br year old man poorly differentiated

1562 1: 80-year-old man, poorly differentiated adenocarcinoma;
1563 case 2: 51-year-old man, poorly differentiated adenocarci-
1565 adenocarcinoma) were obtained from ReproCELL Incorpo-
1566 rated (Yokohama, Japan). Human early gastric adenocarci-
1567 noma tissue specimens were obtained from a 75-year-old
1569 underwent endoscopic submucosal dissection at Keio Uni-
1570 versity Hospital. The pathologic diagnosis of both specimens
1571 was well-differentiated adenocarcinoma according to the
1572 Japanese Gastric Cancer Association classification of gastric
1573 carcinoma (14th edition). Human gastric biopsy specimens
1574 (antrum and body) were obtained via endoscopy. H pylori
1579 mens were examined histologically using H&E. The severity
1580 of neutrophil infiltration was classified into 4 grades (ab-
1581 sent, mild, moderate, and severe) according to the updated 1582 Sydney system.40 CAPZA1 expression was evaluated
1583 immunohistologically. The staining intensity of CAPZA1 per
1584 cell was analyzed using image analysis software (Tissue
1586 1587 Fluorescence Immunocytochemistry
1588 AGS 61036-62-2 were infected with H pylori for 5 hours, incu-
1590 kanamycin for 24 hours, washed in phosphate-buffered sa-
1591 line, fixed with 4% paraformaldehyde, and incubated with 
Alexa Fluor 488–conjugated anti-rabbit IgG (Invitrogen) 1593
was used as the secondary antibody. Nuclei (blue) were 1594
stained with 40,6-diamidino-2-phenylindole. Samples were 1595
examined using an LSM710 Zeiss confocal microscope. Q191596
Immunohistochemistry
Tissue sections were fixed in 4% paraformaldehyde,
depleted of paraffin, and rehydrated using a graded ethanol
series. Thereafter, the sections were subjected to antigen
retrieval by heating for 10 minutes at 105 C in Target
Retrieval Solution (pH 9.0; Dako, Tokyo, Japan) and incu-
bated overnight at 4 C with a primary antibody. Immuno-
reactivity was detected using Alexa Fluor 568–conjugated
anti-rat IgG and Alexa Fluor 488–conjugated anti-rabbit
IgG (Invitrogen). Samples were examined using an
LSM710 Zeiss confocal microscope.
RNA Isolation and Quantitative Reverse-
Transcription PCR
Total RNA was isolated using an SV Total RNA Isolation 1613
System (Promega, Madison, WI). Reverse transcription was 1614
performed using a PrimeScript Reverse Transcription re- 1615
agent Kit (Takara, Ohtsu, Japan) in accordance with the 1616
manufacturer’s guidelines. PCR was performed using a SYBR 1617
Premix Ex Taq Perfect Real Time kit (Takara), a Thermal 1618
Cycler Dice Real Time System (Takara), and the following 1619
primers: CD44total: forward: 5’-CCGCTATGTCCAGAAA 1620
GGA, and reverse: 5’-CTGTCTGTGCTGTCGGTGAT; CD44v9: Q201621 forward: 5’-GGCTTGGAAGAAGATAAAGACC, and reverse: 5’- 1622 TGCTTGATGTCAGAGTAGAAGTTG; SALL4: forward: 5’-TCG 1623 TCTGCTAGCGCTCTTCAGATC, and reverse: 5’-CGGCGGG 1624 CTGAGTTATTGTTCG; KLF5: forward: 5’-TAACCCCGATTTG 1625 GAGAAAC, and reverse: 5’-TGGCTTTTCACCAGTGTGAG; 1626 CAPZA1: forward: 5’-ACAATCTCCTCAGGGAAGGGG, and 1627 reverse: 5’-TGCTTCTTTCCGTAAGTGGTCAAA; and GAPDH: 1628
forward: 5’-GACATCAAGAAGGTGGTGAAGCAG, and reverse: 1629 5’-ATACCAGGAAATGAGCTTGACAAA. GAPDH was used as 1630
the internal control. 1631
NuPAGE gradient gel (Invitrogen). An anti–b-actin antibody
(Sigma-Aldrich) was used as the loading control. Immuno-
reactive bands were detected by chemiluminescence using
ECL Prime (GE Healthcare, Piscataway, NJ). Signals were
quantified using ImageJ software (National Institutes of
Health, Bethesda, MD).
Study Approval 1642
This study was performed in accordance with the prin- 1644
ciples of the Declaration of Helsinki. Isolation of H pylori 1645
strains from participants and biopsy of gastric mucosal 1646
Committee of the Keio University School of Medicine 1648
provided written informed consent. Animal experiments 1650
and procedures were approved by the Keio University An-imal Research Committee (08080-12).
Statistics
All values are expressed as the means ± SD. Differences between 2 groups were statistically evaluated by the Stu-dent t test with JSTAT statistical software (version 8.2). For multiple comparisons, a 1-way analysis of variance was used. P < .05 was considered significant. Correlation anal-ysis was performed using SPSS version 22 for Windows (SPSS Inc, Chicago, IL).