Hindiii

For creating the hairpin of Norway spruce PaBI-1 the corresponding parts of PaBI-1 cDNA were amplified using attB1_AS_BI1_F/AsBI1_R_HindIII and attB2_S_BI1_R/S_BI1_F_HindIII using Phusion DNA polymerase (Thermo Scientific) and cut with HindIII (Thermo scientific), which produced 360 bp and 466 bp fragments of PaBI-1 coding DNA sequences, respectively. The fragments were ligated with T4 DNA Ligase (Thermo Scientific) and recombined into pDONR/Zeocin (Zeo) vector by BP Clonase (Invitrogen) followed by recombination into modified pMDC32 vector containing suspensor-specific NIP (nodulin-like intrinsic protein) promoter [26 (link)] by LR Clonase (Invitrogen). The resulting recombined vector was checked by digestion with restriction enzymes followed by sequencing.

The GPD promoter was amplified from the pYM-N15 plasmid and incorporated upstream of the SSA4 ORF via homologous recombination in the WT and ssa1Δ ssa2Δ backgrounds. Deletion mutants were constructed either by PCR-mediated knockout or sporulation. Transformants and dissected spores were verified by PCR. Plasmids with misfolding GFP-tagged proteins (pguk1-7GFP and pgus1-3GFP) were linearized with restriction enzymes (NsiI) and integrated at the HIS3 locus via homologous recombination. The three chimeric SSA4-SSA1 alleles were constructed (GenScript Biotech Corporation, Nanjing, China) and cloned into carrier vectors. The constructs were then amplified with homologous sequences at the 5’ and 3’-ends to allow for homologous recombination into the pRS415 vector. The SSA1 and SSA4 alleles were amplified from the BY4741 strain with the same homologous sequences. The pRS415 vector was digested with XbaI and HindIII (Thermo Fisher Scientific) and the plasmids were recombined with the constructs in wild type yeast and then extracted and transformed into E. coli to allow for plasmid production. The final vectors were digested with HindIII and SacI (Thermo Fisher Scientific) for insert size control and then sequenced for verification prior to transformation into the intended host strains.

pEMBLyex4 was linearized overnight at 37°C with BamHI, HindIII, SalI and XhoI (Thermo Fisher Scientific, USA) and pXOOY was linearized overnight at 37°C with BamHI and HindIII (Thermo Fisher Scientific, USA) and both were subsequently heated to 80°C for 10 minutes for restriction enzyme inactivation. PAP1500 yeast cells were transformed with overlapping PCR fragments according to [17 (link)] and transformed cells selected on minimal medium containing 2% glucose as well as lysine (20mg/L) and leucine (30mg/L). For rescuing assembled plasmids, total yeast DNA was purified from 10mL cells lysed with lyticase enzyme using the NucleoSpin plasmid DNA purification kit (Macherey-Nagel, Germany).

To generate the transgenic construct pTol2-Cca.actb-GCaMP6s (Figure 1A), Tol2-elavl3-GCaMP6s obtained from Addgene (#59531, Watertown, MA, USA) and pT2/carp_actin_P-Abcc4-Flag-carp_actin_pA constructed in our previous study [61 (link)] were double digested using Hind III and EcoR I from Fermentas (Burlington, NJ, Canada). The desired DNA fragments were recovered through electrophoresis and subsequent gel purification using a kit from Bioer (Hangzhou, China). The common carp β-actin (actb) gene promoter was inserted into the cloning site to replace the elavl3 promoter of Tol2-elavl3-GCaMP6s. The resultant construct was confirmed by double digestion using EcoR I and Xho I (Fermentas, Burlington, NJ, Canada).

pKK233-2 plasmids containing human NQO1*1 (wild-type) and NQO1*2 (C609T) cDNA (NCBI Reference Sequence: NM_000903.3) were a kind gift of Dr. David Ross [15 (link)]. The following primers were used to amplify by PCR the different cDNAs. Forward 5′-ccgaagcttgccatggtcggcagaagagc-3′ and Reverse 5′-ccgggtacctcattttctagctttgatct-3′ (Sigma, St Louis, MO, USA). HindIII and KpnI (Fermentas, Vilnius, Lithuania) were used as restriction enzymes and insert DNA were then cloned into pcDNA3.1 plasmid from Invitrogen (Grand Island, NY, USA). The transfection of MDA-MB-231 cells were done with different plasmids (1 μg), followed by four week-selection of exposure to 1 mg/mL neomycin (Invivogen, San Diego, CA, USA). Both NQO1 enzyme activity and protein levels were used to characterize stable transfecting clones. Only clones with high NQO1 activity and similar NQO1 protein levels were chosen for further studies.