Fig. 1 Supplemental 2

Epiboly could serve as a marker for this screening. (A) Western blot analysis of protein arginine methyltransferase 1 (PRMT1) (upper left) and cytochrome P450 family 11 (CYP11A1) (middle left) protein levels in non-metastatic human cancer cell line (MCF7) and highly metastatic human cancer cell lines (MDA-MB-231, MDA-MB-435, MIA-PaCa2, PC9, HCCLM3, PC3, and SW620); β-actin loading control is shown (bottom left). Preliminary experiments confirmed that epiboly could serve as a marker for this screening assay. Quantification analyses of western blotting bands. The analyses were performed by ImageJ. Signal strength of bands of PRMT1 (Top right) and CYP11A1 (bottom right) was normalized by that of β-actin. (B) Knockdown of PRMT1 or CYP11A1 in MDA-MB-231 cells. MBA-MB-231 cells were transfected with a control short hairpin RNA (shRNA) targeting LacZ, and one of four independent shRNAs targeting PRMT1 (clones #1 to #4), or one of two independent shRNAs targeting CYP11A1 (clones #1 to #4). Reduced PRMT1 and CYP11A1 expression, determined by western blot, in sub-cell lines of MDA-MB-231 cells expressing PRMT1 shRNA (clones #3 and #4) or CYP11A1 (clones #2 and #4), compared with controls (parental cell line MDA-MB-231 and control shRNA cells); β-actin levels shown as a loading control. Quantification analyses of western blotting bands. The analyses were performed by ImageJ. Signal strength of bands of PRMT1 (top right) and CYP11A1 (bottom right) was normalized by that of β-actin. (C) Effect of shRNAs targeting either PRMT1 or CYP11A1 on cell motility and invasion of MBA-MB-231 cells. Parental MDA-MB-231 cells and four sub-cell lines of MDA-MB-231 cells that were transfected with either shRNA targeting either LacZ, two independent shRNAs targeting PRMT1 (clones #3 and #4) or two independent shRNAs targeting CYP11A1 (clones #2 and #4) were subjected to Boyden chamber assays. (D) Zebrafish embryos treated with either vehicle (DMSO), 10 μM niclosamide, or 50 µM vinpocetine. Approximately 20 embryos were treated with either DMSO as a vehicle control, niclosamide, or vinpocetine. The treatment was started at 4 hours post fertilization (hpf) when all of embryos reached sphere stage and ended at 9 hpf when control embryos reached 80–90% epiboly stage. Each experiment was performed at least twice. Statistical analysis was determined by Student’s t test.