Ihh 4

The human PTC cell line, BCPAP, the German Collection of Micro-organisms and Cell Cultures (Braunschweig, Germany). The human PTC cell line, TPC1 and normal thyroid cell line Nthy-ori-3-1were obtained from Chinese Science Institute (Shanghai, China), and the human PTC cell line, K1, were purchased from the European Collection of Authenticated Cell Cultures (ECACC, U.K.). The human PTC cell line, IHH4, was obtained from Health Science Research Resources Bank (Osaka, Japan). Cell culture medium was prepared according to the suppliers’ instructions. The BCPAP and Nthy-ori-3-1 were cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, U.S.A.) supplemented with 10% FBS (Gibco, Carlsbad, CA, U.S.A.). The TPC1 and K1 were maintained in DMEM high glucose (Gibco, Carlsbad, CA, U.S.A.) supplemented with 10% FBS. IHH4 was maintained in a mixture (1:1) of RPMI 1640 and DMEM high glucose medium supplemented with 10% FBS. All cells were cultured at 37°C, in a humidified atmosphere with 5% CO₂.
The plasmids of pcDNA3.1 (control) and pcDNA3.1-GAS8-AS1, GAS8-AS1 siRNA (si-GAS8-AS1) and scramble, miR-135b-5p mimic and negative control (NC), CCNG siRNA (si-CCNG), and scramble were obtained from GenePharma (Shanghai, China). TPC1 and BCPAP were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Human PTC cell lines, TPC-1, K1 and IHH-4 were used; TPC-1 was a gift from Bryan R. Haugen (Division of Endocrinology, Diabetes and Metabolism, University of Colorado Denver, Aurora, CO); K1 was purchased from The Health Protection Agency Culture Collections, UK. The human thyroid cancer cell line IHH-4 was established from a 75-year-old man with a papillary thyroid carcinoma and was obtained from Health Science Research Resources Bank (Osaka, JAPAN). IHH-4 cells were maintained in a 1:1 mixture of DMEM and RPMI-1640 supplemented with 10% fetal bovine serum (Invitrogen). TPC-1 and K1 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum in a humidified incubator at 37 °C and 5% CO₂. Cells were starved in basal medium (devoid of growth factors and serum) for 48 h before stimulation with TSH (10 nM, approximately equal to 2 mU/mL). PAK4- and PAK4-RNAi-lentiviral vectors were purchased from Shanghai GeneChem Company (Shanghai, China). The PAK4 #1 sequence was 5′-GGATGAACGAGGAGCAGAT-3′; the PAK4 #2 sequence was 5′-CTTCATCAAGATTGGCGAG-3′ and the shRNA control sequence was 5′-TTCTCCGAACGTGTCACGTtt-3′. TPC-1 and K1 cells were cultured in a 12-well plate and transfected with lentivirus with 3 mg/ml polybrene. The PKA Cα and TSHR gene–specific siRNA sequence were 5′-AAGTGGTTTGCCACAACTGAC-3′and5′-TCCAAAGAACAGCACTGAT-3′.