Control shrna h lentiviral particles a

YD-10B, HSC-2, HSC-3, Ca9.22 OSCC cells were obtained from the Yonsei University College of Dentistry, Republic of Korea, and all were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (3 : 1 ratio) medium supplemented with 10% FBS, 1 × 10⁻¹⁰ M cholera toxin, 0.4 mg/mL hydrocortisone, 5 μg/mL insulin, 5 μg/mL apotransferrin, and 2 × 10⁻¹¹ M triiodothronine (T3) in a humidified atmosphere of 5% CO₂ at 37°C. PC-3 prostate cancer cells were cultured in RPMI1640 medium with 10% FBS. To establish fascin-depleted YD-10B cell line, fascin 1-specific short hairpin RNA (shRNA) (h) lentiviral particles were transduced in cultured cell with 5 μg/mL polybrene according to the manufacturer protocol (Santa Cruz, Santa Cruz, CA, USA). Continual selection was followed with 1 mg/mL puromycin to establish fascin-depleted stable cell line (Fascin^dep). Control shRNA (h) lentiviral particles-A (Santa Cruz) was also used as a cell control (Mock). The extent of fascin depletion was evaluated by Western blot.

For KNDC1 overexpression, KNDC1 plasmid (GeneCopoeia, Rockville, MD, USA) and blank plasmid (GeneCopoeia, Rockville, MD, USA) were transformed into Escherichia coli cells, and then the cells were selected with 100 µg/mL neomycin. After extraction and purification, the plasmids were transfected into 293T cells using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) when the 293T cells reached 70% confluence, and the cells were harvested 48 hrs after transfection for further experiments. For KNDC1 knockdown, 3AO cells were infected with KNDC1 shRNA (h) lentiviral particles (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) or Control shRNA (h) lentiviral particles-A (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA). The cells were selected with 9 µg/mL puromycin for 7 d, and KNDC1 knockdown efficiency was verified by Western blotting.