C0218

The CMP difference was monitored by bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3)) (B438, Molecular Probes, United States). DiBAC₄(3) stock solution (20 mM, dissolved in dimethylsulfoxide) was diluted in Hank’s balanced salt solution (C0218, Beyotime, China) to a working solution (40 μM). PC12 cells were stained with DiBAC₄(3) working solution in poly-L-lysine-coated 96-well plates (100 μL/well, 1 h) after 0.5, 1, 12, 24 or 48 h of treatment with 200 μM SNPs [23 (link)]. The average fluorescence intensity of DiBAC₄(3) was measured with the HCA system. Cells cultured in medium without SNPs were used as the negative control. Six parallel experiments were conducted at each time point.

Scratch assay and transwell assay to detect the migrated ability. Scratch assay was performed by a 200 μl pipette in 60 mm dishes when cells grew up to 90–100% density. And the floated cells were removed with twice Hank's (Beyotime, C0218) washes, and wound pictures were taken at 0 hour, 24 hours and 48 hours by Olympus IX70 inverted microscope. In transwell assay, bottom chamber was filled with 1 ml RPMI-1640 with 10% fetal bovine serum and 1% penicillin/streptomycin and the top chamber was filled with the same media with 4 × 10⁴ (PC3 cells with ectopic expression of UBE2T) or 2 × 10⁴ (Du145 cells with ectopic expression of UBE2T) or 4 × 10⁴ (LNcaP cells with ectopic expression of UBE2T) cells suspended. The cells were incubated for 48 hours and the fixed in methanol longer than 30 minutes and stained with Giemsa stain after the cells on the top surface of membrane removed by cotton swab. The pictures were taken by Olympus IX70 inverted microscope and the numbers of migrated cells were counted.