Journal: bioRxiv

Article Title: Dopamine receptor D2 confers colonization resistance via gut microbial metabolites

doi: 10.1101/2023.03.14.532647

Figure Lengend Snippet: a , C57Bl/6 mice were pre-treated with antibiotics (ABX) for 7 d, followed by Trp metabolites, I3A (1000 mg/kg), IPyA (2900 mg/kg), or IEt (600 mg/kg) by oral gavage daily for 2 d. The mice were then administered C. rodentium (CR, oral gavage, 10 8 CFU) with continued ABX (except neomycin) and metabolite treatment. Feces were collected before infection and 9 d post-infection to determine Trp metabolite levels (see Extended Data Fig. 1c–e). b, c , Bacterial load in ( b ) feces and ( c ) colon tissue was measured ( b ) at 1–2 d intervals for 24 d post-infection and ( c ) at the peak of infection, 10 d post-infection. d–f , Colon sections were stained with H&E and ( d ) blindly scored as described in . ( e ) Crypt heights were measured. ( f ) Representative images. Scale bar: 50 μm. Data are representative of at least 3 independent experiments, n=10 mice per group, bars = mean, error bars = standard deviation. One-way ANOVA followed by post-hoc Tukey’s test: *p<0.05, **p<0.01, ***p<0.001, n.s. = not significant.

Article Snippet: L-Tryptophan (Trp) was purchased from Chem-Impex International, Inc. Indole-3-aldehyde (I3A) and indole-3-pyruvate (IPyA) were obtained from Biosynth.

Techniques: Infection, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Dopamine receptor D2 confers colonization resistance via gut microbial metabolites

doi: 10.1101/2023.03.14.532647

Figure Lengend Snippet: a , Drd2 fl/fl x Villin (Vil)-Cre or Drd2 fl/fl mice were fed a conventional (2 g Trp/kg diet, ad libitum) or Trp (42 g Trp/kg diet, ad libitum) diet for 7 d or Trp metabolites (shown: IEt (600 mg/kg); see Extended Data Fig. 9 for I3A and IPyA), by oral gavage daily for 2 d, and then infected with C. rodentium (CR, oral gavage, 10 8 CFU) with continued Trp feeding or metabolite treatment. b, c , Bacterial load in ( b ) feces and ( c ) colon tissue was measured ( b ) every 1–2 d for 24 d post-infection and ( c ) at the peak of infection, 10 d post-infection. d–f , Colon sections were stained with H&E and ( d ) blindly scored as described in . ( e ) Crypt heights were measured. ( f ) Representative images. Scale bar: 50 μm. Data are representative of at least 3 independent experiments, n=10 mice per group, bars = mean, error bars = standard deviation. One-way ANOVA followed by post-hoc Tukey’s test: *p<0.05, **p<0.01, ***p<0.001, n.s. = not significant.

Article Snippet: L-Tryptophan (Trp) was purchased from Chem-Impex International, Inc. Indole-3-aldehyde (I3A) and indole-3-pyruvate (IPyA) were obtained from Biosynth.

Techniques: Infection, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Dopamine receptor D2 confers colonization resistance via gut microbial metabolites

doi: 10.1101/2023.03.14.532647

Figure Lengend Snippet: a , C57Bl/6 mice were pre-treated with antibiotics (-/+ ABX, 9 mg/kg each of metronidazole, ampicillin, and neomycin, 4.5 mg/kg of vancomycin) for 7 d, followed by conventional (2 g Trp/kg diet, ad libitum) or Trp (42 g Trp/kg diet, ad libitum) diet for 7 d or Trp metabolites, I3A (1000 mg/kg), IPyA (2900 mg/kg), or IEt (600 mg/kg) by oral gavage daily for 2 d. The mice were then administered C. rodentium (CR, oral gavage, 10 8 CFU) with continued ABX (except neomycin) and Trp feeding or metabolite treatment. b , Drd2 fl/fl x Villin (Vil)-Cre or Drd2 fl/fl mice were fed a conventional (2 g Trp/kg diet, ad libitum) or Trp (42 g Trp/kg diet, ad libitum) diet for 7 d or Trp metabolites, I3A (1000 mg/kg), IPyA, (2900 mg/kg), or IEt (600 mg/kg) by oral gavage daily for 2 d, and then infected with C. rodentium (CR, oral gavage, 10 8 CFU) with continued Trp feeding or metabolite treatment (shown: IEt). c , Caco-2 monolayers ( Drd2 WT vs. KO) were pre-treated with metabolites (100 μM) for 2 d and then infected with EHEC O157:H7 (MOI 50) for 16 h. ( a–c ) Samples were stained with DAPI and Alexa Fluor 647-phalloidin. Pedestal formation = # of pedestals per host or Caco-2 cells (ABX + Trp diet, n = 146–177; ABX + I3A, 121–184; ABX + IPyA, 118–151; ABX + IEt, 118–175; Vil-Cre: Trp diet, n=110–186; IEt, n=100–130, Drd2 WT vs KO Caco-2 cells: IEt, n = 130–188). ( d–o ) Caco-2 cells were pre-treated with the indicated inhibitors (Gβγ inhibitor gallein, 10 μM; PLC inhibitor U-73122, 10 μM; pan-PKC inhibitor sotrastaurin (Sotra), 5 μM; or PKC-8 inhibitor (PKC8i), 5 μM), followed by metabolites (100 μM) for 24 h and then infected with EHEC O157:H7 (MOI 50) for 12 h. ( p–r ) PKC-8 knockdown was performed using two different siRNA duplexes (1 and 2) or negative control siRNA (C). (s–y) Caco-2 cells ( Drd2 WT vs KO) were pre-treated with metabolites (100 μM) for 24 h and then infected with EHEC O157:H7 (MOI 50) for 12 h. ( d–y ) Cell lysates were analyzed by Western blotting with the indicated antibodies. ( e, f, h, i, k, l, n, o, q, r, t, v, x, y ) Densitometry was performed using FIJI, n = 3. z, Model of gut microbial Trp metabolite activation of DRD2 in IECs and reduced pathogen burden. Blue = host actin regulatory proteins. Data are representative of at least 3 independent experiments, n=10 mice per group, bars = mean, error bars = standard deviation. One-way ANOVA followed by post-hoc Tukey’s test: **p<0.01, ***p<0.001, n.s. = not significant.

Article Snippet: L-Tryptophan (Trp) was purchased from Chem-Impex International, Inc. Indole-3-aldehyde (I3A) and indole-3-pyruvate (IPyA) were obtained from Biosynth.

Techniques: Infection, Staining, Knockdown, Negative Control, Western Blot, Activation Assay, Standard Deviation

Journal: International journal of oncology

Article Title: Dimeric Le(a) (Le(a)-on-Le(a)) status of beta-haptoglobin in sera of colon cancer, chronic inflammatory disease and normal subjects.

doi: 10.3892/ijo_00000614

Figure Lengend Snippet: Figure 1. TLC pattern of glycolipids extracted from Colo205 cells. Glycolipids extracted from Colo205 cells were subjected to TLC and stained by orcinol/H2SO4 spraying or blotted by immunostaining using IMH2 and ST421 antibody as described in Materials and methods. (A) Orcinol/H2SO4 spraying; (B) immunostaining using IMH2 antibody; (C) immunostaining using ST421 antibody. Lane 1, mixture of GM1, GM2 and GM3; lane 2, total GSL; lane 3, neutral fraction from total GSL; lane 4, monosialo- gangliodise from total GSL; lane 5, disialogangliodise from total GSL; lane 6, trisialogangliodise from total GSL; lane 7, multi-sialogangliodise from total GSL.

Article Snippet: Colo205 cells (21), originally purchased from ATCC, were cultured and maintained in RPMI-1640 medium with 10% fetal calf serum at 37 ̊C in 5% CO2/95% air.

Techniques: Staining, Immunostaining

Journal: International journal of oncology

Article Title: Dimeric Le(a) (Le(a)-on-Le(a)) status of beta-haptoglobin in sera of colon cancer, chronic inflammatory disease and normal subjects.

doi: 10.3892/ijo_00000614

Figure Lengend Snippet: Figure 4. Effect of O-linked glycosylation inhibition on expression of glycoprotein with dimeric Lea (Lea-on-Lea) structure in Colo205 cells. Colo205 cells were grown with/without the inhibitor of O-linked glyco- sylation, 2 mM benzyl-·-GalNAc, for 72 h, and the cell culture medium was subjected to Western blotting using ST421 antibody as described in Materials and methods. Lane 1, 1/10 diluted fetal calf serum (negative control); lane 2, culture medium without treatment of benzyl-·-GalNAc; lane 3, cultured medium with treatment of benzyl-·-GalNAc.

Article Snippet: Colo205 cells (21), originally purchased from ATCC, were cultured and maintained in RPMI-1640 medium with 10% fetal calf serum at 37 ̊C in 5% CO2/95% air.

Techniques: Glycoproteomics, Inhibition, Expressing, Cell Culture, Western Blot, Negative Control