Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a Map of complement-mediated bactericidal activity. b The proportion of gut bacteria after 16s rRNA analyses of colon contents in normal CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice (pooled samples from six mice). Also see Fig. . c The proportion of gut bacteria after 16s rRNA analyses in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS. Also see Fig. . d The proportion of E. coli (Escherichia-Shigella) in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS ( n = 5). Also see Fig. S3C. e Flow cytometry of feces bacteria after staining using anti-LPS antibodies in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CDKO) mice after 2% DSS. f QPCR of colonic content (upper) and colonic tissues (lower) in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS ( n = 6). g FISH of E. coli in the gut tissues of CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS. One representative from six mice. Scale bar, 40 μM; Scale bar in bottom panel, 5 μM. Two side student’s t test in e and f ; * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Activity Assay, Bacteria, Flow Cytometry, Staining

Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a E. coli clones in the normal human sera (N. Sera, Quidel), C3deficient sera (C3defi, Quidel), C1 deficient sera (C1 defi, Quidel) and inactive sera at different time points. *C3deficint sera vs normal sera. b E. coli clones in the normal sera (N. Sera, Quidel), CFD-deficient sera (CFD defi, Quidel) inactive sera at different time points. *CFD-deficient sera vs normal sera. c E. coli clones in the normal human sera after adding LPS (containing mannose), mannose or peptidoglycan. *LPS vs normal sera. d E. coli clones in the normal human sera after adding rReg4, rREG4 or mutated rREG4. *Reg4 vs normal sera. Only mrReg4 and only hrREG4, E.coli clones in inactive complement serum after adding mrReg4 or hrREG4. e E. coli clones in the normal human sera after adding different concentrations of rReg4. f E. coli clones in the normal human sera (N. sera) or CFD-deficient (CFD defi) sera after adding rReg4 (N. sera/rReg4 or CFDdefi/rReg4). *Normal sera/Reg4 vs normal sera. g E. coli clones in the normal human sera after adding rREG4, IgA or IgA+REG4. *IgA+Reg4 vs Reg4. Around 5 × 10 5 in a – c or 1 × 10 6 in d – g E. coli CFUs were added into 1 ml 10% sera. Bacterial suspension were diluted and plated on MacConkey agar plates. Plates were incubated overnight at 37 °C, and CFU were counted. Analysis of variance test used in a – g . * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance. One representative of three independent experiments.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Clone Assay, Suspension, Incubation

Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a Immunostaining of C3b, C5-9, Reg4, IgA, MASP1/3 and MASP2 on GFP-labeled E. coli in 2% DSS-treated GFP-labeled E. coli infused CFD fl/fl pvillin-cre T (CFDKO/0160) mice (Left) and statistic analyses (right, three images/slide, three slides/mouse, n = 6). Scale bar, 40 μM. No, number; Iso, isotypic antibod. b Flow cytometry of Reg4, IgA, MASP1/3 and MASP2 on the GFP-labeled E. coli in untreated GFP-labeled E. coli infused CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice and statistic analyses ( n = 3). c ELISA for detecting binding of Reg4 with LPS (left), Reg4 with IgA (middle) or IgA with LPS (right) in different concentration of Reg4 (left and middle) or IgA (right) coated plates d ELISA of C3 in different concentration of Reg4 (left) or IgA (right) with LPS coated plates. For complement resources, 5% normal human sera were added. e ELISA of C3b in the LPS, Reg4, IgA, LPS + Reg4 (LPS/Reg4), LPS + IgA (LPS/IgA), Reg4+IgA (Reg4/IgA) and LPS + Reg4+IgA (LPS/Reg4/IgA) coated plates. For complement resources, 5% normal human sera were added. Mann–Whitney U test in a ; Two side student’s t test in b ; ANOVA plus post-Bonferroni analysis in e ; Analysis of variance test in c and d . * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance. In c – e , one representative of three independent experiments.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Immunostaining, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, MANN-WHITNEY