HEK293T cells and the anaplastic thyroid cancer cell lines CAL62, and KHM-5M were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). All cell lines are possese of cell line authentication via Short Tandem Repeat (STR), which is performed through PowerPlex 21 system, turning out that The STR data keep consistent with its in ATCC. HEK293T and CAL62 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, 41965, Life Technologies) supplemented with 10% fetal bovine serum (FBS). KHM-5M cells were cultured in RPMI/1640 medium (42401, Life Technologies) supplemented with 10% FBS. All cells were cultured at 37 °C in a humidified 5% CO2 incubator.
Plasmids and RNA inference
The wild type TRIM11, YAP and their mutant constructs were obtained from Hanbio Biotechnology Co. Ltd. (Shanghai, China). The HA-K6, -K11, -K27, -K29, -K33, -K48, -K63, -K0 and -Ub plasmids were acquired from Addgene. Plasmids were transfected using Lipofectamin 2000 (1662298, Invitrogen). Small interfering RNAs were used for specific gene knocking-down. The TRIM11 siRNA sequences used were: siRNA #1: 5’-CCAACCGCCCGCUUGCUAA-3’; siRNA #2: 5’- GGGUGAGUUCGAGCGUCUU-3’. And YAP siRNA sequences were: siRNA #1: 5’- GCUCAUUCCUCUCCAGCUU-3’; siRNA #2: 5’- CACCUAUCACUCUCGAGAU-3’. SiRNAs were transfected using RNAiMAX reagent (13778150, invitrogen).
RNA extraction and qPCR analysis
The total RNA was extracted from the cancer cells using the RNeasy plus mini kits (Qiagen, Germany). Reverse transcription was performed using the PrimeScript RT Master Mix (Takara, Japan). qRT-PCR was performed using the SYBR green mix (Toyobo, Japan) with the CFX96TM Real-time PCR Detection System (Bio-Rad, USA). The 2−ΔΔCt method was used to calculate the relative expression. 36B4 was used for internal control. All assays were performed in triplicates.
Proliferation, cell cycle and colony formation assay
CAL62 and KHM-5M were treated with SiTRIM11 or SiControl in 6-well plates. Forty-eight hours after transfection, the cells were counted and 2000 cells were seeded in 96-well plates. Cell viability was measured using Cell Counting Kit-8 (CCK8) every 24 hours. Cells transfected with SiTRIM11 or SiControl for 48 hours were stained with propidium iodide (Multisciences, China) and analyzed by flow cytometer (Beckman, USA), The cell cycle phases were determined by relative DNA content. For the clone formation assay, cells were treated with 50 nM TRIM11 siRNA or 50 nM siControl, after 48 hours, the cells were typsinized and seeded (1-1.5×103 cells/well) in 6-well plates and maintained in complete medium for 2 weeks. The cells were fixed with 4% paraformaldehyde for 2 hours, and stained with 1% crystal violet. EdU incorporation assay was performed as our previously described.
Cells were seeded into 6-well plates and transfected with TRIM11 siRNA or siControl. When full confluent, the cell layer was scratched with a 200 μl sterile pipette tip and washed with PBS. Cells were maintained in the medium containing 1% FBS and wound distance was measured every 24 hours.
Transwell migration assay
Migration capability was measured using 24-well transwell chamber systems (Corning, USA) with 8.0 µm pore size. Cells were seeded in upper chamber insert and cultured in serum-free medium. The bottom chambers were filled with 10% fetal bovine serum medium. After incubated at 37°C for 24 hours, the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. The membranes were placed under an inverted phase contrast microscope and imaged to count the migrated cells. Three independent experiments were conducted.
The Dual-Luciferase Reporter kit (Promega, Germany) was used to measure the luciferase activity of YAP-luciferase reporter. The YAP-luciferase reporter and Renilla plasmid were transfected together the into the cells. Luciferase activity was detected after 24 h.
CAL62 and KHM-5M cells cultured on 14 mm slides in 24-well plates were fixed in 4% paraformaldehyde at room temperature for 30 minutes. After washing with PBS for 3 times, the cells were blocked with 10% goat serum and incubated with primary antibodies against YAP (mouse, Santa Cruz), and TRIM11 (rat, Proteintech) at 4 °C overnight. Followed by incubating with FITC- and Cy3-conjugated secondary antibodies. The images were examined with EVOS™ M5000 Imaging System.
Xenograft tumor model
BALB/c nude mice aged 3 weeks were obtained from Beijing HFK Bioscience Co., Ltd. in Beijing, China. 1X106 CAL62 cells were injected to each mouse. The mice were maintained in a temperature and humidity-controlled and specific pathogen-free environment in the laboratory animal facility of Zhongnan Hospital of Wuhan University. Tumor sizes were measured every 5 days until the end of the experiment. The experiments were performed under the protocols approved by ethnic committee of Zhongnan Hospital of Wuhan University.
Total cell lysis of CAL62 were precleared with rabbit IgG for 2 h and the immunoprecipitated with YAP (Proteintech,13584-1-AP) or TRIM11 (Proteintech, 10851-1-AP) antibody overnight as previously described, while rabbit IgG (Santa Cruz) was used as the negative control. The bounded protein was analyzed by Anti-YAP or Anti-TRIM11 antibody.
Protein stability assays
CAL62 and KHM-5M cells were seeded in 24-well plates and transfected with siTRIM11 or siControl. After 24 h, cells were treated with 100 μM cycloheximide (MCE) for indicated time points. Western blot was performed to detect YAP degradation.
Western blot analysis
The anaplastic thyroid cancer cells were lysed with RIPA extraction reagent (Beyotime, China) supplemented with protease inhibitors (Sigma-Aldrich, USA). Total protein was separated using 10-12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.45 μm PVDF membrane (Millipore, USA). Primary antibodies were YAP (Proteintech, 13584-1-AP), TRIM11 (Proteintech, 10851-1-AP), HA (Proteintech, 51064-2-AP), Myc (Proteintech, 60003-2-Ig), Flag (Proteintech, 20543-1-AP), GAPDH (Proteintech, 60004-1-Ig) antibodies. Bands were visualized using an enhanced chemiluminescence (ECL) kit (Boster, China) and detected by ChemiDoc XRS+ Imaging System (Bio-Rad).
RNA sequence analysis
The RNA sequence analysis (siControl and siTRIM11) was performed by Beijing Genomic Institute (BGI). The RNA sequence data are deposited in the SRA database, which a are available at www.ncbi.nlm.nih.gov/bioproject/PRJNA609252/
Student’s t test and one-way ANOVA were used to compare 2 and more groups respectively. Multiple comparison with Bonferroni correction was performed when appropriate. A P value < 0.05 was considered as statistically significant and all tests were two-tailed. All statistical tests were performed with Prism 7.0 (GraphPad, USA).