Hilymax

HeLa cells were cultured in Dulbecco's modified Eagle's medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% heat‐inactivated calf serum at 37 °C under 5% CO₂ atmosphere. Transfections of the expression vectors were performed with polyethyleneimine ‘MAX’ transfection reagent (Polysciences, Inc., Warrington, PA, USA) or Hily Max (DOJINDO, Kumamoto, Japan) according to the protocols supplied by the manufacturers. In the case of CCCP treatment, the cells were exposed to 20 μm CCCP (Wako Pure Chemical Industries, Ltd.) starting at 24 h after cDNA transfection and continuing for the indicated time.

The human prostate cancer cell line C4-2B (American Type Culture Collection CRL-3315) was cultured in DMEM (HyClone) supplemented with 10% FBS (Biological Industries) at 37°C with 5% CO₂.
Transfection with siRNAs was performed using HilyMax (Dojindo). Cells were harvested six hours following transfection and used for studying effects of gene knockdown. Negative control siRNAs and siRNAs targeting ALKBH5, EIF3D, and HNRNPA2B1 were purchased from GenePharma (Shanghai, China). Sequences are listed in Supplementary Table S1.

Cells were seeded in 6-well plates until cell confluence reached 60%–80%. Liposomal cocktails with siRNA (GenePharma, Shanghai, China) (50 nM final) were generated with HilyMax (Dojindo Laboratories, Kumamoto, Japan) in Opti-MEM (Invitrogen, Carlsbad, CA, USA) according to the protocol. The transfected cells were incubated for 48 h and then used for gene expression analysis or other experiments. The sequences of the specific siRNAs are shown in Supplementary Table S1.

Target sites for genome editing were chosen using the web tool CRISPRdirect (https://crispr.dbcls.jp/) to minimize the possibility of off-target cleavage (83 (link)). The information about the chosen target sequences is summarized in Fig. S5. 293T cells were seeded into 24-well plates in Dulbecco's modified Eagle's medium/F-12 (FUJIFILM Wako) containing 10% fetal bovine serum (FBS). After a day, cells were transfected with the SpCas9-2A-Puro expression plasmid and sgRNA expression plasmid using HilyMax (DOJINDO) according to the manufacturer’s instructions. Six to eight hours after transfection, the medium was replaced with a fresh medium. Twenty four hours after transfection, puromycin was supplied at 5 μg/ml. Twenty four hours after the addition of puromycin, live cells were trypsinized, counted, and seeded again on a 10-cm culture dish at 100 to 300 cells per dish. After 15 days, clonal colonies on the culture dish were scratched and transferred to 24-well plates. Genotyping was performed by PCR for each clone to identify the cells carrying biallelic Gα gene KO. The GNAQ/GNA11 double–KO cell was generated by sequentially repeating the above process twice. Thereafter, the GNAQ/GNA11/GNA14/GNA15 quadruple KO cell was produced by one step of the same procedure, involving sgRNA expression plasmids targeting both GNA14 and GNA15.

HEK 293T and HeLa cell lines were maintained in DMEM supplemented with 10% FBS (fetal bovine serum), 100 U/mL penicillin, and 100 μg/mL streptomycin. Transfections were performed by using Hilymax (Dojindo Molecular Technologies) according to the manufacturer’s instructions.

A549 cells derived from human lung adenocarcinoma (RIKEN BRC through the National Bio-Resource Project of the MEXT, Ibaraki, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS, Biological Industries, Israel) as described previously [41 (link)]. In two-dimensional (2D) and three-dimensional (3D) models, the cells were grown on flat-bottomed and PrimeSurface 96U plates (Sumitomo Bakelite, Tokyo, Japan), respectively. CLDN2 promoter construct and internal control pRL-TK vector were transfected into the cells using HilyMax (Dojindo Laboratories, Kumamoto, Japan). The pTRE2 (mock) and CLDN2/pTRE2 mammalian expression vectors were transfected into spheroid cells using ScreenFect A (Fujifilm Wako Pure Chemical Industries).