Journal: PLoS ONE

Article Title: Promotion of Intestinal Epithelial Cell Turnover by Commensal Bacteria: Role of Short-Chain Fatty Acids

doi: 10.1371/journal.pone.0156334

Figure Lengend Snippet: ( A ) Chloroform-treated feces from untreated SPF mice or chloroform-treated PBS as a control were administered orally to 8-week-old GF mice. The resulting exGF and control mice, respectively, were housed separately for 3 weeks in isolators, after which frozen sections of the ileum from both groups of mice (11-week-old) as well as from age-matched SPF mice were immunostained with antibodies to Ki67 (red) and to β-catenin (green). Representative images are shown in the left panels. Scale bar, 20 μm. The number of Ki67-positive cells per crypt was also determined from such sections (right panel). Data are means ± SE for 30 crypts from one mouse and are representative of four mice. *** P < 0.001 (ANOVA and Tukey’s test). ( B ) Lysates of IECs isolated from mice (11-week-old) treated as in (A) were subjected to immunoblot analysis with antibodies to cyclin D1 and to β-tubulin (left panels). The cyclin D1/β-tubulin band intensity ratio was also determined from such blots and expressed relative to the value for GF mice (right panel). Data are means ± SE from three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (ANOVA and Tukey’s test). ( C ) Frozen sections of the ileum were prepared from SPF, GF, and exGF mice (11-week-old) at 2 days after BrdU injection and were immunostained with mAbs to BrdU (red) and to β-catenin (green). Representative images are shown in the left panels. Scale bar, 100 μm. The migration distance for BrdU-positive cells was also determined from such sections (right panel). Data are means ± SE for 30 villi from one mouse and are representative of three mice. *** P < 0.001 (ANOVA and Tukey’s test). ( D ) PCR analysis of 16S rRNA genes of total bacteria, C . leptum , C . coccoides , segmented filamentous bacteria (SFB), or B . fragilis in fecal pellets from GF, exGF, or SPF mice (11-week-old).

Article Snippet: GF mice or exGF mice, the latter of which had been treated orally with chloroform-treated feces of SPF mice, were maintained in vinyl isolators at Sankyo Labo Service after treatment.

Techniques: Control, Isolation, Western Blot, Injection, Migration, Bacteria