Transcript Supplementary Figures 1–9 (ppt 3704K)

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log fold change
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RASP
SEB
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Figure S1: (A) Table shows number of genes that passed Welch’s t-test at different q-values (FDR corrected p-values)
and Fold Change cut-offs. (B) Immune system process was identified as the most enriched biological process upon
functional enrichment using hypergeometric distribution test with FDR correction (q <0.05), and Fisher Exact Test (p <
0.05) on 1396 RASP regulated transcripts. The immune system process was associated with 177 of 1396 transcripts.
Line graph of the177 immune response genes across groups of pre- and post-RASP leukocytes that were also
exposed to SEB toxin. Among the 177 transcripts, 26 (red lines) were up-regulated and 151 (blue lines) were downregulated in post-RASP leukocytes.
A)
B)
C)
Figure S2: Expression changes of genes important for: (A ) pattern recognition receptors , inflammatory response,
chemokinesis and activation of myeloid leukocytes; (B) antigen presentation, T- , B- & NK-cells activations; and
(C) Transcription regulations. We used two different total RNA isolation methods and different microarray platforms:
Trizol RNA isolation and cDNA microarrays (upper panels - purple bar) ; PAXgene RNA isolation and oligonucleotide
arrays (lower panels - blue bar).
Figure S3: Real time PCR array analyses of transcripts involved in chemotaxis, inflammation, and antigen preparation
and presentation pathways . Control (pre-RASP); week 5 (about mid-way of the RASP program); week 8 (post-RASP)
E)
STK4
STAT1
RELB
RELA
REL
NFKB2
NFKB1
IKBKG
Fold change
TNFRSF10B
TLR7
TICAM2
TNFAIP3
TNF
IL8
IL1R1
TLR9
TLR4
TLR3
IFNGR1
IFIT3
Fold change
HLA-A
IFIT3
ICAM2
**
ICAM1
HLA-DQB1
HLA-DMA
CD74
*
CDKN1A
-6
0
-1
-2
-3
-4
-5
-6
-7
***
*
**
CD44
CD8A
ITGAM ITGB2
LYN
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-1
-4
-10
0
-1
-2
-3
-4
-5
-6
-7
-8
-9
CD4
-2
-8
Fold change
D)
0
CD40
Fold change
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C)
TLR2
ACTB
3
2
1
0
-1
-2
-3
-4
-5
CXCL1
Fold change
A)
-2
-3
-4
-5
oligonucleotide microarray
-6
cDNA microarray
real time PCR array
*
Figure S4: Comparisons of fold changes of transcript levels determined by real time PCR array and cDNA and oligonucleotide microarrays.
Shown here are genes significantly associated with: (A) pattern recognition receptors; (B) inflammatory response [to scale the graph, fold
changes of -15.2 and -23.8, labeled * and **, respectively, were assigned a values of ~5 and 6, respectively]; (C) antigen preparation and
presentation (*fold change: -12.3; assigned value ~ -5 for scaling the graph); (D) transcription factors (* fold change: -12.6; ** fold change: 12.3; *** fold change: -14; these were adjusted to around -5 for scaling the graph); (E) T-cell activation, differentiation and proliferations.
Expression profiles of genes shown in panels A-E were assayed using SABiosciences RT² Profiler™ (PAHS 406 and PHAS 25) PCR Arrays, cDNA
microarrays, and oligonucleotide microarrays. Total RNA samples were isolated using Trizol® reagents for cDNA microarray analysis, and total
RNA samples used for PCR and oligonucleotide arrays were isolated from blood samples collected in PAXgene® tubes. (Note: Study subject
for the PCR arrays were the 10 soldiers who started and completed the RASP).
RASP
SEB
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Figure S5: Expression pattern of RASP suppressed 151 immune response genes in pre- and post-RASP leukocytes
which were also exposed to SEB toxin. These is the same data as that of Figure 4A, without linear transformation
against pre-RASP control groups
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B
fold change
Figure S6: (A) Regulatory interaction among stress regulated microRNAs (miRs), important transcription factors
(NFkB1, NR3C1, SATB1), inflammatory cytokines and antigen presenting molecules;
(B) Seven stress-suppressed miRs targeting 48 mRNAs among 288 mRNAs that passed q < 0.001 and 1.5 fold change.
Enriched pathways include IL-17A and IL-8 signaling, and NFkB activation pathways.
fold change
Figure S7: Transcription factors with absolute z-score > 3.0, and their regulatory targets. Targets were
identified among stress altered transcripts (288 transcripts, Fig. 1A) that passed Welch’s t-test with
FDR correction (q < 0.001) and 1.5 fold change cut off. The left panel shows pathways associated
with these genes.
fold change
Figure S8: Transcription factors targeting transcripts which were differentially regulated among RT-PCR assayed
transcripts. Both MYC and NR3C1 were predicted to be activated (according to prediction z-score values, z-score > 2.5).
Top functions associated with these targets were apoptosis of leukocytes, hematopoiesis, proliferation of blood cells,
and immune response. Top pathways are shown in the table (left panel).
Figure S9: Canonical pathways significantly
associated with RASP (battlefield-like stress )
regulated genes that passed Welch’s t-test
and FDR correction (p <= 0.001) and 1.5 Fold
change. Numbers on the right side indicate
total # of genes in the pathway.