Promoter Reporter Assays in HEK293FT and DC2.4 Cells

HEK293FT or DC2.4 (SCC142, Millipore) cells were cultured in DMEM supplied with 10% FBS (GIBCO) and penicillin–streptomycin (Thermo Fisher Scientific). VCAM1, Il1b, Tnf, Il23, Ppara, Rara, IFNG, RORC and IL12RB promoter reporter plasmids expressing gaussia luciferase were acquired from GeneCopoeia; pGud-Luc has been described previously^{91 (link),92 (link)}. Reporters were transfected with Lipofectamine 2000 (11668019, Thermo Fisher Scientific). To evaluate AHR promoter activity, cells were stimulated with vehicle, propyzamide (10 μM), FICZ (0.5 μM) or a combination of propyzamide and FICZ. To evaluate VCAM1 promoter activity, cells were stimulated with vehicle or propyzamide for 2 more days before medium collection. CEBPB plasmid (15738, Addgene) or pGL4.54[luc2/TK] vector (E506A, Promega) were co-transfected to evaluate C/EBPβ binding to the Il1b, Tnf, Il23, RORC, IL12RB1 or IFNG promoters. Luciferase activity was evaluated using the Gaussia Luciferase Flash Assay Kit (16159, Thermo Fisher Scientific).

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Sanmarco L.M., Chao C.C., Wang Y.C., Kenison J.E., Li Z., Rone J.M., Rejano-Gordillo C.M., Polonio C.M., Gutierrez-Vazquez C., Piester G., Plasencia A., Li L., Giovannoni F., Lee H.G., Akl C.F., Wheeler M.A., Mascanfroni I., Jaronen M., Alsuwailm M., Hewson P., Yeste A., Andersen B.M., Franks D.G., Huang C.J., Ekwudo M., Tjon E.C., Rothhammer V., Takenaka M., de Lima K.A., Linnerbauer M., Guo L., Covacu R., Queva H., Fonseca-Castro P.H., Bladi M.A., Cox L.M., Hodgetts K.J., Hahn M.E., Mildner A., Korzenik J., Hauser R., Snapper S.B, & Quintana F.J. (2022). Identification of environmental factors that promote intestinal inflammation. Nature, 611(7937), 801-809.

Publication 2022

SCC142 FBS penicillin–streptomycin Lipofectamine 2000 pGL4.54[luc2/TK] vector Gaussia Luciferase Flash Assay Kit

Assay Cebpb Cells Ficz Ifng Il1b Il23 Lipofectamine 2000 Luciferase Penicillin Pgl4 Plasmid Promega Propyzamide Streptomycin Vector

Corresponding Organization :

Other organizations : Brigham and Women's Hospital, Harvard University, National Yang Ming Chiao Tung University, Woods Hole Oceanographic Institution, Max Delbrück Center, Boston Children's Hospital, Broad Institute

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Variable analysis

independent variables

Vehicle
Propyzamide (10 μM)
FICZ (0.5 μM)
Combination of propyzamide and FICZ
Vehicle or propyzamide for 2 more days
CEBPB plasmid (15738, Addgene)
PGL4.54[luc2/TK] vector (E506A, Promega)

dependent variables

AHR promoter activity
VCAM1 promoter activity
C/EBPβ binding to the Il1b, Tnf, Il23, RORC, IL12RB1 or IFNG promoters
Luciferase activity

control variables

HEK293FT or DC2.4 (SCC142, Millipore) cells
DMEM supplied with 10% FBS (GIBCO) and penicillin–streptomycin (Thermo Fisher Scientific)
VCAM1, Il1b, Tnf, Il23, Ppara, Rara, IFNG, RORC and IL12RB promoter reporter plasmids expressing gaussia luciferase
PGud-Luc
Lipofectamine 2000 (11668019, Thermo Fisher Scientific)

positive controls

PGud-Luc (described previously in Refs. 91 and 92)

negative controls

Vehicle

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