Fig 2: MDM2 induces epithelial-to-mesenchymal transition progression via the B-Raf signaling pathway. (A) Snail, Slug and ZEB1 expression was analyzed by western blotting in stable MDM2-overexpressing U251 cells and control U251 cells. Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a control, and semi-quantitative analysis is shown. (B) Gene Set Enrichment Analysis plot of U251 cells showing the most significantly changed B-Raf signaling pathway set. Note that the B-Raf signaling pathway was significantly upregulated upon expression of MDM2 but downregulated upon expression of empty vector. (C) Western blot analysis of the levels of p-B-Raf, p-MEK, p-ERK, p-P70S6K and p-4EBP1 in stable MDM2-overexpressing U251 cells and control U251 cells. Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH served as a control. (D) E-CAD levels were analyzed by western blotting in control U251 cells and stable MDM2-overexpressing U251 cells following treatment with DMSO, vemurafenib (B-Rafi at 2 µM for 24 h), U0126 (MEKi at 20 µM for 24 h) and FR180204 (ERKi at 10 µM for 24 h). Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a control. (E) Snail, Slug and ZEB1 levels were analyzed by western blotting in control U251 cells and stable MDM2-overexpressing U251 cells treated with FR180204 (ERKi at 10 µM for 24 h). Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a normalization control. (F) E-CAD expression was analyzed by western blotting in stable control U251 cells and stable MDM2-overexpressing U251 cells treated with Vemurafenib, U0126, FR180204 and SL0101 (RSKi at 50 µM for 24 h). Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a control, and the band density was semi-quantified. (G) E-CAD expression was analyzed by western blotting in stable control A549 cells and stable MDM2-overexpressing A549 cells treated with DMSO, vemurafenib (B-Rafi at 2 µM for 24 h), U0126 (MEKi at 20 µM for 24 h), and FR180204 (ERKi at 10 µM for 24 h). Ctrl referred to the A549 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a control. (H) E-CAD levels were analyzed by western blotting in stable control MCF7 cells and stable MDM2-overexpressing MCF7 cells treated with DMSO, vemurafenib (B-Rafi at 2 µM for 24 h), U0126 (MEKi at 20 µM for 24 h) or FR180204 (ERKi at 10 µM for 24 h). Ctrl referred to the MCF7 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a control. For all quantifications, data are presented as the mean ± SD derived from three independent experiments. Statistical significance was analyzed using an unpaired Student's t-test for A, and one-way ANOVA with Tukey's post hoc test was used to compare the indicated group and Ctrl in F. *P<0.05; **P<0.01; ***P<0.001; n.s., not significant vs. Ctrl or as indicated. 4EBP1, eukaryotic translation initiation factor 4E binding protein 1; BRafi, B-Raf inbibitor; Ctrl, control; E-CAD, E-cadherin; ERKi, ERK inhibitor; GFP, green fluorescent protein; IB, immunoblotting; MDM2, MDM2 proto-oncogene, E3 ubiquitin protein ligase; MEKi, MEK inhibitor; p-, phosphorylated; P70S6K, ribosomal protein S6 kinase; RSKi, RSK inhibitor; Slug, Snail family transcriptional repressor 2; Snail, Snail family transcriptional repressor 1; T-, total; ZEB1, zinc finger E-box binding homeobox 1.

Fig 3: MDM2-mediated B-Raf signaling pathway activation via 14-3-3 is dependent on p53. (A) MDM2, p53, E-CAD, N-CAD and VIM protein expression was analyzed by western blotting in control U251 cells and stable MDM2-overexpressing U251 cells transfected with pcDNA3.1-p53. Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a control. (B) Western blot analysis of the expression levels of 14-3-3 in stable MDM2-overexpressing U251 cells and control U251 cells. Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a control. Semi-quantitative analysis data are shown. (C) MDM2, 14-3-3, E-CAD, N-CAD, VIM and p-B-Raf protein expression was analyzed by western blotting in control U251 cells and stable MDM2-overexpressing U251 cells expressing Flag-14-3-3. Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. GAPDH was used as a control. (D) Colony formation assay for control U251 cells, stable MDM2-overexpressing U251 cells and stable MDM2-overexpressing U251 cells treated with FR180204 (ERKi at 10 µM). Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. Data are presented as the mean ± SD percentage of colonies for each group after 14 days (bottom). n=3 independent experiments. (E) Images of the migration of control U251 cells (magnification, ×100), stable MDM2-GFP overexpressing U251 cells (magnification, ×100) and stable MDM2-GFP-overexpressing U251 cells treated with FR180204 (ERKi at 10 µM) (magnification, ×100) at 0, 24 and 48 h after wounding. MDM2 could induce cell migration in U251 cells and FR180204 treatment could rescue this cell migration in scratch wound assays. Ctrl referred to the U251 cell line with stable overexpression of plenti-CMV-puro empty vector. For all quantifications, data are presented as the mean ± SD derived from three independent experiments. Statistical significance was analyzed using an unpaired Student's t-test in B, and one-way ANOVA with Tukey's post hoc test was used to compare the indicated group and Ctrl or two indicated groups in D and E. **P<0.01; ***P<0.001; n.s., not significant vs. Ctrl or as indicated. 14-3-3, tyrosine 3-monooxygenase activation protein ε; Ctrl, control; E-CAD, E-cadherin; ERKi, ERK inhibitor; GFP, green fluorescent protein; IB, immunoblotting; MDM2, MDM2 proto-oncogene, E3 ubiquitin protein ligase; N-CAD, N-cadherin; p-, phosphorylated; T-, total; VIM, vimentin.

Fig 4: CRBN(BRAF)‐24 suppressed cell proliferation of BRAFV600E cells but not BRAFWT cells. A, B, Effect of CRBN(BRAF)‐24 on cell proliferation of homozygous (A375 and SK‐MEL‐28) and heterozygous (COLO205 and RKO) BRAFV600E cells (A) or BRAFWT cells (B). Cells were treated with the indicated concentrations of CRBN(BRAF)‐24, CRBN(BRAF)‐29, or PLX8394 for 72 h. Cell proliferation was evaluated by cell viability assay

Fig 5: PLX8394‐based proteolysis targeting chimeras (PROTACs) induced degradation of BRAFV600E. A‐C, Protein knockdown activities of CRBN(BRAF)s. A375 cells were treated with the indicated concentrations of CRBN(BRAF)s for 24 h. D, CRBN(BRAF)‐24 induced sustained downregulation of BRAF protein levels. A375 cells were treated with the indicated concentrations of CRBN(BRAF)s for the indicated periods. E, CRBN(BRAF)‐24, but not CRBN(BRAF)‐29, led to efficient suppression of BRAF protein levels and pathway signaling. SK‐MEL‐28 cells were treated with the indicated concentrations of CRBN(BRAF)‐24 or CRBN(BRAF)‐29 for 24 h. Whole‐cell lysates were analyzed by Western blotting with the indicated antibodies. Numbers below the BRAF, pMEK1/2, and pERK1/2 panels represent the BRAF/actin, pMEK/MEK, and pERK/ERK ratios, respectively, normalized by designating the level of the vehicle control condition as 100%. Data in the bar graph are the mean (± SD) of two or three independent experiments