Lentivirus

Human normal breast cell line MCF-10A and breast cancer cell lines HCC1937, MDA-MB-231 and MCF-7 were obtained from BeNa Culture Collection (Beijing, China). MCF-10A and HCC1937 cells were cultured in the completely culture medium containing 90% RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) and 10% FBS (Thermo Fisher Scientific); MDA-MB-231 cells were cultured in 90% L-15 cell culture medium (Thermo Fisher Scientific) and 10% FBS; MDA-MB-231 cells and MCF-7 cells were cultured in minimum essential medium (Thermo Fisher Scientific) supplemented with 10% FBS. All cells were maintained in a humidified incubator with 5% CO₂ in a constant temperature of 37°C.
To upregulate or downregulate the expression of PUF60, lentivirus containing PUF60 open reading frame (OE-PUF60) and shRNA targeting human PUF60 gene were purchased from OriGene (Beijing, China). And lentivirus (OE-PTEN) used to upregulate PTEN expression was designed and synthesized by GenePharma (Shanghai, China).

The Lentivirus vectors of three CD36 shRNAs termed as pLent-U6-GFP-Puro-CD36 shRNAs, and one scramble shRNA termed as pLent-U6-GFP-Puro-Scramble shRNA were obtained from Origene (Rockville, MD). The shRNAs were transfected into the cells with X-treme GENE HP DNA Transfection Reagent (Roche, St. Louis, MO) according to the kit instruction. Cells were maintained for another 48h to express the exogenous genes. After testing, the selected CD36 shRNA sequence given the best inhibition effect was 5'-GGACCATTGGTGATGAGAAGGCAAACATG-3'. Then the plasmids (pLent-U6-GFP-Puro) containing the selected CD36 shRNA and scramble-shRNA were packed into Lentivirus respectively by Weizhen Biotechnology Co., Ltd (Shandong, China). The virus particles were used to infect cells and then selected by 1 μg/mL puromycin (Sigma-Aldrich, MO) for 3 days to produce stable transfected cells (designated as NCI-H23-ShCD36, Bet1A-ShCD36, A549-ShCD36, NCI-H23-Scramble, Bet1A-Scramble, and A549-Scramble, respectively). Knockdown efficiency was confirmed at protein levels.