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Interaction and functional association of TOP3A with NSMCE4A, <t>YTHDC2,</t> and NDUFAF7. A , validation of the interaction between TOP3A and NSMCE4A by immunoprecipitation and western blotting. B , validation of the interaction between TOP3A and YTHDC2 by immunoprecipitation and western blotting. C , validation of the interaction between TOP3A and NDUFAF7 by immunoprecipitation and western blotting. D , control small interfering RNA (siRNA) or siRNA targeting NSMCE4A was transfected into wild-type (WT) or BLM-KO cells. After 24 h, cells were seeded into 96-well plates. Proliferation of the indicated cell lines was measured using a CellTiter-Glo assay after 4 days in the presence of the indicated concentrations of CPT, ETO, and HU. Data are presented as mean (±SEM) values (n = 3). E , control siRNA or siRNA targeting YTHDC2 and NSMCE4A was transfected into WT or BLM-KO cells. After 48 h, whole-cell extracts were prepared and subjected to western blotting with the indicated antibodies. CPT, camptothecin; ETO, etoposide; HU, hydroxyurea.
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Interaction and functional association of TOP3A with NSMCE4A, YTHDC2, and NDUFAF7. A , validation of the interaction between TOP3A and NSMCE4A by immunoprecipitation and western blotting. B , validation of the interaction between TOP3A and YTHDC2 by immunoprecipitation and western blotting. C , validation of the interaction between TOP3A and NDUFAF7 by immunoprecipitation and western blotting. D , control small interfering RNA (siRNA) or siRNA targeting NSMCE4A was transfected into wild-type (WT) or BLM-KO cells. After 24 h, cells were seeded into 96-well plates. Proliferation of the indicated cell lines was measured using a CellTiter-Glo assay after 4 days in the presence of the indicated concentrations of CPT, ETO, and HU. Data are presented as mean (±SEM) values (n = 3). E , control siRNA or siRNA targeting YTHDC2 and NSMCE4A was transfected into WT or BLM-KO cells. After 48 h, whole-cell extracts were prepared and subjected to western blotting with the indicated antibodies. CPT, camptothecin; ETO, etoposide; HU, hydroxyurea.

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis of Human Topoisomerases Reveals Their Distinct and Diverse Cellular Functions

doi: 10.1016/j.mcpro.2025.101082

Figure Lengend Snippet: Interaction and functional association of TOP3A with NSMCE4A, YTHDC2, and NDUFAF7. A , validation of the interaction between TOP3A and NSMCE4A by immunoprecipitation and western blotting. B , validation of the interaction between TOP3A and YTHDC2 by immunoprecipitation and western blotting. C , validation of the interaction between TOP3A and NDUFAF7 by immunoprecipitation and western blotting. D , control small interfering RNA (siRNA) or siRNA targeting NSMCE4A was transfected into wild-type (WT) or BLM-KO cells. After 24 h, cells were seeded into 96-well plates. Proliferation of the indicated cell lines was measured using a CellTiter-Glo assay after 4 days in the presence of the indicated concentrations of CPT, ETO, and HU. Data are presented as mean (±SEM) values (n = 3). E , control siRNA or siRNA targeting YTHDC2 and NSMCE4A was transfected into WT or BLM-KO cells. After 48 h, whole-cell extracts were prepared and subjected to western blotting with the indicated antibodies. CPT, camptothecin; ETO, etoposide; HU, hydroxyurea.

Article Snippet: The following antibodies were used for western blotting and immunofluorescent staining: PUM3 (A303-897A; Bethyl Laboratories), hemagglutinin (HA) (2367S; Cell Signaling Technology), YTHDC2 (A303-026A; Bethyl Laboratories), NSMCE4A (HPA037459-100UL; Sigma-Aldrich), TDRD3 (5942S; Cell Signaling Technology), TIPRL (A300-663A; Bethyl Laboratories), TDRKH (13528-1-AP; Proteintech), TOP1 (A302-589A; Bethyl Laboratories), nucleophosmin (32-5200; Thermo Fisher Scientific), TOP3B (ab183520; Abcam), TOM20 (sc-17764; Santa Cruz Biotechnology), FLAG (F3165; Sigma-Aldrich), phosphorylated H2AX (S139, 9718S; Cell Signaling Technology), and tubulin (T6199-200UL; Sigma-Aldrich).

Techniques: Functional Assay, Biomarker Discovery, Immunoprecipitation, Western Blot, Control, Small Interfering RNA, Transfection, Glo Assay