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. 2025 Jul 28;21(11):4895–4907. doi: 10.7150/ijbs.97195

Figure 4.

Figure 4

N78 decreasing N-Myc protein stability by regulating phosphorylation of N-Myc at threonine 58. (A) RT-qPCR analysis of MYCN in SK-N-BE(2) and IMR-32 treatment with N78. The effects of N78 on MYCN mRNA levels after 12 h treatment were evaluated by Real-time PCR. (B-C) SK-N-BE(2) and IMR-32 cells were incubated with N78 (4 μM) for 3 h, following by 50 μg/ml cycloheximide (CHX) treatment. N-Myc levels were detected by western blot analysis (B) and the degradation kinetic curves were plotted (C). (D) The levels of N-Myc protein under the presence and absence of NH4Cl or N78, MG132 or N78 were determined. SK-N-BE(2) and IMR-32 cells were seeded into 6-well plates and treated with 5 μM MG132 for 3 hours, followed by the addition of N78 (4 μM) for an additional 2 hours. Cells were then collected for western blot. (E-F) Phosphorylated N-Myc T58 and S62 in SK-N-BE(2) and IMR-32 treatment with N78 at the indicated time points were detected by western blot (E) and the variation curves of pT58 / total N-Myc and pS62 / total N-Myc were plotted (F). SK-N-BE(2), IMR-32 cells were exposed to N78 (4 μM) for different time, then, cell lysates were collected and analyzed by western blot. (G-H) Western blot of N-Myc in HEK 293T with overexpression of N-My, N-Myc-T58A, N-Myc-S62A after N78 (8 µM) treatment at the indicated time points.