Tpc 1

Thyroid cancer cell lines B-CPAP and TPC-1 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). pCMV-SIX1 transfection plasmid was obtained from Origene. siRNA sequences for Six1 and scramble siRNA were purchased from Dharmacon. Lipofectamine 3000 (Invitrogen) was used for plasmid transfection and Dharmafect 1 (Dharmacon) was used for siRNA transfection.

Human PTC cell lines K1 and TPC-1, the human thyroid follicular epithelial cell line, Nthy-ori3-1, and HEK 293 T cells were purchased from the Shanghai Cell Bank (Shanghai, China). All cell lines were grown in RPMI 1640 medium (BI, Jerusalem, Israel), supplemented with 10% fetal bovine serum (BI), and incubated at 37°C in a humidified incubator containing 5% CO₂. miR-29a-3p mimics, control mimics, specific small interfering RNA (siRNA) for OTUB2, and control siRNA were synthesized by RiboBio Inc (Guangzhou, China) according to the published work.19 (link),20 (link) miRNA mimics and siRNAs with final concentration at 50 nM were transfected into PTC cells using Lipofectamine™ 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. Briefly, nucleic acids, which includes the plasmids and siRNAs, and the Lipofectamine reagent were diluted with RPMI 1640 medium, for 5 minutes. Then the mediums containing nucleic acids and Lipofectamine reagent were mixed and incubated at room temperature to form liposomes. After 15 minutes, each mixture was added into the corresponding culture medium and 6 hours later, the culture medium was removed and replaced with fresh medium.