Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Generation of Atg7 cKO mice. A, expression of Chst4 and Villin1 (Vil1) mRNA in lower gastrointestinal tracts. The relative quantity of each mRNA to that of β-actin was determined by quantitative RT-PCR. Error bars represent the standard deviation (S.D.) of samples within a group. n = 3 per group. B, frozen sections of lower gastrointestinal tracts from GlcNAc6ST-2-Cre+/R26R reporter mice were stained with X-gal solution and Nuclear Fast Red as described previously (26). Bar, 50 μm. C, quantitative RT-PCR analysis of Atg7 mRNA in the colon. Error bars represent the S.D. of samples within a group. An unpaired Student's t test was used to compare WT versus cKO mice. **, p < 0.01. n = 7–9 per group. D, Western blot analysis of whole colonic lysates from WT and cKO mice. The arrowhead in each panel indicates the position of each protein. #, high molecular weight form of p62. E, quantification of the ATG7 protein in the whole colon. Western blot bands (D) were quantified. The error bars represent the S.D. of the samples within a group. An unpaired Student's t test was used to compare WT versus cKO mice. **, p < 0.01. n = 5 per group.

Article Snippet: The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; TaKaRa).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Staining, Western Blot, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Comparison of major microbial populations in fecal samples. A–C, pyrosequencing analysis was used to examine microbial diversity in feces from WT and cKO mice. Sequences were obtained from pooled stool samples of WT (n = 3; 145,188 reads) and cKO mice (n = 3; 111,639 reads). A, comparisons of the diversity at the phylum or class level. Each chart represents the taxonomic composition. B, comparison of the diversity at the family level in Clostridiales. C, comparison of the diversity at the family level in Bacteroidales. D, total bacteria in fecal samples were quantified by quantitative RT-PCR using universal bacterial 16S rRNA genes (WT, n = 4; cKO, n = 6). E, microbial composition of the feces of WT and cKO mice treated with (Abx-cKO) or without (WT and cKO) a combination of metronidazole and ciprofloxacin for 4 weeks was analyzed by quantitative PCR using group- or genus-specific primers of the bacterial 16S rRNA genes. Error bars represent the S.D. of samples within the group (WT, n = 4; cKO, n = 4; Abx-cKO, n = 8). An unpaired Student's t test and a paired Student's t test were used to compare WT versus cKO mice and cKO versus Abx-cKO mice, respectively. **, p < 0.01; *, p < 0.05. n = 4 per group.

Article Snippet: The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; TaKaRa).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Quantitative RT-PCR analyses of antimicrobial peptides, antiparasitic peptides, and cytokines in WT and Atg7 cKO mice. Quantitative RT-PCR analyses of antimicrobial peptides, antiparasitic peptides, and cytokines were performed using total RNA samples from the medial colons of WT and cKO mice treated with (Abx-cKO) or without (WT and cKO) a combination of metronidazole and ciprofloxacin for 4 weeks. A, expression of antimicrobial peptides and antiparasitic peptides. B, expression of Th1/Th2 cytokines. C, expression of epithelium-derived cytokines. Error bars represent the S.D. of samples within a group. An unpaired Student's t test and a paired Student's t test were used to compare WT versus cKO mice and cKO versus Abx-cKO mice, respectively. **, p < 0.01; *, p < 0.05. n = 3–4 per group.

Article Snippet: The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; TaKaRa).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Secretion of colonic mucins in WT and Atg7 cKO mice. Hematoxylin and eosin (A), Alcian blue-PAS (B), and anti-MUC2 (C) staining of the mouse distal colon. D, percentage of mucus-secreting crypts. E, quantification of inner mucus layer thickness. F, detection of fecal MUC2 by ELISA. G, quantitative RT-PCR. The relative quantity of Muc2 mRNA compared with the quantity of β-actin was determined by the ΔΔCt method. Error bars represent the S.D. of samples within a group. Asterisk in A–C, mucus-secreting crypt. Arrows in A–C, mucus-secreting goblet cells. Dashed line in C, mucosal surface. An unpaired Student's t test was used to compare WT versus cKO mice. **, p < 0.01; *, p < 0.05. n = 3 per group.

Article Snippet: The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; TaKaRa).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR