Glutagro

Hepa1-6 and AML-12 murine hepatocyte cell lines were obtained from the ATCC and maintained in DMEM, 4.5 g/L glucose without sodium pyruvate (Thermo Scientific) and supplemented with Pen/Strep and Glutagro (Corning) and DMEM-F12 1:1, (ThermoFisher Scientific) supplemented with Pen/Strep, Glutagro (Corning), insulin-transferrin-selenium (ThermoFisher Scientific) and dexamethasone (100 nM, Sigma Aldrich), respectively.

PC-3 cells were seeded at 1,400 cells/100 μL in 96-well plates in RPMI media (Sigma-Aldrich) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (Pen/Strep), and 1% GlutaGro (Corning). After an overnight incubation at 37°C and 5% CO₂, media in the wells were replaced with fresh RPMI media with 5% charcoal-stripped serum (CSS)-FBS, 1% Pen/Strep, and 1% GlutaGro (Corning). Cells were then grown for 3 days. We then added one of the following three drug groups: 50 nM methotrexate alone, 50 nM methotrexate + CPG2_CP-N89-K177A (100 to 5,000 pM), or 50 nM methotrexate + Pro-CPG2-1-Disulfide (100 to 5,000 pM). Treatment lasted for 6 days with replenishment of the media, drug, and enzymes after 3 days. Cell viability was measured using WST-1 at 1:10 dilution with CSS-FBS media at absorbance of 450 nm (Tecan 1100 Plate Reader).

Doxycycline-inducible human MCL cell lines (Mino, Rec-1, JeKo-1, Z138), originally obtained from the American Type Culture Collection (ATCC), were engineered and authenticated by gene expression profiling to express the bacterial tetracycline repressor as described previously.⁶ (link) Wild-type Granta-519, Maver-1, JVM-13 were provided by Dr. Michael Wang at MD Anderson Cancer Center. Doxycycline (20 ng/mL) was used for inducing the expression of sgDNMT3A of interest. The MCL cell lines were grown in RPMI 1640 media (Hyclone) supplemented with 10% FBS (fetal bovine serum, Atlanta Biologicals), 100 U/ml penicillin, 100 μg/mL streptomycin (Corning Cellgro), 2 mM GlutaGRO (Corning Cellgro), 1×MEM-NEAA (Quanlity Biological, Inc.), and 1 mM Sodium Pyruvate Solution (Hyclone). Human embryonic kidney cell line 293T was cultured in DMEM (Dulbecco’s modified Eagle’s medium, Hyclone) with 10% FBS, 100 U/ml penicillinand 100 μg/mL streptomycin (Corning Cellgro). Cell lines were authenticated using short tandem repeat analysis (Idexx BioAnalytics, Westbrook, ME) and per ATCC guidelines using morphology, growth curves, and Mycoplasma testing within 6 months of use with the e-Myco mycoplasma PCR detection kit (iNtRON Biotechnology Inc, Boca Raton, FL). All cell lines were cultured at 37°C in a 5% CO₂.