Dmem low glucose

Hepa1c1c7 cells (ATCC, Manassas, VA) were cultured in low-glucose DMEM (WAKO Chemicals, Osaka, Japan) containing 10% FBS and 1× antibiotic–antimycotic solution for cell culture and were maintained in a 5% CO₂ incubator at 37 °C. For X8–17 transfection, the cells were seeded 1 day before transfection, at a density of 5000 to 12 000 cells per well in Iwaki 96-well plates (AGC Techno Glass, Shizuoka, Japan), depending on the incubation time. After 24 h, the cells were transfected with X8–17s or ASOs, using Lipofectamine 3000, as per the manufacturer's instructions, and were further grown in high-glucose DMEM containing 10% FBS and 1× antibiotic–antimycotic solution. After transfection and incubation, the cells were harvested and used for assays.

ESCC cell lines, TE-8, TE-9, TE-10, TE-11, and TE-15, obtained from the RIKEN BioResource Center (Tsukuba, Japan), were maintained in RPMI-1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA). Human bone marrow-derived mesenchymal stem cells (MSCs) were purchased from the American Type Culture Collection (ATCC^® PCS-500-012^TM; Manassas, VA, USA). MSCs were maintained in low-glucose DMEM (Wako) supplemented with 10% FBS and 1% antibiotic-antimycotic.

Rat retinal pericyte cell line, TR-rPCT1, from transgenic rats harboring the temperature-sensitive SV40 large T-antigen gene, was provided by Fact Inc. (Sendai, Japan) [22 (link)]. Pericyte migration was evaluated with an Oris 96-well collagen coated cell migration assay kit (Platypus Technologies, Madison, WI, USA), according to the manufacturer’s protocol. Briefly, cells were seeded in each well and cultured in low glucose DMEM (Fujifilm Wako Pure Chemicals, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) at 33 °C and 5% CO₂. Cells were then starved with DMEM low glucose medium supplemented with 0.5% FBS, for 24 h. After pretreatment with APB5 antibody (0.1 μg/mL, 1 μg/mL and 10 μg/mL) or 10 μg/mL IgG control for 30 min, cells were then treated with PDGF-BB recombinant protein (R&D Systems, Minneapolis, MN, USA) with 10 μg/mL aphidicolin (Fuji Film Wako Pure Chemical) to inhibit cell division. The stoppers were removed to allow cells to migrate into the detection zone. Cells were then incubated for 24 h and stained with PBS containing calcein AM (Dojindo, Kumamoto, Japan) for 1 h. Micrographs were taken with a microscope system (Biorevo). The area of the stained cells that had migrated into the detection zone was measured.

SH-SY5Y and U251 cells (ATCC, Manassas, VA, US) were maintained on tissue culture dishes coated with type I collagens (Nitta Gelatin, Tokyo, Japan) and grown in low glucose DMEM (Wako, Tokyo, Japan) added with 10% fetal bovine serum (Thermo Fisher Scientific, Tokyo, Japan) and appropriate antibiotics (Wako, Tokyo, Japan). For transfection, SH-SY5Y and U251 cells were inoculated into 24-well tissue culture plates and then transfected with NC, miR-125a precursor, or ET1 siRNA by Fugene 6 HD (Promega, Madison, WI). At 48 h after the transfection, transfected cells were harvested to assay the expression levels of ET1. Data were averaged from 3 repeated experiments.

To understand at which stage of the cell cycle SKI–p21 signaling affects CC growth, we performed flow cytometry on two complex phases, G0/G1 and S/G2/M. KKU100 or OZ cells (5 × 10⁴ cells/well) were seeded in a 6‐well plate 1 day before transfection. KKU100 cells were transfected with 500 ng·mL⁻¹ of SKI plasmid using Lipofectamine 3000 (ThermoFisher Scientific), and OZ cells were transfected with 10 nm of SKI siRNA using Lipofectamine RNAiMAX (ThermoFisher Scientific). After 48–58 h of transfection, the cells were starved in low‐glucose DMEM (Fujifilm Wako Pure Chemical Corporation) supplemented with 0.5% fetal bovine serum (ThermoFisher Scientific), followed by trypsinization and centrifugation at 72‐h post‐transfection. The supernatant was discarded, and cells were suspended in phosphate‐buffered saline and incubated with fluorescent particle allophycocyanin and a cyanine dye (APC‐Cy7 A; Dojindo) for 15 min. Diploid and tetraploid fractions of SKI‐overexpressing KKU100 cells (Fig. S4A) or SKI‐knockdown OZ cells (Fig. S4B) were detected with an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).