Fig. 4.

MSDNc degrades MDM2 and restores p53 levels, thereby effectively killing tumor cells in vitro. (A) Schematic diagram of degradation of MDM2 by MSDNc through home-protac mechanism. (B&C) After 1650 cells were pretreated with MG132 or PYR-41 for 12 h, and incubated with MSDNc at a dose of 100 nm for 48 h, western blot was performed (B) and the relative MDM2 level were quantified (C) to analyze the expression of MDM2 proteins, and GAPDH was used as an internal reference protein. (D) After MSDNc treatment, the volcano plot of the differential changes in gene expression in 1650 cell line was shown. The absolute value of the base 2 logarithm of the genetic difference is required to be>1.3, and the adjusted p value should be>0.05. (E&F) GSEA result (E) and hierarchical clustering of genes (F) for p53 signaling pathway. (G-J) GSEA results for KEGG apoptosis, REACTOME cell cycle, REACTOME cell cycle checkpoints and REACTOME cell cycle mitotic. (K) After 1650 cells were treated by ^PEGN, ^CtrlMSDNc, and MSDNc at a dose of 100 nm for 48 h, and then western blot was performed to analyze the expression of p53, p73 and p21 proteins, and GAPDH was used as an internal reference protein. (L&M) Immunofluorescence staining (L) and quantitative analysis (M) of p53 were performed in the 1650 cell line after above treatment. (N) The cell viability was measured by MTT after treated by ^PEGN, ^CtrlMSDNc and MSDNc at different concentration in 1650 cell line. (O&P) The cell cycle (O) and apoptosis (P) were analysed by flow cytometry after treated by ^PEGN, ^CtrlMSDNc and MSDNc at a dose of 100 nm for 48 h.