Journal: PLOS Pathogens

Article Title: Th2-dependent STAT6-regulated genes in intestinal epithelial cells mediate larval trapping during secondary Heligmosomoides polygyrus bakeri infection

doi: 10.1371/journal.ppat.1011296

Figure Lengend Snippet: Bone marrow derived macrophages (BMDM, purity >95%) were generated and splenic B cells (purity > 90%) isolated from mice that do express STAT6 only under control of the LysMCre promoter (Mac-STAT6), STAT6 knockout mice (STAT6ko) and BALB/c wild-type (WT) controls. A) Western blot of unstimulated (w/o) and IL-4-stimulated BMDM and B cell lysates of the indicated genotypes for phosphorylated STAT6 (pSTAT6), total STAT6 and β-actin as loading control. Western blot is representative for three to five mice per genotype from two independent experiments. B) qRT-PCR of BMDM from either Mac-STAT6, STAT6ko or WT mice stimulated with 20 ng/ml IL-4 for 48 hrs. Bars show mean expression + SEM of Arg1 , Retnla and Pdcd1lg2 normalized on Hprt of three to four mice per genotype out of two experiments. Statistical significance was determined by Two-Way-ANOVA with Holm-Sidak post-hoc testingon log-transformed data. ***p < 0.001; *p < 0.05.

Article Snippet: Membranes were blocked with 3% BSA in TRIS-buffered saline-tween buffer (TBS-T) and phospho-STAT6 (Y641), STAT6 (Rabbit Ab) or beta-actin (13E5) were applied as primary antibodies (all Cell Signaling Technology, Danvers, MA) at 4°C overnight.

Techniques: Derivative Assay, Generated, Isolation, Control, Knock-Out, Western Blot, Quantitative RT-PCR, Expressing, Transformation Assay

Journal: PLOS Pathogens

Article Title: Th2-dependent STAT6-regulated genes in intestinal epithelial cells mediate larval trapping during secondary Heligmosomoides polygyrus bakeri infection

doi: 10.1371/journal.ppat.1011296

Figure Lengend Snippet: A) Schematic timeline: Mac-STAT6 mice, STAT6ko mice or BALB/c (WT) control mice were primary infected with Hpb , infection was cleared by Pyrantel pamoate after two weeks and four to six weeks later, mice were secondarily infected. 5 μg/mouse IL-4c in 100 μl PBS or PBS for control groups was injected i . p . on days 1, 3, 5 and 7 after secondary infection. B) Mean + SEM of granuloma with or without (w/o) larvae on day 9 after secondary infection. Statistical analysis was performed separately for granuloma with or w/o larvae by Kruskal-Wallis with Dunn’s post-hoc testing. *p<0.05. Data are pooled from three independent experiments with five to seven mice per group. C and D) Histological stainings and quantifications of small intestinal cross-sections day 9 after secondary Hpb infection for detection of Relm-α (red) and CD68 (green) (C), or DAPI (blue) and Arg1 (green) (D). Quantification plots display Mean + SEM of percentage of Relm-α + or Arg1 + of DAPI + CD68 + macrophages, respectively. Representative picture and quantification for four to seven mice per genotype. Scale bar is 100 μm.

Article Snippet: Membranes were blocked with 3% BSA in TRIS-buffered saline-tween buffer (TBS-T) and phospho-STAT6 (Y641), STAT6 (Rabbit Ab) or beta-actin (13E5) were applied as primary antibodies (all Cell Signaling Technology, Danvers, MA) at 4°C overnight.

Techniques: Control, Infection, Injection

Journal: PLOS Pathogens

Article Title: Th2-dependent STAT6-regulated genes in intestinal epithelial cells mediate larval trapping during secondary Heligmosomoides polygyrus bakeri infection

doi: 10.1371/journal.ppat.1011296

Figure Lengend Snippet: A) Schematic timeline for the CD4 depletion experiment performed with mice with constitutively active STAT6 in IECs (VillinCre_STAT6vt) and Cre - littermate controls (STAT6vt): Primary Hpb infection was cleared by oral gavage with twice 1 mg Pyrantel pamoate per mouse. 4–6 weeks later, mice were secondarily infected with Hpb and anti-CD4 or isotype antibody (AB) was injected i . v . on day 0 and 4 of secondary infection. B) Number of counted granuloma with or without (w/o) larvae in the submucosa on day 9 or 10 after secondary Hpb infection. Bars show mean count + SEM of four to eleven mice per group from two independent experiments. C) Eggs per gram feces counted on day 14 after secondary Hpb infection. Data show mean count + SEM of three to seven mice per group from two independent experiments. D) Relative expression of Arg1 , Retnla , Retnlb , Chil3 , Pdcd1lg2 , and Mrc1 , calculated by normalisation to Hprt . Bars show mean + SEM of four to eleven mice per group from two independent experiments. E) Histological staining and quantification of Relm-α (red) and CD68 (green) in tissue sections from small intestine day 9 after secondary Hpb infection in the left panel and for DAPI (blue) and Arg1 (green) in the right panel. Mean + SEM of percentage of Relm-α + or Arg1 + of DAPI + CD68 + cells, respectively. Representative picture for one mouse per genotype and quantification for all five to ten mice per group from two independent experiments. Scale bar corresponds to 100 μm. B-E) Statistical significance was determined by Two-Way ANOVA with Holm-Sidak post-hoc testing. ***p < 0.001; **p < 0.01; *p < 0.05.

Article Snippet: Membranes were blocked with 3% BSA in TRIS-buffered saline-tween buffer (TBS-T) and phospho-STAT6 (Y641), STAT6 (Rabbit Ab) or beta-actin (13E5) were applied as primary antibodies (all Cell Signaling Technology, Danvers, MA) at 4°C overnight.

Techniques: Infection, Injection, Expressing, Staining