Vehicle dimethylsulfoxide

Cell lines, including AGS, NCI-N87, MGC-803 and MKN1, were treated with a demethylating agent (5-Aza) and histone deacetylases inhibitor.^{47 (link)} For 5-Aza (Sigma, St Louis, MO, USA) treatment group, the cells were treated with 10 μm 5-Aza for 3 days. For TSA (Sigma) treatment group, 100 nm TSA was added to cells for 24 h. For combination, we treated the cells with 5-Aza for 4 days. In the following 24 h, TSA was added at 100 nm concentration. Control cultures were treated with an equal amount of vehicle dimethylsulfoxide (Sigma).

For EdU incorporation, mitotic G2 and cell viability analysis, myoblasts were seeded in 96-well glass bottom microplates (BD Falcon) at a density 1.5 × 10³ cells/well. After 48 h, media was replaced containing 0.01, 1, 100 nM 1α,25(OH)₂D₃ (vitamin D; Sigma-Aldrich) or vehicle (dimethyl sulfoxide) (Sigma-Aldrich) in either low- or high-serum media and stimulated for 24 and 48 h. Following stimulation myoblasts were incubated with 10 μM EdU 60 min prior to fixation in 4% paraformaldehyde (PFA) in PBS for 15 min at RT. Fixed cells were washed with PBS and permeabilised with 0.3% Triton X-100 in PBS (PBST) and then incubated 30 min at room temperature with 2 μM Alexa 647-conjugated Azide, 10 mM ascorbic acid, 2.5 mM CuSo₄ in PBS. DAPI staining was performed by a 1-h incubation at 4°C with 10 μg/mL RNAse, followed by 10 min of 1 μg/mL DAPI in PBS at room temperature. All plates were imaged on an ImageXpress imaging system (Molecular Devices) at 10× magnification, and segmentation of nuclei and measurement of fluorescence intensities were performed in CellProfiler (29 (link)).