Lipofectamine 200

The si-negative control (si-NC), and si-METTL14-1/2/3 were designed and synthesized by GENEray Biotechnology (Shanghai, China), as well as the pcDNA3.1(+)-METTL14 plasmid (oe-METTL14) and pcDNA3.1(+) plasmid (oe-NC) were prepared and purchased from Sangon Biotech (Shanghai, China). The methods of cell transfection were described as previously [20 (link)]. Briefly, the A549 cells in good condition were inoculated into a 24-well plate at a density of 4 × 10⁴ cells/well, and cultured overnight. On the next day, the cell medium was changed to the serum-free medium. The A549 cells were transfected with 15 pmol si-NC or si-METTL14-1/2/3, and 1 μg pcDNA3.1(+)-METTL14 plasmid or pcDNA3.1(+) plasmid using Lipofectamine 200 (Thermo Fisher Scientific) based on the protocols of the manufacturer. After 6 h of transfection, the medium was replaced with the complete medium, and the cells continued to culture for 48 h. The total RNA was extracted from the cells with different transfection, and the expression of METTL14 was determined by western blot to assess the cell transfection efficiency.

The rBMVECs were acquired from Procell Life Science & Technology Co., Ltd (Wuhan, China) and were cultured in Dulbecco’s modification of Eagle’s medium (Gibco, USA), which contained 10% fetal calf serum (Gibco) and 1% penicillin/streptomycin (Gibco). The cells were stored in an incubator that was supplemented with 5% CO₂ at 37°C and passed when the cells grew to 80–90% confluence.
The pcDNA3.1(+)-WTAP plasmid (oe-WTAP) and pcDNA3.1(+) plasmid (oe-NC) were prepared and synthesized by Yanzai Biotechnology (Shanghai) Co. Ltd; the cell transfection methods were the same as those reported previously [30 (link)]. The rBMVECs in good condition were harvested and inoculated into a six-well plate (6 × 10⁵ cells/well). Following overnight incubation, the serum-free medium was replaced with the original medium, and either 2.5 μg of pcDNA3.1(+)-WTAP plasmids or pcDNA3.1(+) plasmids were transfected into the rBMVECs using Lipofectamine 200 (Thermo Fisher Scientific). After 6 h of transfection, the complete medium was added and cultured for another 48 h. The RNA and proteins were extracted from the differently treated cells, whereas the WTAP mRNA and protein expressions were assessed using RT-qPCR and western blotting, to estimate the cell transfection efficiency. The WTAP sequences are displayed in Table 1.

U2OS cells were grown using standard cell culture conditions as described in Higashitsuji9 (link) and with the same FLAG-gankyrin overexpression vector (Agilent Technologies, Santa Clara, CA) kindly provided by Dr J. Fujita (Japan). Cells were transiently transfected with 2 μg of this construct using Lipofectamine 200 (Life Technologies) with 1 × 10⁶ cells per transfection. Gankyrin, p53, p21 and β-actin expression levels were subsequently assessed at 24–72 h by Western blot both with and without incubation of varying concentrations of cjoc42 that had been freshly dissolved in 0.5% DMSO (final). DNA damage, where necessary, was induced with 0.2 μM etoposide treatment, 24 hours post gankyrin transfection. Control transfections were performed simultaneously using 2μg pmaxGFP expression vector (Amaxa) to determine transfection efficiency.

CHO and HeLa cells were purchased from ATCC (Manassas, VA). DMEM, Advanced DMEM, DMEM/F12, Penicillin, Streptomycin, Geneticin, Lipofectamine 200 and Alexa546-conjugated transferrin were all obtained from Life Technologies (Gaithersburg, MD). pCI vectors were from Promega (Madison, WI). Fluorescent constructs were obtained from OriGene (Rockville, MD) and AddGene (Cambridge, MA). Antibodies used were LMTK2 (Rabbit monoclonal, Sigma Aldrich, St Louis MO; note the antibody recognizes a region distal to the putative di-acidic motif [22 (link), 29 (link)]), TGN46 (Rabbit Polyclonal, Abcam, Cambridge MA), EEA1 (Rabbit monoclonal, Cell Signaling, CA), PDI (Rabbit polyclonal, Santa Cruz, CA), GAPDH (rabbit polyclonal; Santa Cruz). Secondary antibodies were goat anti-rabbit IRDye 680RD conjugates (Licor, Lincoln, NE), and all images were collected on a Licor Odyssey SA imager. For immunofluorescence microscopy, labeled secondary antibodies (cy3 and alexa 488 conjugates) were from Jackson Laboratories (West Grove, PA). All other reagents were obtained from Sigma and were of reagent grade.