Fig 2: ZNF582 inhibits NPC cell adhesion, migration, and invasion via Nectin-3 and NRXN3. (A-B) The cell adhesion, migration, and invasion assays were performed in SUNE1 and HONE1 cells stably overexpressed vector or ZNF582 and co-transfected with plasmids encoding empty vector or Nectin-3. (C-D) The cell adhesion, migration, and invasion assays were performed in SUNE1 and HONE1 cells stably overexpressed vector or ZNF582 and co-transfected with plasmids encoding empty vector or NRNX3-shRNA (#1 and #2). Scale bar: 100 µm. All data are presented as mean ± standard deviation of at least three independent experiments. Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: NPC, nasopharyngeal carcinoma; shControl, control shRNA; shRNA, short-hairpin RNA.

Fig 3: ZNF582 regulates the transcription of adhesion molecules Nectin-3 and NRXN3. (A) KEGG analysis of ZNF582 candidate transcriptional targets identified by ChIP-sequencing. (B-C) qRT-PCR analysis of Nectin-3 and NRXN3 mRNA expression in HONE1 and SUNE1 cells overexpressing ZNF582. (D) ZNF582-binding motif (up) and ZNF582-binding sites in Nectin-3 and NRXN3 promoters (down). (E-F) ChIP-qPCR analysis of Nectin-3 (E) and NRXN3 (F) promoter expression in SUNE1 and HONE1 cells transfected with plasmids encoding ZNF582. (G) Wild-type or mutant ZNF582 target sequence in Nectin-3 and NRXN3 promoters. (H-I) Luciferase reporter assays of Nectin-3 or NRXN3 activity in SUNE1 and HONE1 cells co-transfected with plasmids encoding control vector or ZNF582 and wild-type or mutant luciferase reporter. All data are presented as mean ± standard deviation of at least three independent experiments. Student's t-test, *P < 0.05, **P < 0.01; ***P < 0.001. Abbreviations: ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time polymerase chain reaction; KEGG, Kyoto Encyclopedia of Genes and Genomes; WT, wild-type; Mut, mutant; D-Mut, double mutant; NS, no significance.

Fig 4: Lack of MYO6 in sv/sv spermatids disrupts the structural integrity of the nectin-3-positive endocytic compartment. Confocal microscopy images of stage VII sv/+(A) and sv/sv (B) spermatids representing dynamics of TBC-related endocytic compartment immunostained for nectin-3 (red) and F-actin (green). Arrows highlight the position of the TBCs and dotted squares indicate areas magnified in images marked with prime and double prime. All bars 1 µm.

Fig 5: ZNF582 suppresses NPC cell invasion and metastasis in vivo. Popliteal lymph node metastasis model was employed by injecting SUNE1 (3 × 105) cells stably expressing shZNF582 or control shRNA (shControl) into the footpads of nude mice (n = 8 per group). (A) A representative image of primary footpad tumor and metastatic popliteal lymph nodes. (B) The H&E staining shows that the primary footpad tumor invaded in the skin and muscle. Scale bar: 100 µm. (C) Image of metastatic popliteal lymph nodes. The volume of the popliteal lymph nodes was greater in the ZNF582 knockdown group than in the shControl group. (D-E) The immunohistochemical staining of pan-cytokeratin-positive tumor cells in popliteal lymph nodes. Scale bar: 50 µm. (F-I) Lung metastasis (F), H&E staining (G), density (H), number (I) of tumors metastasized in the lungs of nude mice at day 55 after intravenous injection with SUNE1 cells (1 × 106) stably transfected with plasmids encoding vector or ZNF582 (n = 6, per group). (J) Survival analysis of nude mice intravenously injected with SUNE1 cells (1 × 106) stably transfected with plasmids encoding vector (n = 9) or ZNF582 (n = 10). (K) Immunohistochemical staining for ZNF582, Nectin-3, NRXN3, and IgG in lung metastases. Scale bar: 100 µm. (L-M) IHC staining and score for ZNF582, Nectin-3, NRXN3, and IgG expression in NPE (n = 7) and NPC (n = 11) tissue sections. Scale bar: 100 µm. All data are presented as mean ± standard deviation of at least three independent experiments. Student's t-test, *P < 0.01, ***P < 0.001. Abbreviations: LNs, lymph nodes; IHC, immunohistochemistry; IgG, immunoglobulin G; NPC, nasopharyngeal carcinoma; shControl, control shRNA; shRNA, short-hairpin RNA.