SiRNAs or anti-sense nucleotides (ASO) specifically targeting circPGR were purchased from RiboBio (siRNA targeting sequence: CCAGGGCAGCACAACUACU dTdT; ASO targeting sequence: GCCAGGGCAGCACAACUAC dTdT). miR-301a-5p mimic and inhibitor were also purchased from RiboBio. SiRNA transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Plasmid transfections in HEK293T cells were performed using Polyethyleneimine (PEI, Polysciences) according to the manufacturer's protocol. Plasmids transfection in MCF7 cells were performed using Lipoplus reagent (Sagecreation) according to the manufacturer's protocol.
For Lenti-virus packaging: HEK293T cells were seeded in culture plates coated with poly-D-lysine (0.1% (w/v), Sigma, P7280) and transfected with lenti-viral vectors together with packaging vectors, pMDL, VSVG and REV, at a ratio of 10:5:3:2 using Polyethyleneimine (PEI, Polysciences) for 48 h according to the manufacturer's protocol. Virus were collected, filtered and added to MCF7 cells in the presence of 10 μg/mL polybrene (Sigma, H9268), followed by centrifugation for 30 min at 1,500 g at 37 °C. Medium was replaced 24 h later.
Copyright and License information: ©2021 The author(s)
This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.