2.1 Cell Culture and Transfection
The study utilized human UVM cell lines 92.1, OCM-1, Mel202, and Mel270, as well as human retinal pigment epithelial cells (HEM-d). These cells were grown in Dulbecco's Modified Eagle Medium (DMEM) enhanced with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin, and maintained at 37°C in a 5% CO2 humidified atmosphere. For gene overexpression experiments, we transfected the cells with Myc-tagged OTULIN, both wild-type and mutant Flag-OTULIN, and Myc-IGSF11 plasmids using Lipofectamine 2000 as per Invitrogen's protocol. Post-transfection (24 hours later), cells underwent treatment with 50 μg/ml cycloheximide (CHX, Sigma-Aldrich) for the specified durations and were subsequently lysed to conduct Western blot analysis. The efficiency of transfection was verified through Western blotting 48 hours after the transfection process.
2.2 shRNAs and siRNAs construction and gene transfection
Lentiviral shRNAs targeting OTULIN (shOTULIN) and a non-targeting control (shNC) were sourced from GenePharma (Shanghai, China), and prepared using a conventional protocol. Similarly, siRNAs directed against IGSF11 (siIGSF11), TET3 (siTET3), along with a non-targeting control (siNC), were obtained from Guangzhou Ribobio (Guangzhou, China). Additional siRNAs targeting DNMT1, DNMT3a, DNMT3b, TET1, and TET2 were synthesized by GenePharma. These siRNAs were transiently transfected into HEK293T and 92.1 cells utilizing Lipofectamine 2000 (Invitrogen), following the manufacturer's guidelines. The sequences for siRNA and shRNA can be found in Supplementary Table S1.
2.2 Quantitative Real-Time PCR (qRT-PCR)
Total RNA was isolated from cells utilizing TRIzol reagent (Invitrogen), followed by cDNA synthesis with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative RT-PCR (qRT-PCR) was conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems) and SYBR Green Master Mix. Gene expression was quantified relative to β-actin as a normalization control. Primer sequences can be provided upon request.
2.3 Western Blot and Immunoprecipitation
Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein levels were quantified with the BCA Protein Assay Kit. Proteins were then subjected to SDS-PAGE, transferred to PVDF membranes, and these membranes were blocked. Primary antibodies used included OTULIN (#ab151117, Abcam), IGSF11 (#14003-1-AP, Proteintech), TET3 (#ab231785, Abcam), and β-actin (#ab8226, Abcam), followed by HRP-linked secondary antibodies. Protein bands were detected using the ECL system. For immunoprecipitation, lysates were treated overnight at 4°C with antibodies against OTULIN, IGSF11, Flag, or Myc, then bound to protein A/G beads. The complexes were washed, eluted, and subjected to Western blot analysis.
2.4 Immunoprecipitation-coupled mass spectrometry (IP-MS)
HEK293T cells were transfected with control or Flag-OTULIN plasmids using Lipofectamine 2000 (Invitrogen), and lysates were collected 24 hours later for immunoprecipitation with anti-Flag antibodies. Proteins retrieved from the immunoprecipitation were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Bands indicative of Flag-tagged proteins were then excised for LC-MS/MS analysis to determine their peptide and protein identities. The resulting LC-MS/MS spectra were processed using MaxQuant software, referencing the UniProt human proteome database, with protein identifications confirmed by at least two distinct peptides.
2.5 Cell Viability Assay
Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8, Dojindo). Cells were cultured in 96-well plates and subjected to the conditions outlined in the figure legends. At designated intervals, CCK-8 reagent was introduced to each well, and the resultant absorbance was recorded at 450 nm using a microplate reader.
2.6 Transwell assay
In transwell assays, cells were seeded in the upper chamber with matrigel and incubated for 24 hours. Post-incubation, non-invasive cells were carefully removed with a cotton swab. Cells on the lower surface of the chamber were fixed with methanol for 10 minutes and stained with 0.5% crystal violet. Images of five random fields were then captured using a Leica microscope.
2.7 Wound healing assay
Cells were seeded in 24-well plates and grown to confluence over 24-48 hours. Wounds were then created with 200 µl micropipette tips, followed by rinsing the wells twice with 500 µl of PBS each. After adding 500 µl of serum-free medium to each well, images were taken at 6-hour intervals for 24 hours. Image analysis was subsequently performed using ImageJ software.
2.7 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Apoptotic cell death was assessed using the TUNEL assay with the In Situ Cell Death Detection Kit, Fluorescein (Roche) according to the manufacturer's protocol. Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for one hour at room temperature and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for two minutes on ice. This was followed by incubation with the TUNEL reaction mixture for one hour at 37°C in a dark, humidified environment. After incubation, cells were washed thrice with phosphate-buffered saline (PBS). Nuclei were then stained with DAPI for five minutes at room temperature to highlight apoptotic cells for fluorescence microscopy analysis. The proportion of TUNEL-positive cells was determined relative to the total cell count, ensuring reproducibility through at least three independent experiments.
2.8 Xenograft Tumor Growth
Prior to injection, 92.1 cells or OTULIN-deficient 92.1 cells were harvested at 80% confluency, washed with PBS, , and resuspended in a Matrigel basement membrane matrix (Corning) to a final concentration of 1x10^6 cells per 100 μL. The cell-Matrigel mixture was kept on ice until injected to prevent premature gelation. Mice were anesthetized with isoflurane during the procedure to minimize distress. Mice were sacrificed, and tumors were harvested for further analysis. Tumor growth was monitored biweekly, and caliper measurements were taken to calculate tumor volume using the formula: ½×length×width2. The study adhered to ethical standards for animal care and use, with protocols approved by the Institutional Animal Care and Use Committee (IACUC). The protocol was approved by the Committee on the Ethics of Animal Experiments of China Medical University.
2.9 Immunohistochemistry (IHC)
Tumor sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Sections were blocked and incubated with primary antibody against OTULIN overnight at 4°C. Following washing steps, sections were incubated with HRP-conjugated secondary antibody and developed using DAB. Nuclei were counterstained with hematoxylin.
2.10 Immunofluorescence(IF)
Cells grown on coverslips were fixed, permeabilized, and blocked before incubation with primary antibodies against OTULIN and IGSF11. After washing, cells were incubated with Alexa Fluor-conjugated secondary antibodies. Nuclei were stained with DAPI. Coverslips were mounted, and images were captured using a confocal microscope.
2.12 Dual-luciferase reporter assays
92.1 cells underwent transfection with OTULIN regulatory sequence fragments and the Renilla internal control vector using Lipofectamine 2000 (Invitrogen), adhering to the provided protocol. After thirty-six hours, the cells were processed for luciferase assays via the Dual-Luciferase® Reporter Assay System (Promega). Normalization of firefly luciferase activity was achieved relative to Renilla expression for each analyzed sample.
2.13 Statistical Analysis
Data were presented as mean ± SD. Statistical significance was determined using Student's t-test or one-way ANOVA followed by a post-hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001). Analyses were performed using GraphPad Prism software(9.0).