Figure 3.

4-Me targets Nox4 to cause apoptosis of MDA-MB-231 cells. (a) Flowchart of the strategy to identify cellular interacting partners of 4-Me. 4-Me with an alkyne group (4′-alkyne) was used as a bait. (b) Immunodetection of Nox 4 from immunoprecipitates of 4′-alkyne conjugated to biotin–azide. 4′-alkyne–azide–biotin was affinity pulldown using streptavidin Sepharose beads. (c–e) Representative sensograms of three independent experiments showing binding profiles between immobilized-full-length (FL) Nox4 and indicated concentrations of 4-Me (c); between immobilized-FL Nox4 and 500 nM of 4-Me, 3-Cl, 4-CF₃, and H (d); between either immobilized-FL Nox4, ΔFAD/NADPH or ΔHEME with 4-Me (e). Sensograms were corrected against a reference flow cells with no immobilized proteins. (f) Relative mRNA and protein levels of Nox4 in various human cell lines. HaCaT is a non-tumorigenic skin line, A-5RT3 is a metastatic skin line, and MKN28 is a gastric carcinoma line. The mRNA data (means±S.D.) are from two independent qPCR experiments performed in triplicates. The ribosomal protein 18S gene serves as a reference housekeeping gene and β-tubulin serves as a loading and transfer control in immunoblot. ***P<0.001. (g) Percentage of suspension-induced apoptotic (Annexin V positive) cells treated with indicated concentrations of 4-Me for 0.5 h as analyzed by FACS (5000 events). Vehicle-treated cells served as control. The sum of Annexin V⁺/PI⁻ (early apoptosis) and Annexin V⁺/PI⁺ (late apoptosis) cells were considered apoptotic. Results are representative of three independent experiments. See Supplementary Figures S3D and S4A for FACS data. Error bars: S.E.M. Comparison was performed against cognate vehicle controls. n.s. indicates not significant; ***P<0.001