Fig 2: FBXO2 is overexpressed in PTC tissues and cell lines. (A) The mRNA expression levels of FBXO2 in normal tissues (n = 59) and human THCA tissues (n = 505) from the TCGA database. (B) The mRNA expression levels of FBXO2 in normal tissues (n = 338) and human THCA tissues (n = 512) from the TCGA and GTEx database. (C) Expression of FBXO2 in THCA based on nodal metastasis status. Normal vs. N0 or N1; N0 vs. N1. (D) According to qRT-PCR results, PTC tissues expressed significantly more FBXO2 mRNA than adjacent healthy tissues. (E) Staining of samples with immunohistochemistry (IHC) revealed the levels of FBXO2 protein expression. The tumor sample shown here is stage T2N1aM0. (F–H) qRT-PCR (F) and western blotting analysis (G,H) were performed on Nthy-ori3-1 and THCA cell lines (HTh-7, B-CPAP, TPC-1, KTC-1 and CAL62) to determine the mRNA and protein levels of FBXO2. (I) Immunofluorescence (IF) showed that FBXO2 protein stained red was localized in cytoplasm of PTC cells. Nuclei were stained blue with DAPI (scale bar, 10 μm). Three independent replicates are used to calculate the mean ± SEM. *P < 0.05, **P < 0.01, ∗∗∗p < 0.001 vs. Normal group (Student’s t test).

Fig 3: FBXO2 targets p53 for ubiquitination and degradation. (A,B) Gene set enrichment analysis (GSEA) of FBXO2 mRNA and thyroid carcinoma signaling pathways was performed. FDR < 25% and P < 0.05 were considered significant. (C,D) qRT-PCR (C) and western blotting (D) were carried out to detect the expression of p53 in TPC-1 and KTC-1 cells. (E) Western blotting to detect the effect of siFBXO2 on the expression of downstream target genes of p53-related proteins. (F) Ectopic expression of FBXO2 promoted ubiquitination of p53 in TPC-1 cells analyzed by in vivo ubiquitination assays. Ub, ubiquitin. (G) 293T cells were transfected with either FLAG-Con or Flag-FBXO2 for 36 h. Four hours prior to harvesting, MG132 was added. The cells were then lysed, and immunoprecipitation was performed using Flag M2 beads. Proteins bound to the beads were eluted with Flag peptide and analyzed by immunoblotting with anti-FLAG and anti-p53 antibodies. (H) The cell lysates of TPC-1 cells were subjected to immunoprecipitation with IgG or anti-FBXO2 or anti-p53 antibody and then detected by immunoblotting with indicated antibodies. (I) 293T cells transfected with HA-p53 were lysed and then incubated with either GST alone or GST-FBXO2 immobilized on GST-Sepharose beads. The bound proteins were eluted using SDS loading buffer and detected by immunoblotting with the specified antibodies. (J) Cell lysates from siNC or siFBXO2 cells treated with 20 µg/ml cycloheximide (CHX) were subjected to western blotting with the p53 antibodies. (K) Cell lysates from siNC or siFBXO2 cells treated with 10 µM MG-132 were subjected to western blotting with the p53 antibodies. Three independent replicates are used to calculate the mean ± SEM (Student’s t test).

Fig 4: Knockdown of FBXO2 inhibits PTC cells proliferation and promotes PTC cells apoptosis. (A,B) qRT-PCR (A) and western blot analysis (B) were performed on TPC-1 and KTC-1 cells. (C) Cell viability was then assessed using CCK-8 assays. (D) The proliferation of cells was detected by EdU assays. (E) Cell cycle arrest at G0/G1 phase was observed in TPC-1 and KTC-1 cells following knockdown of FBXO2 compared to their negative controls. (F) Western blotting were performed to determine the expression of cyclin D1 in TPC-1 and KTC-1 cells after knockdown of FBXO2. (G) Tumors collected from mice were exhibited, and the weights and volumes of xenograft tumors were measured and analyzed. (H) Cell apoptosis rates in siFBXO2 cells and corresponding control cells were detected by flow cytometry. Data are presented as mean ± SEM from three independent replicates. **P < 0.01 (Student’s t test).