Cell culture
Esophageal carcinoma cell lines (EC9706 and KYSE150) were cultured in RPMI 1640 Medium (Biological Industries) containing 10% fetal bovine serum (Biological Industries), 1% penicillin/streptomycin (Invitrogen). HEK-293 cells were cultured in DMEM Medium supplemented with 10% FBS and 1%penicillin/streptomycin. EC9706 and KYSE150 cells were authenticated by short tandem repeat profiling (STR). STR profiling of our KYSE150 cells was found to be 100% consistent with the STR data of the KYSE150 from China Infrastructure of Cell Line Resources (Supplementary figure 1). EC9706 cell STR profiling data was not accessible in public databases including ATCC. Cells were regularly tested for mycoplasma using Lookout Mycoplasma PCR detection kit (MP0035, Sigma) and only used when negative.
Generation of knockout
The PARK2 sgRNA was designed, synthesized, and cloned to the pX460 cloning vectors. Then, 100 million ESCC cells were electroporated with 4 μg of pX460 plasmid containing sgRNA using the Nucleofector™ 2b Device (Lonza). After the electroporation, cell population was sorted by flow cytometry. Single cell can be obtained and then added into 96-well plates. Further 10 days' culture was allowed for the single cell clone expansion. The clones were collected for PCR amplification and sequencing to analyze the gene mutation in the PARK2 sgRNA recognizing site. The PARK2 sgRNA sequences or siRNA sequence were shown in supplementary table 1. The PARK2 KO sequence was shown in supplementary Figure 2.
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted with RNeasy Plus Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s specifications. Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit(Thermo, Lithuania). qRT-PCR was carried out using GoTaq® qPCR Master Mix (Promega, USA) and 7500 Fast Real-Time PCR System (Applied Biosystems, Singapore). GAPDH was used as an internal control. The sequence of the primers for qPCR was listed in Supplementary Table 2.
Western blot
Standard western-blot assays were used to analyze protein expression in cells. The following antibodies were used for assays: anti-Flag-M2 (A8592, Sigma, 1:1000), anti-HA (2013819001, Roche, 1:1000) anti-Myc (9E10, Santa Cruz, 1:1000), anti-GAPDH (0411, Santa Cruz, 1:1000), anti-PARK2 (Abcam,ab15954), anti-YAP (63.7, Santa Cruz, 1:1000), Protein signals were detected with an ECL kit ( Millipore Co., Billerica, Massachusetts, USA)
Immunofluorescence (IF) staining
Cells on the coverslips were fixed with 4% paraformaldehyde and incubated with the primary antibody against PARK2 (Santa cruz,sc-32282), YAP (CST,14074) at 4 °C overnight. After washing with PBS, cells were then incubated with fluorescence-conjugated secondary antibody (Invitrogen, Carlsbad, CA), and subsequently counterstained with DAPI (Life Technology). Images were captured after staining with anti-fade DAPI solution using a confocal laser-scanning microscope (Leica TCS SP8 STED).
Co-IP assays
Co-IP assays were performed according to standard protocols. For the co-IP of Flag-PARK2 and Myc-YAP proteins, anti-Flag (A2220, Sigma) and anti-Myc ((9E10, Santa Cruz) agarose beads (30 µl) were used to pull down Flag-PARK2 and Myc-YAP, respectively. Beads were washed with PBST three times, and bound protein was denatured with 2× SDS sample buffer. The supernatants were collected and proceded to SDS-PAGE western blot analysis.
Wound healing and Transwell assays
For the wound healing assay, cells were cultured in a 6-well plates until confluent and then wounded with a sterile tip. The cells were captured at the indicated time points after scratching. The distances between the two edges of the scratched wound were measured using Image J software. The trans-well system (8 μm pore size, Corning) was employed for cell migration and invasion assays. For migration assays, cells in serum-free medium were seeded into the upper chambers. For invasion assays, the upper chambers were coated with matrigel (BD Biocoat, USA).After 24 h, cells that had migrated through to the bottom of the insert membrane were fixed, stained with crystal violet and counted under ×20 objective lens. The experiments were repeated thrice.
In vivo ubiquitination assays
For in vivo ubiquitination assays, cells were transfected with vectors, including expressing Myc-YAP, Flag-PARK2 and HA-Ub, respectively, for 24 h. Cells were then treated with MG132 (10 μM) for 6 h, and the levels of Myc-YAP ubiquitination was determined by IP with an anti-Myc antibody followed by western-blot assays with an anti-HA antibody (2013819001, Roche,1:1000)
In vivo tumorigenesis and metastasis assay
For in vivo tumorigenic experiment, EC9706 cells (4×106) were injected into the right dorsal flank of 4-week-old female BALB/c nude mice. Tumor formation in nude mice was monitored over a 4-week period. The tumor volume was calculated by the formula: tumor volume = 0.5 × length × width2 .For in vivo metastasis assays, each experimental group consisted of 5 4-week-old female BALB/c nude mice. Briefly, 2×106 cells were injected intravenously through the tail vein into mouse. The mice were killed 8 weeks after injection. Tumor nodules formed on the lung surfaces were macroscopically determined and counted. The lungs were excised and embedded in paraffin. Further, the tissue sections were stained with H&E to visualize the structure.
Luciferase reporter assays
For TEAD luciferase activity assays, cells with ectopic HA-PARK2 expression and their control cells were transfected with the TEAD luciferase reporter vector for 24 h. Cells were then harvested for assays. Luciferase reporter assays were performed using the dual luciferase assay kit (Promega). The pRL-null vector expressing renilla luciferase (Promega) was used as an internal control to normalize the transfection efficiency.
Cycloheximide assay
Cycloheximide was added into culture medium with the final concentration of 100 μmol/L. Cell lysis were collected at 1.5, 3 and 4.5h after the treatment of cycloheximide.
Tissue microarray (TMA) and immunohistochemistry (IHC)
223 cases of ESCC were selected for the TMA construction. All of these tissue samples were obtained from the First Affiliated Hospital of Xinxiang Medical University. All patients signed informed consent. No patients recruited in the study received preoperative treatments. The ESCC samples used in this study were authorized by the Committees for Ethical Review of Research at Xinxiang medical University. IHC was performed according to a standard streptavidin-biotin-peroxidase complex method Signals in tumor cells were visually quantified using a scoring system from 0 to 9. The scores were obtained by multiplying the intensity of signals with the percentage of positive cells (signal: 0 = no signal, 1 = weak signal, 2 = intermediate signal, and 3 = strong signal; percentage: 0 = 0%, 1 ≤ 25%, 2 = 25-50%, and 3 ≥ 50%). Low and high expression were defined as scores of< 6 and ≥ 6, respectively.
Cell proliferation assay
Cell proliferation was assessed by EdU incorporation and flow cytometry. Proliferating cells were determined by using the 5-ethynyl-20-deoxyuridine (EdU) assay kit (Ribobio, Guangzhou, China). For quantification analysis of the images, each data point represents the positive fluorescence area calculated from a minimum of five randomly chosen fields from three individual experiments. EdU incorporation flow cytometry assay was carried out according to the manufacturer’s instructions. The experiments were performed in triplicate.
Statistical analysis
No specific statistical tests were used to predetermine the sample size. Statistical analysis was performed using GraphPad Prism 7 software or SPSS version 23.0 (SPSS, Inc., IL). Data were expressed as mean ± s.e.m. Differences between two independent groups were tested with Student’s t-test. Kaplan −Meier analysis with log-rank test was applied for survival analysis. The relation between PARK2 expression and clinicopathological characteristics was analyzed by Pearson χ2 test. Univariate and multivariate Cox proportional hazard regression models were used to evaluate the survival hazard using Cox proportional hazard model with a forward stepwise procedure. Differences were considered to be statistically significant when P <0.05 (*P < 0.01; **P < 0.001).