Pcdna3.1 empty

HEK293T cells, cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific) with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and SH-SY5Y cells, cultured in DMEM: F12 (Thermo Scientific) with 15% FBS and 1% penicillin and streptomycin, were used. The cell lines were transfected with 0.5 μg/μl pcDNA3.1-CG4928-Flag (Invitrogen, 17AD6RSP), pcDNA3.1-CG4928-eGFP (Invitrogen, 17ADONXP) (for sequences, see Supplementary Dataset S3), or pcDNA3.1-empty (Invitrogen) plasmids using Lipofectamine 3000 (Thermo Scientific) and Fugene HD transfection reagent [Promega, membrane preparation and solid-supported membrane (SSM)-based electrophysiology].

GBM01 cells stably overexpressing RRM2 were generated by wet reverse transfection using the X-tremeGENE HP DNA Transfection Reagent (Roche, cat. no. 06 366 244 001). pcDNA3 RRM2 was a gift from Edward Whang (Addgene plasmid #13796) and pcDNA3.1 empty vector was used as control (Invitrogen). Protocol in brief, 6 μl transfection reagent and 2 μg plasmid DNA of interest was incubated for 15 min at room temperature and then spread on the bottom of a well in a 6 well plate. 200,000 GBM01 cells were seeded on top of the transfection solution in 2 ml complete medium and cultured overnight before plating in selection medium.

The cDNA sequence of SNHG1 or PTEN was amplified and inserted into pcDNA3.1 vector (Invitrogen) to create SNHG1-overexpressing plasmid (pcDNA-SNHG1) or PTEN-overexpressing plasmid (pcDNA-PTEN), with pcDNA3.1 empty vector (pcDNA) as the negative control. siRNA antagonistic to SNHG1 (si-SNHG1), siRNA antagonistic to PTEN (si-PTEN), and scrambled negative control (si-NC) were obtained from GenePharma (Shanghai, China). miR-153-3p mimics (miR-153-3p), miR-153-3p antagonist (anti-miR-153-3p), and scrambled oligomer sequences (miR-NC, anti-miR-NC) were designed and synthesized by Sangon Biotech (Shanghai, China). SH-SY5Y cells (1 × 10⁵/ml) were inoculated on the 6-well plates and incubated to 80% confluence. Transfection with plasmids and/or oligonucleotides was carried out using Lipofectamine 2000 (Invitrogen), followed by treatment with 1 mM MPP⁺ for 48 h.