Puromysin

The RNAi target site was designed by using the website of GeneSil (http://www.genesil.com/business/products/order2.htm), and the target sequences are listed at Supplementary Table S2. The hairpin oligonucleotides were synthesised in Beijing Genomics Institute (BGI, Beijing, China) and cloned into the pLKO.1 lentivirus vector. Human full-length Bcl-w (GenBank: NM_004050.4) cDNA was obtained by using PCR (primers were shown in Supplementary Table S3), and the DNA fragments were then inserted into PCDH-CMV-MCS-EF1-puro vectors to generate the recombinant plasmids. Vectors were transfected into 293FT cells to generate lentiviruses by using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, the lentiviruses were infected into cells according to the manufacturer’s protocol from Invitrogen and the cells were selected with puromysin (5 μg ml⁻¹, Sigma) for at least 72 h.

The BMSCs were purchased from Cyagen Biosciences Co., Guangzhou, and cultured as previously described (22 (link)). BMSCs-OE-FNDC5 were established by transfection with lentiviruses, and a standard protocol was used to generate viruses. Briefly, lentiviruses were prepared using the VsVg and PAX2 lentiviral package system (Invitrogen, Carlsbad, CA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in HEK293FT cells. Supernatants containing viral particles were collected 3 days after transfection and concentrated using centrifugal cutoff filters (Merck, Darmstadt, Germany).
After dissociation into single-cell suspensions, the BMSCs were replaced with concentrated lentiviral particles. Twenty-four hours after infection, a fresh culture medium was slowly replaced. Puromysin (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1–5 µg/mL was added to the culture medium 4 days after transduction. Five days later, the conventional culture medium without Puromysin was replaced. Ultimately, BMSCs-OE-FNDC5 and BMSCs-OE-NC were obtained.