Human tagged orf clone

AKR1C3 encoding vector (RC200210–AKR1C3 (NM_003739) Human Tagged ORF Clone) was purchased from OriGene (Rockville, MD, US). A total of 25 × 10⁴ cells/well were transfected with 1 µg of total DNA in six-well plates by using Lipofectamine LTX (Invitrogen) for 8 h, as recommended by the supplier. At 24 h after transfection, cells were trypsinized, plated at 30 × 10⁴ cells/well in six-well plates and treated as indicated.

Site-directed mutagenesis was performed using 50 ng of KAT3A/CBP (CREBBP) (NM_004380) Human Tagged ORF Clone (OriGene) as the dsDNA template. This was carried out using the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s protocol with the following exception: One Shot™ Stbl3™ Chemically Competent E. coli (Invitrogen) was used for transformation rather than the XL10-Gold Ultracompetent Cells supplied with the kit because of the dependency on chloramphenicol selection already found in the full-length CREBBP vector. Mutagenic oligonucleotide primers were designed using Agilent QuickChange Primer Design program and purchased from Sigma-Aldrich with PAGE purification with the following sequences:
5′-tggatgcagcgctagatgctcagccgg-3′
5′-ccggctgagcatctagcgctgcatcca-3′
Following transformation, single colonies were selected on LB plates containing 34ug/mL chloramphenicol, expanded in LB broth containing 34ug/mL chloramphenicol overnight, and extracted with a QIAfilter midi kit (Qiagen). Sanger sequencing was utilized to confirm the presence of the desired mutation. Mutant plasmids were directly transfected into cell lines using GeneJet transfection reagent (SignaGen Labs) or packaged in 293T for lentiviral infection.

For plasmid construction, full-length coding sequences of human B3GALT5 from B3GALT5 (NM_006057) Human Tagged ORF Clone (Origene, MD, USA) were cloned with pLenti6/V5 Directional TOPO Cloning Kit (ThermoFisher, CA, USA) according to the manufacturer’s instructions. Viral supernatant was collected 48 h after transfection and then filtered with 0.45-μm filters. MDA-MB-231 cells were transduced with lentiviruses carrying vector overexpressing B3GALT5 or control vector at a multiplicity of infection (MOI) of 10 in the presence of 8 μg/ml polybrene (Sigma-Aldrich, MO, USA) for 12 h. Cells were selected with 10 μg/ml blasticidin (Catalog #A1113903, ThermoFisher, CA, USA) for more than 1 month to generate stable clones of blasticidin-resistant cells.

Site-directed mutagenesis was performed for shCOX17#2 hairpin using Q5^® Site-Directed Mutagenesis Kit (NEB) and the following primers: mutantF (5′-cccgaaagccaagaaaaaaagccgctgaagccc-3′) and mutantR (5′-cggggcagggtttgagtcaaccag-3′) for inducing silent mutations in wild-type COX17 (NM_005694) Human Tagged ORF Clone (Origene) to make it resistant to shCOX17#2. This shCOX17#2 mut (6.25 ng) was transfected into LM2 cells stably expressing shCOX17#2 using lipofectamine and used for invasion assays.

cDNAs were prepared with the Transcriptor First Strand cDNA Synthesis Kit (Roche) from total RNA from passages p4-p8 (NucleoSpin RNA Kit, Macherey-Nagel), and qRT-PCR analyses were performed using the SYBR Premix Ex Taq (Takara) and a Light Cycler 480 instrument (Roche). AnyGenes custom panels (BioNova) were used to validate some gene expression results from the microarrays. Expression was normalized with respect to the mean level of expression of the housekeeping genes ACTB1, TFAP2E, ACBD3, and LETMD1. Reactions were performed in triplicate.
To investigate the effect in gene expression upon introduction of control NIPBL (Supplementary Fig. 3), patient 3-derived fibroblast were nucleofected (Amaxa® Human Dermal Fibroblasts Nucleofector Kit (Lonza)) with 2 µg of the commercial plasmid IDN3 (NIPBL-GFP) (NM_015384) Human Tagged ORF Clone (Origene) in triplicate, followed by qRT-PCR after 24 h.