P3000

HEK293 cells grown in 24-well plates were transfected with 1.5 µg of recombinant pBlueScript plasmid DNAs, using the empty pBlueScript vector or lipofectamine reagent as the controls. Lipofectamine® 3000, P3000^TM reagents and Opti-MEM® (Invitrogen Life Technologies) were used according to the protocol manual. At 24, 48 and 72 h post-transfection, the HEK293 cells were harvested and centrifuged at 4000 rpm for 5 min, respectively, with supernatants transferred to new Eppendorf tubes labelled “supernatant”, and pellets re-suspended in phosphate-buffered saline (PBS) in tubes labelled “cells” and partly spotted onto acetone-cleaned slides. Nucleic acids were extracted using Trizol® reagent (Invitrogen Life Technologies) from each 125 µl sample labelled as “cell” or “supernatant”.

The coding regions of the HBoV1 VP3 gene (nt 3,443–5,071 of ABK32030.1) (Schildgen et al., 2008 (link)) were inserted into the eukaryotic expression vector pcDNA3.1 (Invitrogen, USA) and transfected into HEK293 cells using Lipofectamine® 3000, P3000TM reagents, and Opti-MEM® (Invitrogen Life Technologies) according to the protocol manual. At 48 h of post-transfections, the HEK293 cells were harvested and centrifuged at 4,000 rpm for 5 min. Pellets were resuspended in phosphate-buffered saline (PBS) and spotted onto acetone-cleaned slides. The slides were acetone fixed and kept at −20°C, which would be used as an antigen in IFA.

We generated RNF126, RNF126-NT, RNF126-CT, PTEN, PTEN-NT, and PTEN-CT plasmids according to the ClonExpress^® II One Step Cloning Kit (Vazyme Biotech Co., Ltd). The primers of all plasmid constructions were listed in Supplementary Table S1. The cDNA of RNF126, RNF126-NT, and RNF126-CT were cloned into a 3×FLAG-pcDNA3.1 vector. The cDNA of PTEN, PTEN-NT, and PTEN-CT were cloned into a GFP-pcDNA3.1 vector. The DNA constructs sequences were systematically verified by DNA sequencing.
The RNF126-si1: 5’-GCCGGAUUAUAUCUGUCCATT-3’, RNF126-si2: 5’-GCAUCUUCGAUGACAGCUUTT-3’, RNF126-si3: 5’-CCAACGGCCUGGAUGCCAUTT-3’, siPTEN: 5’-ATCGATAGCATTTGCAGTATA-3’ and siNC: 5’-UUCUCCGAACGUGUCACGUTT-3’ were purchased from GenePharma Co., Ltd, Shanghai, China. The siRNAs and plasmids were transfected into cells with Opti-MEM culture medium, Lipofectamine^® 3000 and P3000^TM (Invitrogen).
The lentiviral shRNA expression vectors for human RNF126, human PTEN and control were also purchased from GenePharma Co., Ltd. The shRNF126-1: 5’-CCAACGGCCTGGATGCCAT-3’, shRNF126-2: 5’-GCCTCACGGGACAGAACACTT-3’, shPTEN: 5’-ATCGATAGCATTTGCAGTATA-3’ and shNC: 5’-TTCTCCGAACGTGTCACGT-3’ were also purchased from GenePharma Co., Ltd. The lentiviral shRNA expression vectors were transfected into cells with 6 μg/ml polybrene. After transfection for two days, the infected cells were selected by treatment with 2 μg/ml puromycin.

For the QKI-6 knockdown experiment, we transfected GSCs with plasmids containing a short hairpin RNA directed against the human QKI-6 gene (pGPU6-shQKI-6) or the negative control (pGPU6-shQKI-6-NC) (GenePharma). In other experiments, the QKI-6 gene or a mutant QKI-6 gene lacking the WTAP binding site (V157E) was cloned into the pIRES2-EGFP plasmid vector by SalI to allow overexpression of QKI-6 (pIRES2-QKI-6) or its mutant (pIRES2-QKI-6-Mut) (GenScript, Nanjing, China). The same negative control (pIRES2-QKI-6-NC) was used for pIRES2-QKI-6 and pIRES2-QKI-6-Mut. Stable transfection was performed with Lipofectamine 3000 and P3000 (Life Technologies Corporation, Carlsbad, CA, USA) per the manufacturer's instructions in 24-well plates with cells at about 80% confluency. The stably transfected cells were selected with culture medium containing 0.5 mg/mL G418 (Sigma-Aldrich, St Louis, MO, USA). G418-resistant cell clones were established after approximately four weeks. The transfection efficacy was determined by Western blot analysis.

The two cell lines, SGC-7901 and AGS were respectively cultured in Dulbecco’s Modified Eagles Medium (DMEM) with 10% Fetal Bovine Serum(FBS) and Ham's F-12K(Kaighn's) medium (F-12K) with 20% FBS in a humidified incubator at 37°C with 5% CO₂. Cell lines used in the experiments were authenticated using short tandem repeat (STR) profiling in the Genomics core facility of Capital Medical University on an annual basis, with last authentication in April, 2020, and passaged less than 5 times at any given time. When SGC-7901 and AGS cells grew to 50-60% confluency, the Rictor and Cav 1 plasmids and vectors were used to infect cells using lipofectamine 3000 and P3000 (Life Technologies, China), according to the manufacturer’s instructions. Rictor-siRNA and Cav 1-siRNA were transfected into two the cell lines by using lipofectamine 3000 according to the provided protocols. The transfection efficacy was determined by western blot.