Lipofectamine 2000 3000

Delivery of modified mRNA was optimized for use in cultured primary human hepatocytes. Lipofectamine™ 2000, 3000, RNAiMax (Thermo Fisher Scientific, Inc., MA) and TransIT® -mRNA transfection kit (Mirus BIO, LLC, WI) were tested for efficiency and Lipofectamine™ 2000 transfection reagents proved best suited. Modified mRNA was allowed to thaw and mixed with Opti-MEM® I (Thermo Fisher Scientific) reduced serum media. Lipofectamine™ 2000 was also diluted in Opti-MEM® I and incubated at room temperature for 5 minutes. Diluted Lipofectamine™ 2000 and modified mRNA were mixed at a ratio of 3:1, where 9 µl Lipofectamine™ 2000 reagent was used for 3 µg modified mRNA and allowed for lipoplex formation for 15 minutes at room temperature. Lipoplexes were added and mixed into culture media of cultured hepatocytes. Lipofection using enhanced green fluorescent protein (eGFP) encoding modified mRNA was used as control of transfection. Empty liposomes without mRNA content acted as vehicle control, this was selected as minor cytotoxic effect was observed using eGFP as control. Cellular cytotoxicity of GFP has been previously reported^{38 (link),39} .

Transient transfections were performed with Lipofectamine 2000/3000 (Invitrogen Life Technologies) for NIH-3T3 and E14 cells and JetPei (Polyplus transfection) for U2OS, HeLa and RPE1 cells according to the manufacturer’s instructions. siRNA transfections were performed with Lipofectamine RNAi max, according to the manufacturer’s instructions. For siRNAs used see key resources table and Table S2.

SiRNAs were transfected into MOLM13, THP1, MV4-11, and RS4-11 cells at a final concentration of 50 nM with Neon™ Transfection System 10 μL Kit using the Neon Transfection System (Invitrogen, USA). HEK293T cells were transfected using the Lipofectamine 2000/3000 (Invitrogen, USA). Cells were collected 48 or 72 h after transfection. The siRNA sequences were listed in Additional file 1: Table S4.

The cDNA encoding the CDS of NFATC3 and full length of linc00423 was PCR-amplified by Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix (Thermo) and subcloned into the EcoRI and XhoI sites, BamHI and EcoRV sites of the pcDNA3.0 vector (Invitrogen) respectively, named NFATC3 and linc00423. The siRNAs specifically targeting linc00423 and NFACT3, and control siRNA were synthesized by GenePharma (Shanghai). Cells were transfected with the plasmids or siRNAs using Lipofectamine 2000/3000 (Invitrogen) according to the manufacturer’s protocol.

The BRD4 inhibitor i-BET151 (Selleck, S2780) was treated in MV4-11 cells at a concentration of 1 μM and the cells were collected after 12 h. The FTY720 (Selleck, S5002) was treated in MV4-11 and RS4;11 cells and the cells were collected after 24 h. The doxycycline (Selleck, S4163) was treated in doxycycline-inducible SERINC2 expression MV4-11 and RS4;11 cells at a concentration of 4 μM. SiRNAs or SERINC2-expressed plasmid were transfected into 4 × 10⁵ cells at a final concentration of 50 nM or 50 ng/μL with Neon™ Transfection System 10 μL Kit using the Neon Transfection System (Invitrogen, USA). HEK293T cells were transfected using the Lipofectamine 2000/3000 (Invitrogen, USA). Cells were collected 48 or 72 h after transfection. The siRNAs sequences were shown in Additional file 6: Table S5.

The method used to construct circRNA overexpression vector was as described previously [32 ]. In brief, we used the introns from SUZ12 to facilitate circRNA product. All intron sequences were cloned in pCDH-CMV-MCS-EF1-Puro-copGFP vector. Sequences of exon 3-4 of AF4 gene were inserted into the vector. We named this vector for circAF4 overexpression as PCDH-circAF4. For stable overexpression of circAF4 in leukemia cell RS4;11, PCDH-circAF4 vector was packaged into lentiviruses using Lentivector Expression Systems (System Biosciences, Germany), consisting of pPACKH1-GAG, pPACKH1-REV, and pVSV-G vectors, which were co-transfected in 293T cells using the Lipofectamine 2000/3000 (Invitrogen, USA) system according to the manufacturer’s guidelines. Finally, the lentiviruses were transformed into RS4;11 cells, and the transformed cells were then selected with puromycin.

Specific shRNAs against LINC01503, SFPQ, FOSL1, ALX3, and AR were designed by the BLOCK-iT^™ RNAi Designer (http://rnaidesigner.thermofisher.com) and then synthesized according to sequences shown in Supplementary Table 5. All shRNA sequences were cloned into the pLKO.1 vector (Addgene, Cambridge, MA, USA). The full-length sequences of LINC01503, SFPQ, FOSL1, and AR were amplified and cloned into the pHAGE-6tag-puro vector (Addgene). The wild type and mutant LINC01503 and FOSL1 promoters were cloned into the pGL3 vector (Addgene). Specific primers were listed in Supplementary Table 5.
All of the plasmids were confirmed by DNA sequencing, transiently transfected into NPC cells with Lipofectamine 2000/3000 reagents (Invitrogen), and the transfected cells were then harvested for assays 48 h later. The shLINC01503 (sh1503) or its scramble control (shCtrl) plasmids were co-transfected into HEK293T cells with the lentivirus packaging plasmids pMD2G and psPAX2 (Addgene). After 48 h incubated, the lentivirus supernatant was collected and used to infect HK1 and SUNE1 cells, and stably transfected cells were selected and maintained using puromycin.