Download 413 13 23 400 377 117 Suppl. Figure S1 99 18

Suppl. Figure S1
B
A
patients prospectively recruited
patients selected for analyses
patients analyzed
H.pylori+
H.pylori+
anti-CagA-IgG- anti-CagA-IgG+
69
413
30
13
6
exclusion criteria
cagA DNAcagA DNA+
400
63
23
31
missing blood/test
vacA s2m2
vacA s1m1/2
377
32
9
H.pylori infected patients
cagA RNA+
117
23
18
11
failed H.pylori cultivation
patients with H.pylori isolates
99
no/low cagA RNA
(5 low quality)
unexplained
11
Immunoblot
potentially positive
(1 missing)
Suppl. Figure S2
A
r
P (two-tailed)
0.025
0.9167
<0.0001
IL-8 / -actin
0.020
0.015
0.010
0.005
0.000
0
500
1000
1500
2000
2500
IL-8 (pg/ml)
C
s1m1
s1m1
s1m2
s1m2
vacA
vacA
B
s2m2
r
P (two-tailed)
control
0.000
0.005
0.010
0.015
IL-8 / -actin
0.7634
0.0007
0.020
0.025
s2m2
r
P (two-tailed)
control
0
500
1000
1500
IL-8 (pg/ml)
0.8148
0.0002
2000
2500
Suppl. Figure S3
B
600
400
200
0
C
800
ViraStripe CagA-IgG
ViraStripe CagA-IgG
800
600
400
200
0
0
20
40
60
80 100 120
CagA-IgG Omega Genesis
400
ViraStripe CagA-IgG
A
300
200
100
0
0
20
40
60
80 100 120
CagA-IgG Omega Genesis
0
2
4
6
8
CagA-IgG Omega Genesis
Suppl. Figure S1. Study design. (A) Number of patients included in the study. (B)
Schematic explanation of H.pylori+ anti-CagA-IgG negative samples.
Suppl. Figure S2. Correlation between IL-8 mRNA and IL-8 protein expression and
vacA polymorphisms in AGS co-culturing model. (A) IL-8 mRNA expression of AGS cells
was measured using qPCR and normalized to β-actin expression. IL-8 protein was measured
in supernatant of AGS cell co-cultivated with H.pylori strains. IL-8 mRNA (B) and IL-8 protein
expression (C) were correlated with vacA polymorphisms. Spearman’s test was used for
correlation analyses. AGS cells co-cultivated in similar conditions but without H.pylori were
considered as control.
Suppl. Figure S3. Validation of anti-CagA-IgG ELISA using Immunoblot. To confirm the
anti-CagA-IgG titer we performed independent measurement of anti-CagA-IgG using
ViraStripe® CagA IgG immunoblot-based method. (A) Correlation between the tests. (B) All
samples positive in ELISA showed strong signal with values above 200% of IU, while only
few samples with negative results in ELISA showed varying intensities on immunoblot (C).
Box highlights the cut offs according to each test (≥6.25 U/ml for ELISA and 80% of IU in
Immunoblot).
Suppl. Table S1. Primer used for qualitative PCR and quantitative real-time PCR.
Primer Sequence (5’3’)
Genes
cagA*1
EPIYA
Motifs*2
F
GATAACAGGCAAGCTTTTGAGG
R
CTGCAAAAGATTGTTTGGCAGA
F
ACCCTAGTCGGTAATGGGTTA
R
GTAATTGTCTAGTTTCGC
Annealing
temperatur
e
Product
56°C
349
50°C
AB 500,
(bp)
ABC 600,
ABCC 700,
ABCCC 800
More than one
fragment = mixed
infection
vacA s*3
vacA m*4
glmM5
cagE*6
virB11*7
β-actin8
F
ATGGAAATACAACAAACACAC
R
CTGCTTGAATGCGCCAAAC
F
CAATCTGTCCAATCAAGCGAG
R
GCGTCTAAATAATTCCAAGG
F
AGGCTTTTAGGGGTGTTAGGGGTTT
R
AAGCTTACTTTCTAACACTAACGC
F
TTGAAAACTTCAAGGATAGGATAGAGC
R
GCCTAGCGTAATATCACCATTACCC
F
TTAAATCCTCTAAGGCATGCTAC
R
GATATAAGTCGTTTTACCGCTTC
F
56°C
s1: 259
s2: 286
56°C
m1: 570
m2: 645
56°C
293
53°C
508
49°C
491
CATGCCATCCTGCGTCTGGACC
ACATGGTGGTGCCGCCAGACA
60°C
400
F
CTTCCTGATTTCTGCAGCTCTG
57°C
193
R
GAGCTCTCTTCCATCAGAAAGC
R
IL-89
F = forward, R = reverse; *-qualitative PCR.
References: 1 Peak et al. 1995, 2 Yamaoka et al. 1998, 3,4 Ryberg et al. 2008, 5 Shahamat et al. 2004,
6,7 Tomasini et al. 2003, 8 Wex et al. 2004, 9 Al-Sammak et al.2013