Fig 2: Structure of drugs tested. SB590885 inhibits BRAF; GDC-0879 inhibits P-ERK; and AZD6244 inhibits MEK and ERK1/2 phosphorylation. As per MCLP drug–protein relationship, Nutlin induces p21 and induces differentiation (Bortezomib is an FDA-approved cancer drug, proteosome inhibitor. These molecules were tested in equipotent concentrations to compare the relative biological activities.

Fig 3: Involvement of BRAF/ANXA7 axis with p21 and combination therapy. (a) The protein expression level of p21 in different cancer cell lines. Data show the lowest expression in prostate cancer cell lines. (b) Expression of RB/p21-related protein expression level in four different thyroid cancer cell lines. (c) The p21 and drug relationship. Data indicating that the Nutlin will increase the expression of p21 and sensitize the apoptosis of the cancer cells. (d) A series of drugs response to 8505C cell apoptosis at 24 h incubation. (e) Crystal violet staining of the 8505C cells after incubating with different drugs for 24 h. The cells are resistant to GDC and AZD treatment alone. Cells are mildly sensitive to Bortezomib (BIZ) treatment in a dose-dependent manner. The Nutlin (Nut) inhibits growth and differentiates the cells. The Nut-only panel shows the more differentiated morphology by absorption of dendritic processes and contact inhibition. GDC + AZD treatment has some additive effect which increases the cell death but is not enough to completely overcome the resistance and complete apoptosis. Nut + AZD treatment is more potent—it induces differentiation and sensitizes the cells to chemotherapy. Nut + BIZ treatment completely overcomes the chemoresistance and all cells are killed and eradicated, as seen by the negative toward crystal violet staining. The panel shows no viable cells and only dense apoptotic bodies are visible. Cell ghosts are faintly visible in the background. Abbreviation: BIZ = Bortezomib; AZD = AZD6244 (1 µM, 1×); GDC = GDC0879 (0.1 µM; 1×); SB = SB590885 (1 µM; 1×).

Fig 4: BRAF can bind to PA28γ and stabilize its protein level.A–C Western blot assays showed that the expression levels of BRAF were upregulated in a dosage-dependent and time-dependent manner. D, E A coimmunoprecipitation assay showed that exogenous Myc-BRAF and Flag-PA28γ bind to each other in 293T cells. F, G Coimmunoprecipitation experiments showed that endogenous BRAF and PA28γ interact with each other in epithelial cells. H After transfecting the Myc-BRAF plasmid into 293T cells, coimmunostaining showed that Myc-BRAF (red) colocalized with PA28γ (green). Scale bar: 50 μm. I Immunoprecipitation assays showed that exogenous BRAF and MEK could simultaneously bind to PA28γ. Immunoprecipitation of PA28γ was performed with the Flag antibody. J Exogenous BRAF and PA28γ were detected in immunoprecipitates performed with the HA antibody. K Exogenous MEK1 and PA28γ can be detected in immunoprecipitates performed with the Myc antibody. L Transfection of 293T cells with the Myc-BRAF plasmid in a dose-dependent manner. Western blot assays showed that the expression levels of PA28γ, p-MEK1, and p-ERK were significantly upregulated. M Immunoprecipitation shows that silencing BRAF expression reduces MEK1 binding to PA28γ. N Western blot assays showed that knocking down PA28γ inhibited BRAF-dependent expression of p-MEK1 and p-ERK.

Fig 5: BRAF inhibited the ubiquitination level of PA28γ.293T cells were constantly transfected with vector (2 μg), Flag-PA28γ (2 μg), or Myc-BRAF plasmid (2 μg) 24 h later, and then serum-starved 293T cells were pretreated with A CHX (100 μg/ml) or B MG132 (10 μM) for the indicated periods of time. Data represent the means ± s.d. of three independent experiments. Quantification of Flag-PA28γ levels relative to GAPDH is shown. Statistical analysis was performed using Student’s t test (**p < 0.01). C The ubiquitylation assay showed the ubiquitination degradation process of the PA28γ protein. D, E BRAF inhibited the PA28γ ubiquitination level at both the K48 and K63 sites.