Download Supplement_2

Transcriptional response to muscle ischemia in rats with CKD
SUPPLEMENT
Genes
Primer-sequence 5'-3'
18S
fw: TTGATTAAGTCCCTGCCCTTTGT
rv: CGATCCGAGGGCCTCACTA
fw: AACGAAAGCGCAAGAAATCC
rv: GCTCACAGTGAACGCTCCAG
fw: CGACACTCTTTTGGCTCCTTCTAAC
rv: TGACAGGTAGTCCGTCTTTACTTCG
fw: CCACCCCAGAAATGTACCAAAC
rv: AAAACGCGGGTCTCTGGTT
fw: TGGAAGTGGATCCACGCGCCCAAGG
rv: GCAGGAGCGCACGATCATGTTGGAC
fw: CTCGTCCATGGTGCTGACCAAG
rv: CCGCTGCGAGTCGTTGAAGTAG
fe: TATGGAGCACCACTGATGGA
rv: TCAGCCAGGCTTTGAGATTT
fw: CCTCCACCACTATGCAGGTCTC
rv: GCACGTGGATGCTACAGGC
fw: AAACCACACGGCCACCAT
rv: TGGATTTCAAGACGGGATGT
fw: GACCAGTGGGCATCGCTACG
rv: CATTGTCCGAATCCTTGTGCT
fw: CAAGCCATCTGCGGCATCTC
rv: CAGGACACCTTTTTGCAGA
fw: CAGACCTCAAGTATGAGTGCAGAG
rv: GGTGTTGGCAGCACACTG
fw: AAAGTGGCTGAGTTTGGCAG
rv: AAGTGGCTACAGCATCTGAGTGT
Probe (5' FAM, 3' TAMRA): TCAGAGGAGAAGGCGCATTACAGCA
fw: GCCACGGAATCCAGATTTGT
rv: GGGAAGGCTTCGGGTCAT
fw: CTGGCTCACGCCATTCG
rv: CTGTACTTCTTGTCCTCATAGCTTGTG
fw: GCCCATGCATTTAATTTGATCA
rv: TGAGGATCACAAGCCATGACA
fw: AGGGCTGTGTCTGCAAAGGT
rv: GCAGCACTGTTCGTCACTTCAG
fw: CTCCCGCTTTGATGTCATTCA
rv: CCCACACTCATGTCTAGGTGGTT
fw: TGTGGGCCCAACCAGATG
rv: CTTGGGAATCTTTTGTTTCTTGACA
fw: TGCCAGGGATTCACCAGTGT
rv: GGCAGGTGCTATGACGAGAAG
fw: TCCCCCATTCCATGTGACA
rv: CAACAGGTACGGCTGCCATA
fw: TCAGCCTACAAAGGACACAATCTC
rv: GAGCTGTTGGGAAAGGACATTTT
Vegfa
Flt1
Kdr
Tgfb1
Hsp70
Pgf
Ccl2
Angpt1
Angpt2
Thbs1
Thbs2
Spp1
Timp1
Gstm2
Gprc51
Mt1a
Prkag3
Ccl7
Csrnp1
E2f8
Hoxd10
Supplement 1 Primer for real-time RT-PCR
Transcriptional response to muscle ischemia in rats with CKD
Symbol
Gene title
Probeset ID
p-value
FC
Prkab2
protein kinase. AMP-activated. beta 2 non-catalytic subunit
1369271_at
0.020
2.02
Klhdc3
kelch domain containing 3
1392378_at
0.014
2.02
Rbfox1
RNA binding protein. fox-1 homolog (C. elegans) 1
1378371_at
0.016
2.14
Tpm1
tropomyosin 1. alpha
1395794_at
0.031
2.17
Myod1
myogenic differentiation 1
1398655_at
0.010
2.19
Col7a1
procollagen. type VII. alpha 1
1374226_at
0.047
2.35
Eml1
Echinoderm microtubule associated protein like 1
1390174_at
0.031
2.36
Gstm2
glutathione S-transferase mu 2
1370952_at
0.038
2.42
similar to germinal histone H4 gene
1369728_at
0.001
2.48
Camk2a
Calcium/calmodulin-dependent protein kinase II alpha
1381637_at
0.013
2.68
Neu2
sialidase 2 (cytosolic sialidase)
1368127_at
0.026
2.79
Hist4h4
Supplement 2 All upregulated genes of SNX I vs. Sham I (FC > 2, p < 0.05), I = ischemic, NI = nonischemic, FC = Fold Change
Ratio
Gene
Symbol
Title
PCR
Microarray
PCR
Microarray
PCR
Microarray
Sham I/NI
Sham I/NI
SNX I/NI
SNX I/NI
SNX I/Sham I
SNX I/ Sham I
Vegfa
vascular endothelial growth factor A
2.05
1.75
1.67
1.38
0.46
0.54
Flt1
vascular endothelial growth factor receptor 1
1.50
1.24
1.31
1.10
0.68
0.85
Kdr
vascular endothelial growth factor receptor 2
2.04
1.72
1.96
1.89
0.64
0.88
Angpt1
angiopoietin 1
1.04
0.84
1.00
0.89
1.01
1.17
Angpt2
angiopoietin 2
2.81
2.53
2.90
2.29
0.75
0.69
Ccl2
chemokine (C-C motif) ligand 2
68.80
53.13
30.57
29.14
0.16
0.31
Ccl7
chemokine (C-C motif) ligand 7
28.36
43.09
18.80
11.50
0.33
0.22
Hsp70
heat shock 70kD protein 1A
3.93
2.55
4.23
3.50
0.42
0.63
Il6
interleukin 6
40.25
2.88
11.10
1.02
0.37
0.36
Mt1a \\\
Ttr
metallothionein 1a /// transthyretin
15.39
40.58
7.43
11.68
0.26
0.22
Timp1
TIMP metallopeptidase inhibitor 1
12.59
12.81
10.04
10.60
0.44
0.40
E2f8
E2F transcription factor 8
10.24
5.59
9.20
3.29
0.18
0.25
Pgf
placental growth factor
1.67
2.76
1.09
1.21
0.59
0.33
Tgfb1
transforming growth factor. beta 1
3.56
1.39
3.04
1.04
0.79
0.73
Thbs2
thrombospondin 2
1.67
1.54
2.12
1.05
0.66
0.65
Spp1
osteopontin
2.46
13.13
8.67
35.02
0.85
0.45
Gstm2
glutathione S-transferase mu 2
1.51
0.58
0.82
0.81
1.25
2.42
1.11
0.49
0.88
0.60
1.14
1.79
Prkag3
Csrnp1
protein kinase. AMP-activated. gamma 3 noncatalytic subunit
cysteine-serine-rich nuclear protein 1
8.54
8.57
3.18
3.63
0.17
0.14
Gprc5a
G protein-coupled receptor. family C. group 5.
member A
10.13
6.19
13.20
2.39
0.60
0.38
Hoxd10
homeo box D10
1.03
0.49
1.50
1.10
1.85
2.55
Supplement 3 Overview of all ratios for the relative expression changes in both analytic procedures
(PCR and Microarray), I = ischemic, NI = non-ischemic.
Transcriptional response to muscle ischemia in rats with CKD
Symbol
Gene title
Probeset ID
p-value
FC
Spp1
secreted phosphoprotein 1
1367581_a_at
0.003
35.02
Ccl2
chemokine (C-C motif) ligand 2
1367973_at
0.000
29.14
Ccl7
chemokine (C-C motif) ligand 7
1379935_at
0.000
11.5
Timp1
TIMP metallopeptidase inhibitor 1
1367712_at
0.002
10.6
Tnfrsf12
tumor necrosis factor receptor superfamily. member
12A
1371785_at
0.001
7.08
Cd44
CD44 molecule (Indian blood group)
1387952_a_at
0.003
5.8
Apln
apelin
1389651_at
0.000
5.79
Hmox1
heme oxygenase (decycling) 1
1370080_at
0.004
5.57
Junb
jun B proto-oncogene
1387788_at
0.000
5.41
Runx1
runt-related transcription factor 1
1368914_at
0.003
5.02
Serpine1
serpin peptidase inhibitor. clade E
1368519_at
0.004
4.95
Myc
v-myc myelocytomatosis viral oncogene homolog
(avian)
1368308_at
0.002
4.29
Col8a1
collagen. type VIII. alpha 1
1393891_at
0.009
4.01
Lgals3
lectin. galactoside-binding. soluble. 3
1386879_at
0.014
3.65
Fn1
fibronectin 1
1370234_at
0.003
3.6
Alcam
activated leukocyte cell adhesion molecule
1370043_at
0.021
3.52
Tnc
tenascin C
1373401_at
0.002
3.48
Klf5
Kruppel-like factor 5 (intestinal)
1368363_at
0.002
3.43
Anxa2
annexin A2
1367584_at
0.02
2.97
Icam1
intercellular adhesion molecule 1
1387202_at
0.002
2.7
F2r
coagulation factor II (thrombin) receptor
1367899_at
0.000
2.63
Ccnd1
cyclin D1
1371643_at
0.006
2.54
Col4a1
collagen. type IV. alpha 1
1372439_at
0.000
2.47
Gadd45a
growth arrest and DNA-damage-inducible. alpha
1368947_at
0.019
2.45
Sema3b
sema domain. immunoglobulin domain (Ig)
1377336_at
0.003
2.44
Aangpt2
angiopoietin 2
1374207_at
0.000
2.29
Igfbp3
insulin-like growth factor binding protein 3
Ednrb
endothelin receptor type B
Stat3
1386881_at
0.001
2.26
1387146_a_at
0.009
2.22
signal transducer and activator of transcription 3
1371781_at
0.002
2.14
Vim
vimentin
1367574_at
0.003
2.13
Hspb1
heat shock 27kDa protein 1
1367577_at
0.017
2.08
Ehd4
EH-domain containing 4
1374399_at
0.043
2.08
Rgcc
regulator of cell cycle
1368080_at
0.016
2.07
Ctsb
cathepsin B
1367646_at
0.029
2.06
G6pd
glucose-6-phosphate dehydrogenase
1367856_at
0.019
2.06
Cxcl12
chemokine (C-X-C motif) ligand 12
1387655_at
0.009
-2.03
Supplement 3 All up- or down-regulated angiogenesis related genes of SNX I vs. SNX NI (FC > 2 and
FC < -2, p < 0.05), I = ischemic, NI = non-ischemic, FC = Fold Change
Transcriptional response to muscle ischemia in rats with CKD
Symbol
Gene title
Probeset ID
p-value
FC
Ccl2
chemokine (C-C motif) ligand 2
1367973_at
0.000
53.13
Ccl7
chemokine (C-C motif) ligand 7
1379935_at
0.000
43.11
Timp1
TIMP metallopeptidase inhibitor 1
1367712_at
0.002
12.81
Spp1
secreted phosphoprotein 1
1367581_a_at
0.035
13.13
Junb
jun B proto-oncogene
1387788_at
0.000
11.11
Serpine1
serpin peptidase inhibitor. clade E
1368519_at
0.000
10.44
Hmox1
heme oxygenase (decycling) 1
1370080_at
0.003
7.69
Itgb2
integrin. beta 2
1383131_at
0.029
6.75
Myc
v-myc myelocytomatosis viral oncogene homolog (avian)
1368308_at
0.001
6.39
Runx1
runt-related transcription factor 1
1368914_at
0.004
5.56
Col8a1
collagen. type VIII. alpha 1
1393891_at
0.010
4.60
Tnfrsf12
tumor necrosis factor receptor superfamily. member 12A
1371785_at
0.015
4.27
Lgals3
lectin. galactoside-binding. soluble. 3
1386879_at
0.014
4.19
Efna1
ephrin-A1
Cd44
CD44 molecule (Indian blood group)
Adm
Apln
1372844_at
0.000
3.92
1387952_a_at
0.034
3.60
adrenomedullin
1387219_at
0.004
3.57
apelin
1389651_at
0.013
3.48
S1pr3
sphingosine-1-phosphate receptor 3
1376150_at
0.000
3.22
Klf5
Kruppel-like factor 5 (intestinal)
1368363_at
0.008
3.11
F2r
coagulation factor II (thrombin) receptor
1367899_at
0.000
2.89
IL6
interleukin 6 (interferon. beta 2)
1369191_at
0.028
2.88
Igfbp3
insulin-like growth factor binding protein 3
1367652_at
0.000
2.87
Stat3
signal transducer and activator of transcription 3
1370224_at
0.001
2.74
Gadd45a
growth arrest and DNA-damage-inducible. alpha
1368947_at
0.022
2.65
Ptafr
platelet-activating factor receptor
1383372_at
0.022
2.58
Cxcl9
chemokine (C-X-C motif) ligand 9
1373544_at
0.047
2.55
Angpt2
angiopoietin 2
Ednrb
endothelin receptor type B
Icam1
Ehd4
Nab2
NGFI-A binding protein 2 (EGR1 binding protein 2)
Cxcr4
chemokine (C-X-C motif) receptor 4
Ctsb
G6pd
1374207_at
0.000
2.53
1387146_a_at
0.007
2.53
intercellular adhesion molecule 1
1387202_at
0.007
2.53
EH-domain containing 4
1372106_at
0.015
2.42
1374925_at
0.000
2.38
1373661_a_at
0.049
2.28
cathepsin B
1367646_at
0.030
2.23
glucose-6-phosphate dehydrogenase
1367856_at
0.024
2.15
Tnfrsf1
tumor necrosis factor receptor superfamily. member 1A
1367715_at
0.000
2.13
Ptgs2
prostaglandin-endoperoxide synthase 2
1368527_at
0.004
2.08
Ccnd1
cyclin D1
1371150_at
0.046
2.06
Fgr
Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene
homolog
1368693_at
0.049
2.04
Supplement 4 All up-regulated angiogenesis related genes of Sham I vs. Sham NI (FC > 2, p < 0.05), I
= ischemic, NI = non-ischemic, FC = Fold Change
Transcriptional response to muscle ischemia in rats with CKD
Symbol
Gene title
Probeset ID
p-value
FC
Gem
GTP binding protein overexpressed in skeletal
muscle
1382351_at
0.004
-3.7
Serpine1
serpin peptidase inhibitor. clade E
1368519_at
0.029
-3.29
Ccl2
chemokine (C-C motif) ligand 2
1367973_at
0.031
-3.28
Myc
v-myc myelocytomatosis viral oncogene homolog
1368308_at
0.015
-3.18
Cxcl9
chemokine (C-X-C motif) ligand 9
1373544_at
0.019
-2.95
Il6
interleukin 6 (interferon. beta 2)
1369191_at
0.025
-2.79
Ctgf
connective tissue growth factor
1367631_at
0.024
-2.28
Adm
adrenomedullin
1387219_at
0.041
-2.2
Nr4a1
nuclear receptor subfamily 4. group A. member 1
1386935_at
0.027
-2.17
Junb
jun B proto-oncogene
1387788_at
0.009
-2.17
Cxcl14
chemokine (C-X-C motif) ligand 14
1388485_at
0.000
-2.08
Efna1
ephrin-A1
1372844_at
0.004
-2.05
Ptgs1
prostaglandin-endoperoxide synthase 1
1368259_at
0.008
-2.04
Supplement 5 All down-regulated angiogenesis related genes of SNX I vs. Sham I (FC > 2 and FC < 2, p < 0.05), I = ischemic, FC = Fold Change
Abbreviation
Explanation
ANOVA
analysis of variance
cDNA
complementary deoxyribonucleic acid
CKD
chronic kidney disease
cRNA
complementary ribonucleic acid
dl
deciliter
FC
fold change
g
gram
GEO
Gene Expression Omnibus
h
hour
I
ischemic
IPA
Ingenuity Pathway Analysis
IPKB
Ingenuity Pathways Knowledge Base
mg
millilgram
mRNA
messenger ribonucleic acid
NCBI
National Center for Biotechnology Information
NI
non-ischemic
nm
nanometer
RIN
RNA-Integrity-Number
RNA
ribonucleic acid
RT-PCR
realtime polymerase chain reaction
SNX
5/6 nephrectomy
vs
versus
Supplement 7 Overview of used abbreviations and their explanations.