Pcdna expression vector

To construct the stable cell lines, lentiviral vector containing short hairpin RNA (shRNA) of circRERE (sh-circRERE) and that containing shRNA of negative control (sh-NC) were provided by GenePharma (Shanghai, China). MiR-152-3p mimic, mimic NC, miR-152-3p inhibitor and inhibitor NC were also from GenePharma. The pcDNA expression vector and the pcDNA-CD47 recombinant vector were obtained from Invitrogen (Carlsbad, CA, USA). Lipofectamine™ 3000 (Invitrogen) was used for the transfection of RNAs or vectors in 8226R5 and MM1.R cells, following the user guide supplied by the manufacturer.

The majority of expression vectors were generated using Gateway cloning technology (Invitrogen). Briefly, complementary DNA (cDNA) was amplified using PCR and then subcloned into the pENTR-D/TOPO vector. Each cDNA in the TOPO vector was recombined into the pcDNA expression vector, which contained various N-terminal or C-terminal tags (Invitrogen). The procedures were followed according to the manufacturer’s descriptions and modified as previously described (54 (link)). pcDNA expression vectors containing N or C terminus 3xFLAG-3xHA (3F3H) or C terminus 3xHA were used in these studies. Each PCR product encoding a GPCR conjugated with KKTN sequences at the C terminus was amplified from pcDNA-GPCR-3F3H constructs using cloning primers listed in SI Appendix, Table S2 and then was inserted into the KpnI and XbaI restriction sites of the pcDNA-PROKR2-GFP plasmid. The expression plasmids for FLAG-tagged gp78 and His/Myc-tagged HRD1 were kindly provided by Yihong Ye (National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD) (55 (link)). All mutations were generated by site-directed mutagenesis (QuikChange II Site-Directed Mutagenesis Kit; Agilent) using primers listed in SI Appendix, Table S2.

Small interfering RNAs (si-circ_0035483#1, si-circ_0035483#2, and negative control si-NC), miRNA mimics (miR-31-5p and miR-NC), miRNA inhibitors (anti-miR-31-5p and anti-miR-NC), and lentiviral short hairpin RNA (shRNA) vectors (sh-circ_0035483 and sh-NC) were bought from GenePharma (Shanghai, China). In addition, an empty pcDNA expression vector was acquired from Invitrogen (Carlsbad, CA, USA) and the recombinant pcDNA-HMGA1 vector was constructed for HMGA1 overexpression. After 786-O and CaKi-1 cell density reached 60%, the mixture of oligonucleotides or vectors, Lipofectamine™ 3000 Reagent (Invitrogen) and Opti-MEM™ I Reduced Serum Medium (Gibco) was added following the operating instruction for users.