Lipofectamine 3000

MA104-shRNA-NSP2 cell line was generated using the PiggyBac system. Briefly, 10⁵ MA104 cells were co-transfected with the plasmid pCMV-HyPBase encoding the hyperactive variant of PiggyBac transposase Yusa et al., 2011 (link); Li et al., 2013 (link); Hubstenberger et al., 2017 (link) along with the plasmid pPB[shRNA]-EGFP:T2A:Puro-U6 harbouring shRNA targeting RVA NSP2 gene using Lipofectamine 3000 (Sigma-Aldrich), following the manufacturer’s instructions. The cells were maintained in DMEM supplemented with 10% FBS for 3  days, and then the cells were subjected to selection in the presence of 5  μg/ml puromycin (Sigma-Aldrich) for 4  days, prior to further selection by FACS sorting for EGFP expression.

LN18 or U87-MG at approximately 70% confluence were transfected with each of PZFN1 and PZFN2 CompoZr^™ Knockout Zinc Finger Nuclease plasmids targeted to BCL6 (Sigma Aldrich, USA), using Lipofectamine^® 3000. After both 24 hours and 72 hours, the medium was collected and the debris and detached cell fraction was pelleted by centrifugation, and the supernatant aspirated. The adherent cells were then harvested and pelleted by centrifugation, or combined with the non-adherent fraction before pelleting by centrifugation. Cell pellets were stored at -80° C. gDNA was extracted from ZFN transfected cell pellets using the Quick-gDNA^™ MiniPrep kit (Zymo Research, USA) as per the manufacturers protocol. gDNA was quantified by fluorometry before PCR amplification of a 381 bp fragment of the BCL6 using Phusion^® High-Fidelity DNA Polymerase, and BCL6 locus forward primer (5’-GAAGAGTTCCTCAACAGCCG-3’), and reverse primer (5’-TTCTGGGATTGTTTCCTTGG-3’), using the following parameters: 1 cycle of 98 °C, 30 s. 30 cycles of 98 °C, 10s; 57 °C, 30 s; 72°C, 10 s. Nuclease activity was detected by CEL1 endonuclease digestion of heteroduplex DNA using the Surveyor^® Mutation Detection Kit (IDT, USA) using the manufacturers protocol.

The cells cultured in 100-mm-diameter dishes (Thermo Fisher) were transfected with indicated plasmids using Lipofectamine 3000, and 36 h later, the cells were lysed with lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, 0.5% Triton X-100, 1 mM EDTA, 0.1% sodium deoxycholate, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail [Sigma-Aldrich]) on ice for 30 min. The cell lysate supernatant was collected by centrifuging and then precleared by incubation with protein G/protein A agarose for 1 h at 4°C. The supernatant was incubated with indicated antibodies overnight at 4°C and precipitated with protein G agarose/protein A agarose for 30 min at room temperature. Then, the precipitated protein G agarose/protein A agarose was centrifuged at 2,000 × g for 10 s and washed three times with PBS. Finally, the bound proteins were eluted by boiling for 10 min in 2 × loading buffer, followed by SDS-PAGE and immunoblotting. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL) reagents (Bio-Rad).

We seeded H9c2 cells into six-well plates. When the cell growth density reached 30%, we used Lipofectamine 3000 reagent (Sigma-Aldrich, St. Louis, MO, USA) to transfect NC plasmid and FOXC2 overexpression plasmid into H9c2 cells. RT-PCR was used to detect the transfection efficiency.

LNAs (Qiagen) were designed to target SNORA73 and the SNHG3 lncRNA, or GFP as a control. pGFP-C-shLENTI lentiviral shRNA constructs (Origene TR30023) were designed to target hamster SNHG3 lncRNA or GFP as a control. See Supplementary Table 2. For LNA knockdown experiments in NIH 3T3 cells, 4.0 × 10⁴ cells were seeded per well in a 6-well dish and transfected the following day with 25 nM LNA using Lipofectamine 3000 (ThermoFisher L3000015). SNHG3 and SNORA73 expression and palmitate-induced cell death were analyzed 48 h following transfection. For LNA knockdown in CHO cells, 2.5 × 10⁵ cells were plated in 6-cm dishes and transfected the next day with 25 nM LNA using Lipofectamine 3000. For LNA knockdown in primary human fibroblasts, 5.0 × 10⁴ cells were plated in 6-well plates, or 1.5 × 10⁵ cells were seeded in 6-cm dishes. Cells were transfected with 25 nM LNA the following day using Lipofectamine 3000. For shRNA transduction experiments, virus was harvested from HEK293T cells (ATCC CRL-3216) that were transfected using Lipofectamine 3000 with shRNA constructs and Mission helper plasmids (Sigma SHP001). 5.0 × 10⁵ CHO cells were seeded in 6-cm dishes, grown for 24 h, and transduced with shSCR or shSNHG3 lentiviral particles. After expansion of the population, the top 20 percent of cells were flow-sorted and maintained as the transduced population.