Quickchange lightning kit

cDNA coding human wild-type DNMT3A was a kind gift from Dr. F. Fuks (Free University of Brussels). DNMT3A open reading frame was PCR amplified and subcloned into pMIGR1 (MSCV-IRES-GFP) retroviral vector. R882H substitution was introduced by site-directed mutagenesis using QuickChange Lightning kit (Agilent Technologies) according to manufacturer’s instructions. Viral particles were produced in 293T cells using pPAX2 packaging system and pseudotyped with VSV-G. Virus-containing supernatants were concentrated by PEG-8000 precipitation. Target cells transduced with DNMT3A^wt or DNMT3A^R882H viral particles were FACS-sorted based on GFP expression. SUPT16H (a gene coding SPT-16) targeting constructs were cloned in the pLKO.1 backbone from the TRC 2.0 collection: TRCN0000293281, TRCN0000293313, TRCN0000293348, TRCN0000293349, TRCN0000293350. Viral particles were generated as described above and used to transduce U2OS cells. Transduced cell populations were selected on puromycin for 7 days. Knock-down efficiency was assessed by immunoblotting.

pCDH-ESR1 plasmid was mutated at alanine 569 of ER to serine with Quick Change Lightning Kit (Agilent Technologies). Cells were obtained from the Tissue Culture Shared Resource at the Lombardi Comprehensive Cancer Center (Georgetown University, Washington, DC) in 2001. Cells were never passaged more than 15 times, were free of mycoplasma contamination (most recent testing, May, 2015) and identity was confirmed by short tandem repeat (most recent testing, July, 2014). Lentivirus containing the p.A569S ESR1 transgene was packaged in 293T cells. 24 hours after plating, cells were transfected with 8ug of pCDH-ESR1-A569S plasmid, 5ug psPAX2 and 2ug pMD2.G plasmids. MCF-7 cells were virally transduced with 1ml of viral supernatant supplemented with 4ug/ml polybrene for 8 hours. Steroid-depleted parental and ESR1 p.A569S MCF-7 cells were seeded into 96-well plates and treated with 17β-estradiol (Sigma Aldrich) or ethanol control alone or in combination with tamoxifen, 4-hydroxytamoxifen, endoxifen or fulvestrant. Cell number was assessed by crystal violet stain five days after hormone treatment as previously described (30 (link)) (Supplementary Materials and Methods).

Animal studies were performed in accordance with the University of Pennsylvania, institutional review board (IRB). LDLR^-/-, APOBEC-1^-/- double knockout (DKO) and LDLR^-/-, APOBEC-1^-/-, human ApoB100 transgenic (LAHB) mice were injected via tail vein for vector administration and serum collected by retro-orbital bleeds. At end of study animals were sacrificed and livers harvested for analysis. Vector were obtained from the Vector Core at the University of Pennsylvania and expressed cDNAs for hLDLR, hPCSK9 or hIDOL driven from a thyroxine binding globulin promoter (TBG). Mutations were introduced into wild type hLDLR using the Quick Change lightning kit (Agilent). Serum cholesterol levels were analyzed on a MIRA analyzer (Roche). Western blotting was done using precast mini gels (Invitrogen) and probed using a polyclonal hLDLR antibody. In vitro LDLR assay was performed by transiently transfecting HEK293 cells and pulsing the cells the following day with BODIPY labeled LDL (Invitrogen). Data were analyzed using one-way Analysis of Variance models with pair-wise group differences in mean cholesterol level assessed using Tukey's post-hoc tests.