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
6.
Figure 10

Figure 10. Comparative analysis of group cluster gene correlation scores to astrocytic and neurophysiological activity terms.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

(A) Representation of the mean transcript LSI score for the four group cluster genesets analyzed with nine separate LSI GeneIndexer interrogation terms. The numerical value of each bar was calculated by the multiplication of the number of implicitly correlated genes for each group cluster (ONE-FOUR)-interrogation term (1–9) pair, with the number of implicitly-correlated genes present for each group cluster-interrogation term pair. Statistical significance is indicated as follows: TWO, THREE, FOUR compared to ONE, p≤0.05 = a, p≤0.01 = a′, p≤0.001 = a″; THREE, FOUR, compared to TWO, p≤0.05 = b, p≤0.01 = b′, p≤0.001 = b″; FOUR compared to THREE, p≤0.05 = c, p≤0.01 = c′, p≤0.001 = c″. (B) Mean cumulative transcript LSI scores (± standard error) for all of the nine interrogation terms for each complete group cluster. Statistical significance was estimated with a Student's t-test using GraphPad Prism version 5.02 (GraphPad Software Inc.). p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***.

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
7.
Figure 7

Figure 7. Astrocyte oxygen tension-dependent transcript group cluster structure.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

Panels (A) (group ONE), (B) (group TWO), (C) (group THREE) and (D) (group FOUR) represent the mean ± standard error, group cluster z ratios generated from k-means clustering across the three experimental oxygen tensions. (E) Coomassie-stained gel demonstrating equal total protein loading from astrocyte cultures exposed to the multiple oxygen tensions. Representative western blots for exemplar proteins from each group are depicted and additionally quantified in panels (FI). Protein expression is represented via measurement of immunoreactive absorbance units with background subtraction per square pixel ((AU-B)/px2). Statistical significance was estimated with a Student's t-test using GraphPad Prism version 5.02 (GraphPad Software Inc.). p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***.

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
8.
Figure 5

Figure 5. K-means clustering analysis of oxygen tension-dependent transcriptional responses in rat primary astrocytes.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

(A) Hierarchical K-means clustering of gene transcriptional responses in astrocytes to 24 hour exposure to 1, 4 or 9% O2 conditions relative to the transcriptional expression levels at 20% O2 tension. K-means clustering attempts to group together transcriptional activity with similar trends across the multiple O2 tensions. Colored blocks and the associated dendrogram structure indicate the gross nature of the identified hierarchical k-means functional activity clusters. (B) Histogram represents the numbers of significantly regulated genes in the most distinct gene transcriptional activity clusters numbered 1 to 40. Hierarchical clustering was performed and mathematically assessed using DIANE 6.0/JMP across the three oxygen tensions.

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
9.
Figure 9

Figure 9. Latent Semantic Indexing analysis of group clusters ONE, TWO, THREE, FOUR with astrocytic and neurophysiological activities.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

Significantly regulated transcripts from the four respective group clusters (ONE-FOUR) were individually analyzed by latent semantic indexing (LSI) using GeneIndexer with the following user-defined interrogation terms: 1-neurodegeneration; 2-Alzheimer's; 3-aging; 4-ischemia; 5-neuroprotective; 6-cognition; 7-hyperoxia; 8-hypoxia; 9-astrocyte. (A) Block matrix of the ten highest LSI correlating genes from group ONE according to each interrogation term (numbered 1–9 atop the block). Four selected genes were verified from each group cluster LSI block. The quantitative and statistical analysis of at least three separate westerns for each chosen protein is indicated in each panel. Statistical significance of the variation of the 1% and 9% datapoints from the 4% O2 datapoint is demonstrated in a manner similar to that previously performed (Figure 7A–D). This similar form of statistical analysis was subsequently performed for panels B–D. (B) Block matrix of the ten highest LSI correlating genes from group TWO. (C) Block matrix of the ten highest LSI correlating genes from group THREE. (D) Block matrix of the ten highest LSI correlating genes from group FOUR. Input interrogation terms not implicitly correlating with any significantly regulated transcripts in panels (C) and (D) were omitted from the matrix. Statistical significance was estimated with a Student's t-test using GraphPad Prism version 5.02 (GraphPad Software Inc.). p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***.

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.
10.
Figure 1

Figure 1. Primary transcriptional oxygen tension-dependent responses in astrocytes.. From: Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes.

(A) Principal component analysis of triplicate transcriptomic datasets generated from primary astrocytes exposed to 1 (z 1% O2 # 1, 2, 3), 4 (z 4% O2 # 1, 2, 3), 9 (z 9% O2 # 1, 2, 3) or 20% (z 20% O2 # 1, 2, 3) ambient oxygen conditions for 24 hours. Inset panel presents an RNA electropherogram of isolated RNA from the various samples (kilo-basepair (kbp) mass – M; control – C; 1% O2 – 1%; 4% O2 – 4%; 9% O2 – 9%) as well as a representative 28S(green)/18S(pink) fluorescence quantification trace employed for RNA quality control. (B) Left panel, individual gene transcriptional z ratios (with associated heatmap key) and gene selector scores (based on criteria mentioned in Methods: right panel) for gene array datasets from astrocytes experiencing 1, 4, 9% O2 conditions relative to the expression level at 20% O2. Gene selector scores of ±3 indicate compliance with stringent limitations of z ratio, FDR and probability. (C) z ratio expression profile of significantly regulated transcripts in astrocytes exposed to 1% (red circles), 4% (blue) or 9% (green) ambient O2 conditions. (D) Mean ± standard deviations of z ratios for significantly, up- or down-regulated, genes after exposure to 1, 4, or 9% O2 conditions. The specific numbers of up or downregulated genes are indicated in white in each histogram bar. Statistical analysis, Student's t-test, was performed using GraphPad Prism version 5.02 (GraphPad Software Inc.). p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***. Non-significant differences are indicated by numerical p values.

Wayne Chadwick, et al. PLoS One. 2011;6(6):e21638.

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