Lipofectamine 2000 reagent

A pool of four Hmrhl specific siRNAs targeting the conserved region of Hmrhl and scrambled negative control siRNA were obtained from Dharmacon. K562 cells were transfected with 100 nM siRNA diluted in a serum-free media with lipofectamine 2000 reagent (Thermo Fischer) in a six-well plate as per the manufacturer's protocol. The transfected medium was replaced with complete medium (10% FBS and 1% penicillin/streptomycin) after 9 h of transfection. Cells were harvested for RNA isolation after 36 h of post-transfection, and the knockdown efficiency for Hmrhl was verified by real-time PCR.
For co-transfection experiment, lipofectamine 2000 reagent (Thermo Fischer) was used and instructions from manufacture was followed. Briefly, K562 cells were co-transfected with 1μg of PDGRF-β expression vector (Origene, Cat. #RC206377) and 30 pmol of siRNA-Hmrhl, diluted in serum free media with lipofectamine in a six-well plate. The transfected medium was replaced with complete medium (10% FBS and 1% penicillin/streptomycin) after 9 h of transfection. Cells were harvested for RNA isolation after 48 h of post-transfection, and real-time PCR was done to check the expression level of PDGRF-β and Hmrhl. Knockdown and transfected cells were used further for various phenotypic assays.
The list of all RT-PCR primers, si-RNA and probes used for the experiments are listed in Supplementary Table S1.

This experiment was divided into four groups—Blank, blank; plasmid containing the APOBEC3B gene, APOBEC3B; empty plasmids, vector; and APOBEC3B knockdown, si-APOBEC3B. Based on the APOBEC3B gene sequence in GenBank (NM004900; https://ncbi.nlm.nih.gov/nuccore), the plasmid containing the APOBEC3B gene and empty vector plasmids were constructed by Shanghai Jikai Co., Ltd. Plasmids were transfected into GC cell lines using Lipofectamine 2000 reagent (Thermo Fisher Scientific Inc.), following the manufacturer's instructions, for 2 days at 37 °C. After transfection for 48 h, the infected cells were cultured with neomycin for 30 days at 37 °C to yield stably infected cells for mouse xenograft studies. For APOBEC3B knockdown, GC cells were transfected with APOBEC3B siRNA (Ruibo Biotechnology) using Lipofectamine 2000 reagent (Thermo Fisher Scientific Inc.), following the manufacturer's instructions, for 2 days at 37 °C. The following sequences were used: genOFFTM st-h-APOBEC3B-001, AGACCTACTTGTGCTATGA; genOFFTM st-h-APOBEC3B-002, CAGTACCACGCAGAAATGT; and genOFFTM st-h-APOBEC3B-003, TGGGCTTTCTATGCAACGA. Transfected cells were collected for subsequent experiments.

HCT116 cells were transfected with the empty pcDNA3.1 vector (METTL3-EV), METTL3-expressing pcDNA3.1 vector (METTL3-OE), METTL3-con, METTL3-si, circ-EV, circ-OE, circ-con, circ-si, mimics, mimics NC, inhibitor and inhibitor NC using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. miR-let-7b mimic sense (5′-UGAGGUAGUAGGUUGUGUGGUU-3′), its negative control (5′-UCACAACCUCCUAGAAAGAGUAGA-3′), inhibitor (5′-AACCACACAACCUACUACCUCA-3′) and inhibitor NC (5′-UCUACUCUUUCUAGGAGGUUGUGA-3′) were synthesized by Wuhan GeneCreate Biological Engineering Company. The RNA concentrations of mimics and inhibitor were 0.32 and 0.26 µg/µl, respectively. HCT116 cells (1×10⁴) were seeded in 6-well plates and maintained at a confluence of 70–80% in complete culture medium. Then, 2.5 µg plasmid or 100 pmol siRNA oligo was added to 250 µl Opti-MEM (Thermo Fisher Scientific, Inc.), and 5 µl Lipofectamine 2000 reagent was added to 250 µl Opti-MEM. The diluted plasmid or siRNA oligo was mixed with diluted Lipofectamine 2000 reagent and kept at room temperature for 20 min. After 500 µl serum-free medium was added to each well, the Opti-MEM mixture was added. After transfected cells were incubated at 37°C for 4 h, the culture medium was changed to regular DMEM supplemented with 10% FBS. After 48 h, the subsequent experiments were performed.

COS-7 cells were seeded on a 6-well dish at a density of 1.8 × 10⁵ cells/well in DMEM with 10% fetal bovine serum at 37°C in an air/5% CO₂ atmosphere at constant humidity. MDCK II cells were cultured in DMEM with 10% calf serum at 37°C in an air/5% CO₂ atmosphere at constant humidity. Transfections were performed using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. For shRNA knockdown, the following complementary set of single-stranded oligonucleotides was synthesized coding Scrib target shRNAs: shScrib #2, 5′-GCACTTCAAGATCTCCAAG-3′ (nucleotides 1,836–1,854); and shScrib #6, 5′-GCAACGAGCTGGAAGTACT-3′ (nucleotides 539–557). The selected sequences were submitted to a BLAST search against the canis lupus familiaris genome to ensure that only the Scrib gene was targeted. To produce retroviral supernatants, GP2-293 packaging cells were transfected with 24 µg of control or Scrib shRNA-containing pSUPER-retro-puro vector and 4 µg of pVSVG using Lipofectamine 2000 reagent in collagen-coated 100-mm cell culture dishes containing Opti-MEM (Thermo Fisher Scientific). The medium was replaced 24 h later, and virus-containing supernatants were harvested 48 h after transfection and used for subsequent infection of MDCK II cells. Selection was performed using 2 µg/ml puromycin 2 d after infection.