Cell culture and plasmids

Normal thyroid follicular epithelial cell line Nthy-ori 3–1; papillary thyroid carcinoma (PTC) lines KTC-1, B-CPAP, and TPC-1; follicular thyroid carcinoma (FTC) cells K-1; and anaplastic thyroid carcinoma (ATC) cells CAL-62 were obtained from the Shanghai Bank of Cell Culture (Chinese Academy of Sciences, Shanghai, China). 293 T and HeLa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All the cells were maintained in DMEM or RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) in a humidified atmosphere of 5% CO2 at 37°C. All the cells were routinely checked to exclude mycoplasma contamination. The plasmids encoding Flag-tagged SYT12, Myc-tagged cullins (Cullin1, Cullin 2, Cullin 3, and Cullin 4A), His-tagged ubiquitin, Myc-tagged SPOP, Keap1, KLHL2, KLHL25, HA-tagged SYT11, and SYT12 were purchased from ZhiAO Biotech Ltd. (Hangzhou, China). The point mutation and truncate mutation constructs used in the study were generated via a PCR-based site-directed mutagenesis kit (Thermo Fisher Scientific, Waltham, MA, USA). The primer information is provided in Table 1

Table 1 Primers for site-directed muatgenesisChemicals and antibodies

CHX (cycloheximide) (Cat# S7418), MG132 (Cat# S2619) and MLN4924 (Cat# S7109) were obtained from Selleck Chemicals (Houston, TX, USA), and λ-ppase (Cat# HY-P0015), LY2584702 (Cat# HY-50876), rapamycin (Cat# HY-10219) and TWS119 (Cat# HY-13422) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). All other routine chemicals were obtained from Sigma‒Aldrich (St. Louis, MO, USA). Anti-SYT12 (Cat# SAB2107403) was ordered from Merck (Darmstadt, Germany), anti-Cul3 (Cat# ab108407), anti-SPOP (Cat# ab168619), and anti-GST (Cat# ab307273) antibodies were obtained from Abcam (Cambridge, MA, USA), and anti-Flag (Cat# 14,793), anti-Myc (Cat# 2276) and anti-ubiquitin (Cat# 3936) antibodies were obtained from Cellular Signaling Technology (Danvers, MA, USA). Anti-GAPDH (Cat# AC027), anti-Cyclin E (Cat# A0112), anti-HA (Cat# AE008) and anti-His (Cat# AE003) antibodies were obtained from ABclonal Technology (Wuhan, Hubei, China). All primary antibodies were diluted at 1:1000 except for GAPDH at 1:10,000. Secondary antibodies: HRP-conjugated anti-rabbit/mouse IgG (Cat# AS014/AS003, ABclonal; 1:5,000).

Transfection, RNAi, and CRISPR/Cas9

Transient transfection: 293 T cells were transfected with plasmids (1–4 μg DNA/6-well) using Lipofectamine 3000 (Thermo Fisher). Stable transduction: Lentiviral shRNAs (shCul3, shSPOP) or sgRNAs were packaged in 293 T cells using psPAX2 and pMD2.G (Addgene, Watertown, MA, USA). Viral supernatant (0.45 μm-filtered) was used to infect cells with 8 μg/ml Polybrene. The viral supernatant was collected and filtered through a 0.22 µm filter.The single guide RNA (sgRNA) sequences were as follows: SPOP, F: 5'-CACCGGATTGCTTCAGGCGTTTGCG-3'; R:5'-AAACCGCAAACGCCTGAAGCAATC-3' SYT12, F: 5'-CACCG ATGAGCGCAACGTCAGCACG-3'; R: 5'-AAACCGTGCTGACGTTGCGCTCAT-3'. Scramble, F: 5’-CACCACGGAGGCTAAGCGTCGCAA-3’; R: 5’-AAACCTACCAGCTGCCACCG-3’. Off-target effects were predicted using CHOP online tools (https://chopchop.cbu.uib.no). To confirm successful editing, genomic DNA was extracted from puromycin-selected single clones. The target region flanking the SPOP sgRNA binding site was PCR amplified and subjected to Sanger sequencing (Sangon Biotech Inc, Shanghai China). Knockout efficiency was confirmed by western blots.

In vivo ubiquitination assays

To investigate the ubiquitination of SYT12 in a physiological cellular context, we performed in vivo ubiquitination assays. This method enables detection of target protein ubiquitination under physiological cellular conditions, reflecting endogenous interactions and cellular regulatory mechanisms. Vectors expressing Myc-Cul3/Myc-SPOP, Flag-SYT12 or its variants were cotransfected with or without HA-Ubi into 293 T cells. Thirty-six hours later, the cells were treated with 10 μM MG132 for 12 h and washed with PBS three times. The cells were lysed on ice with buffer A (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, and 10 mM imidazole (pH 8.0)) for 30 min. The supernatant was subsequently collected and incubated with nickel beads (Ni–NTA) (Life Technologies, Carlsbad, CA, USA) for 3 h at room temperature. The beads were washed with buffer A two times, buffer A/TI three times (buffer A:buffer TI = 1:3), and buffer TI once (25 mM Tris–HCl and 20 mM imidazole (pH 6.8)). The proteins were then subjected to western blot analysis.

In vitro ubiquitination assays

To further assess whether SYT12 is a direct substrate of SPOP-mediated ubiquitination, we carried out in vitro ubiquitination assays using purified components. Unlike in vivo assays, this approach eliminates cellular complexity and allows a direct biochemical demonstration of ubiquitin transfer.

293 T cells were transfected with a vector expressing Myc-SPOP for 24 h, and Myc-SPOP was purified via Myc affinity precipitation. Bacteria expressing GST-SYT12 (0.5 μg) were incubated with purified SPOP along with E1, E2, and ubiquitin in reaction buffer (In vitro ubiquitination kit, Cat# BML-UW9920, Enzo Life Sciences, Farmingdale, NY, USA) at 37°C for 1 h. Then, 1% SDS was added to stop the reaction. The products were then subjected to western blot analysis.

Phos-tag gel electrophoresis

Phosphorylation of SYT12 was evaluated by phos-tag gel electrophoresis as previously described [14]. Briefly, 293 T cells were transfected as indicated for 24 h. Then, the cells were lysed with NP-40 lysis buffer on ice for 30 min. The total cellular lysates (20 μg) were loaded onto a Phos-tag™ acrylamide gel (Wako Chemical, Osaka, Japan). The proteins were subsequently transferred to a PVDF membrane (0.45 µm, Millipore, USA) following the manufacturer’s instructions. Phosphorylated SYT12 was detected with corresponding antibodies.

Cell viability assay

Cellular viability was measured with a CCK-8 assay kit (Cat# CK04, Donjido, Kumamoto, Japan) as described previously [15].

Western blotting and immunoprecipitation

After different transfections for 24 h, the cells were collected and washed with PBS three times. Then, the cells were lysed with RIPA lysis buffer (NCM Biotech, Suzhou, China) supplemented with protease inhibitor cocktail (NCM Biotech) on ice for 20 min. The protein concentrations were measured via the Bradford reagent (Beyotime Biotechnologies, China). Equal amounts of protein (20 μg) were separated via 10% SDS‒PAGE (NCM Biotech) and transferred to a PVDF membrane. The membrane was incubated with various primary antibodies overnight at 4 °C. The membrane was subsequently washed three times with TBS-T and incubated with secondary antibody at room temperature for 1 h. The results were visualized via an enhanced chemiluminescence (ECL) assay kit. For immunoprecipitation, the cells were collected after different transfections for 24 h. The cells were washed three times with PBS and lysed with IP lysis buffer (25 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol). The protein concentration was assayed via the Bradford reagent. Then, the cellular lysates (1 mg) were incubated with primary antibody-conjugated beads at 4°C for 1 h. The beads were washed three times with IP lysis buffer, boiled and subjected to western blot analysis. The density of the western blotting bands was quantified via ImageJ software (ImageJ v1.53, NIH, Bethesda, MD, USA).

Measurement of protein stability

After different transfections, the cells were incubated with 50 μg/mL CHX for different durations (0, 1, 3 and 4 h). Next, the cellular lysates were subjected to western blot analysis. The results were analyzed with ImageJ software.

Colony formation assay

The cells (1000 cells/well) were seeded onto 6-well plates and cultured for 2 weeks. Then, the colonies were fixed with 4% formaldehyde for 30 min and stained with 0.1% crystal violet for 1 h. The number of colonies was counted and normalized to that of the control group.

Statistical analysis

All the statistical analyses were conducted via Prism 10.0 software (GraphPad, San Diego, CA, USA). Unpaired t tests were used to compare differences between two groups, whereas one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. The data are presented as the means ± SDs (standard deviations) of three independent experiments. *P < 0.05 was considered statistically significant.