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Raw data VS. Residual value
A
B
25000
Gene number
20000
766
1898
44
15000
Normal
10000
Steatosis
NASH
5000
0
0
0.2
0.4
Coefficient of variation
0.6
C
495
2169
80
Supporting Fig. 1. Neither age nor gender were major drivers of expression differences
between the control and NAFLD groups. (A) The cumulative curve of coefficient of
variation of gene expression. (B) Overlap between differentially expressed genes using
either the residual values from age or the raw data. (C) Overlap between differentially
expressed genes using either the residual values from gender or the raw data.
Scaled expression values
-3
1
3
Age (years)
0
35
70
Supporting Fig. 2. Gene expression in NAFLD. Heat map of top 200 genes variant
expressed between control, NAFL and NAFLD samples. Scaled expression values are
color-coded according to the legend on the left. The top bar shows the disease status:
red, NASH; green, NAFL; black, control. The bottom bars show additional variables for
each sample: age and gender (black, female; grey, male; green, missing value). The
scale for age is shown on the left.
Color key
0.9
1
Supporting Fig. 3. Hierarchical clustering of microarray samples based on inter-array
correlation showing distinct clustering of NASH, NAFL and control samples. The
heatmap shown the square inter-array correlation matrix(IAC). IAC coefficients were
calculated using Pearson correlation. The scale for IAC was shown on the left. The
upper bar show age for each sample (green, female; blue, male; white, missing value).
The scale for age is shown on the left side(white, missing value).
Eigengene expression
-0.2 0.0 0.2
Eigengene expression
0.2 0.4
0.0
-0.2
A
B
Control
NASH
Control
NASH
Control
NASH
Eigengene expression
-0.4 0.2 0.0 0.2 0.4
C
Supporting Fig. 4. Expression within NASH modules N1, N8 and N14 is shown in the heatmap and summarized with the module. The visualization of these modules was performed
using VisANT to plot the 100 strongest connections within each module. Genes that are
positively correlated are connected by red lines, whereas genes that are inversely correlated
are connected by blue lines. (A) The module N1 is involved in chromosome organization. (B)
The module N8 contains genes participate in protein degradation. (C) The module N14
contains genes involved in immune response.
Eigengene expression
0.3
0.0
-0.3
Eigengene expression
-0.4 0.2 0.0 0.2
A
B
NAFL
NASH
NAFL
NASH
-0.2 0.0 0.2 0.4
Eigengene expression
Eigengene expression
-0.4 0.2 0.0 0.2
C
D
NAFL
NASH
NAFL
NASH
Eigengene expression
0.2 0.0 0.2 0.4
Eigengene expression
-0.2 0.0 0.2 0.4
E
F
NAFL
NASH
NAFL
NASH
NAFL
NASH
Eigengene expression
-0.2 0.0 0.2 0.4
G
Supporting Fig. 5. Expression within progression modules P1(A), P2(B), P5(C), P7(D), P9(E),
P13(F) and P14(G) is shown in the heat-map and summarized with the module. The
visualization of these modules was performed using VisANT to plot the 100 strongest
connections within each module. Genes that are positively correlated are connected by red
lines, whereas genes that are inversely correlated are connected by blue lines.
Connectivity correlation
1.2
1
0.8
0.6
0.4
0.2
0
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14
Module
Supporting Fig. 6. To test the stability of modules, intramodular connectivity in 1000
module gene sets generated by sampling 13 of the 26 array samples were calculated
for each module. Then, correlations between the true module gene connectivity
values and those from the 1000 randomly selected sets were calculated.
Module-miRNA relationships
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
-0.52
0.45
(0.006) (0.02)
0.3
(0.1)
0.27
(0.2)
0.29
(0.2)
0.38
(0.06)
0.43
(0.03)
0.42
(0.03)
0.38
(0.06)
0.33
(0.1)
0.16
(0.4)
0.11
(0.6)
0.19
(0.4)
0.33
(0.1)
0.26
(0.2)
-0.04
(0.8)
0.52
0.53
0.51
0.46
(0.006) (0.006) (0.008) (0.02)
0.49
(0.01)
0.29
(0.1)
0.2
(0.3)
0.32
(0.1)
0.51
0.44
(0.008) (0.02)
0.41
(0.04)
0.35
(0.08)
0.46
(0.02)
0.44
(0.02)
0.1
(0.6)
-0.058
(0.8)
0.067
(0.7)
-0.019
(0.9)
-0.2
(0.3)
-0.082
(0.7)
-0.18
(0.4)
0.12
(0.6)
-0.21
(0.3)
0.024
(0.9)
0.21
(0.3)
-0.1
(0.6)
0.062
(0.8)
-0.21
(0.3)
0.3
(0.1)
-0.56
0.73
0.49
0.73
0.67
0.7
0.59
0.62
0.43
(0.003) (2e-05) (0.01) (2e-05) (2e-04) (7e-05) (0.002) (7e-04) (0.03)
0.36
0.6
0.37
(0.07) (0.001) (0.07)
0.21
(0.3)
0.45
(0.02)
0.5
(0.01)
-0.18
(0.4)
0.47
(0.02)
0.27
(0.2)
0.53
0.39
(0.005) (0.05)
0.44
(0.02)
0.31
(0.1)
0.21
(0.3)
0.078
(0.7)
0.29
(0.2)
0.55
(0.003)
0.34
(0.09)
-0.2
(0.3)
-0.21
(0.3)
-0.087 -0.17
(0.7)
(0.4)
-0.36
(0.07)
-0.072 -0.56 -0.022 -0.064 0.053
(0.7) (0.003) (0.9)
(0.8)
(0.8)
-0.11
(0.6)
-0.18
(0.4)
-0.43
(0.03)
0.086
(0.7)
-0.26
(0.2)
-0.25
(0.2)
-0.087
(0.7)
0.26
(0.2)
0.17
(0.4)
0.56
0.34
(0.003) (0.09)
0.53
-0.55 -0.44
(0.005) (0.004) (0.02)
-0.49 -0.57
-0.5
(0.01) (0.002) (0.01)
-0.52 -0.24
(0.006) (0.2)
-0.32
(0.1)
-0.37
(0.06)
-0.16
(0.4)
-0.27
(0.2)
0.65
-0.64 -0.48
(3e-04) (5e-04) (0.01)
-0.48 -0.61 -0.57 -0.53 -0.82 -0.49
(0.01) (9e-04) (0.003) (0.005) (3e-07) (0.01)
-0.35
(0.08)
-0.23
(0.3)
-0.3
(0.1)
-0.35 -0.51
-0.2
(0.08) (0.008) (0.3)
0.7
-0.33
(6e-05) (0.09)
-0.17
(0.4)
-0.19
(0.4)
-0.37
(0.06)
-0.2
(0.3)
-0.21
(0.3)
-0.63 -0.28
(5e-04) (0.2)
-0.061 0.019
(0.8)
(0.9)
-0.071 -0.033 -0.043 0.001
(0.7)
(0.9)
(0.8)
(1)
0.14
(0.5)
0.24
(0.2)
0.39
(0.05)
0.22
(0.3)
0.28
(0.2)
0.45
(0.02)
0.07
(0.7)
0.22
(0.3)
0.14
(0.5)
0.23
(0.3)
0.054
(0.8)
0.14
(0.5)
0.27
(0.2)
0.0075 0.0083
(1)
(1)
0.049
(0.8)
-0.29
(0.2)
-0.2
(0.3)
-0.35
(0.08)
-0.26
(0.2)
-0.41
(0.04)
-0.28
(0.2)
-0.16
(0.4)
0.0033 -0.22
(1)
(0.3)
-0.39
(0.05)
-0.079
(0.7)
-0.12
(0.5)
-0.41
(0.04)
-0.32
(0.1)
0.28
(0.2)
-0.19
(0.3)
0.048
(0.8)
-0.2
(0.3)
-0.13
(0.5)
-0.011
(1)
-0.31
(0.1)
-0.13
(0.5)
-0.26
(0.2)
-0.23
(0.3)
0.021
(0.9)
0.13
(0.5)
-0.16
(0.4)
-0.096
(0.6)
0.68
-0.49
(1e-04) (0.01)
-0.27
(0.2)
-0.4
(0.04)
-0.39
(0.05)
-0.49
(0.01)
-0.38 -0.64
-0.3
(0.05) (4e-04) (0.1)
-0.037 -0.34
(0.9) (0.09)
-0.21
(0.3)
-0.079 -0.51 -0.19
(0.7) (0.007) (0.4)
0.4
(0.04)
-0.14
(0.5)
-0.39
(0.05)
-0.27
(0.2)
-0.27
(0.2)
-0.33
(0.1)
0.02
(0.9)
-0.12
(0.5)
0.054
(0.8)
-0.33
(0.1)
-0.23
(0.3)
-0.085
(0.7)
0.12
(0.6)
-0.5
(0.01)
-0.32
(0.1)
-0.28
(0.2)
1
0.5
0
-0.5
-1
Supporting Fig. 7. Module-miRNA associations in progression network. Each row corresponds
to a module eigengene, column to a miRNA. Each cell contains the corresponding correlation
and P-value (bracket). The table is color-coded by correlation according to the color legend.
A
B
C
D
E
F
G
H
I
J
K
L
M
Supporting Fig. 8. The individual gene expression of the candidates between control,
NAFL and NASH groups were shown. MT1DP(A), MT1X(B), PDIA6(C), SRPRB(D), TCF4(E),
RPL8(F), IGHG1(G), NANS(H), VIM(I), PTGS2(J), PAN3(K), YIF1A(L) and SRRM2(M)
NAFL NASH
**
A
21 37 55
B
**
21 16 70
C
2 12 2
**
D
**
19
93
7
GO category/KEGG pathway
S1
N5
Sterol biosynthetic process
6.3E-29
3.4E-27
Lipid biosynthetic process
2.8E-20
8.9E-24
Endoplasmic reticulum
3.4E-4
5.8E-6
Microsome
6.2E-4
8.0E-5
GO category/KEGG pathway
S2
N6
Oxidation reduction
9.9E-8
8.6E-15
Microsome
2.1E-13
2.4E-13
Endoplasmic reticulum
1.5E-8
3.8E-8
Drug metabolism
1.4E-20
1.1E-16
GO category/KEGG pathway
S4
N7
Ion homeostasis
2.6E-2
8.0E-2
Cadmium ion binding
3.5E-16
3.3E-19
Copper ion binding
2.8E-13
4.1E-15
Zinc ion binding
4.1E-4
2.8E-4
GO category/KEGG pathway
S6
N13
M phase
1.5E-56
1.4E-52
Spindle
3.6E-30
1.4E-26
Chromosome
2.2E-23
6.1E-25
P53 signaling pathway
2.1E-2
1.4E-3
E
*
892
20 61
F
**
42
70 43
G
**
424 77
392
GO category/KEGG pathway
S9
N11
RNA processing
2.4E-11
7.8E-4
DNA repair
8.8E-5
NS
Spliceosome
4.1E-6
NS
Ubiquitin mediated proteolysis
7.7E-3
NS
GO category/KEGG pathway
S10
N14
Innate immune response
1.9E-7
9.6E-10
Adaptive immune response
1.9E-5
2.3E-5
MHC class II receptor activity
1.2E-2
NS
Cell adhesion molecules (CAMs)
6.5E-3
1.1E-4
GO category/KEGG pathway
S13
N8
Negative regulation of protein ubiquitination
2.8E-19
1.0E-11
Positive regulation of protein ubiquitination
8.2E-18
9.4E-12
Regulation of protein modification process
7.3E-7
4.9E-5
Proteasome
6.7E-21
6.3E-16
Supporting Fig. 9. Modules from NAFL analysis overlap significantly with modules from
NASH analysis, as measured by both gene number and gene ontology categories
enrichment P value from DAVID. *P<0.001; **P<10-9. P values were obtained using a
hypergeometric distribution.