Gateway cloning kit

The wild-type human CYP1B1 cDNA was purchased from Open Biosystems, and was cloned in a pDONR201vector using the Gateway Cloning kit (Invitrogen). An attB-flanked PCR product of CYP1B1 cDNA was cloned into the pDONR201 entry vector and then transferred into destination vector pSD-69 using the Gateway Cloning kit (Invitrogen). The pSD-69 plasmid contains the human phosphoglycerate kinase (PGK) promoter, a Gateway attR cassette (Invitrogen), the mouse PGK promoter and the puromycin acetyltransferase gene cloned into pRRLhPGK.GFP.SIN18.^{14 (link)} The variant cDNA was obtained by site-directed mutagenesis through replacement of G¹⁶⁹⁷ by C¹⁶⁹⁷, using the Site-Directed Mutagenesis kit (Stratagene). Constructions were verified by sequencing, and nucleotide replacement was checked by pyrosequencing. The pER51CYP1B1-WT (G allele: WT) and pER51CYP1B1-VAR (C allele: VAR) plasmid DNAs were then used for the generation of viruses and the infection of cell lines. The tdTomato-CAL27 and -CAL33 isogenic cell lines were produced by transduction of the cells with a second lentivirus carrying the tdTomato reporter gene (PGK-tdTomato), which was a generous gift from Pr François Moreau-Gaudry (University of Bordeaux).

The coding region of Trx h2 gene was amplified using the primers, TRXH2-F_GW (5′-aaaaagcaggctttatgggaggagctttatcaactgtg-3′), TRXH2-R_GW (5′-agaaagctgggtttgctctgagtttgctaactttcttc-3′), attB1 adapter (5′-ggggacaagtttgtacaaaaaagcaggct-3′) and attB2 adapter (5′-ggggaccactttgtacaagaaagctgggt-3′). The DNA fragment was first subcloned into pDONR221 (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) and then transferred into pJCV52 (VIB, Ghent, Belgium) using a GATEWAY cloning kit (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Arabidopsis plants were transformed by following the floral dip method [48 (link)]. The transformed plants were grown on the medium containing 50 mM of kanamycin. The survived plants were used to propagate seeds for the selection of homozygous transgenic lines.