PMC

Effects of dabrafenib on the Ser358 phosphorylation of MLKL and the interaction between RIP3, RIP1 or MLKL in HT29 cells. (a) Dabrafenib (10 μM) inhibited the Ser358 phosphorylation of MLKL induced by TSZ at the indicated times. (b) Effects of dabrafenib on the interaction between RIP3, RIP1 or MLKL in the cells exposed to the indicated combinations for 6 h. con, control; D, dabrafenib (10 μM); N, necrostatin-1 (10 μM); S, Smac mimetic (100 nM); Z, z-VAD-fmk (20 μM); T, TNF-α (20 ng/ml). The data were representative of three independent experiments

Dabrafenib alleviates acetaminophen-induced hepatotoxicity in mice. Mice were treated with 300 mg/kg acetaminophen (i.p.), with or without pretreatment with 300 mg/kg or 100 mg/kg dabrafenib (p.o.). Plasma alanine aminotransferase (ALT) (a) and aspartate aminotransferase (AST) (b) were determined. Data were expressed as mean±S.D.; n=10. In a, *P=0.015 and **P=0.0007; and in b, *P=0.03; **P=0.002. (c) The histological changes in mouse liver tissues shown by hematoxylin and eosin (H&E) staining. (d) TUNEL staining for nuclear DNA fragmentation in mouse liver cells. The typical images were captured under an optical microscope at × 20 (c) or × 40 (d) magnification

Dabrafenib inhibits RIP3 independently of its effect on the B-Raf family members. (a) The B-Raf^V600E inhibitors GDC-0879 (GDC), vemurafenib (VEM) and dabrafenib (DAB) inhibited the phosphorylation of MEK and ERK in B-Raf^V600E-expressed A375 cells and HT29 cells. con, control. The data were representative of three independent experiments. (b) The cell viability of the cells exposed to TSZ in the presence or absence of the indicated B-Raf^V600E inhibitors for 24 h was examined. All the B-Raf^V600E inhibitors were used at 1 μM in A375 cells while at 10 μM in both shRIP3 and shNC N cells. (c) MLKL was silenced with the specific siRNA in HT29 cells. The protein levels were examined by western blotting. The cell viability of the cells exposed to the indicated combinations for 24 h was examined. Dabrafenib was used at 10 μM. *P=0.00002; **P=0.0002. (d) B-Raf was silenced with the specific siRNA in HT29 cells. The protein levels were examined by western blotting (left). The cell viability of HT29 cells exposed to the indicated combinations for 24 h was examined

Dabrafenib selectively inhibits RIP3 enzymatic activity by binding to the enzyme protein. (a) The inhibition of dabrafenib against RIP1∼RIP3 and RIP5 was examined by the radioactive assay (Reaction Biology Corporation). Column, the mean of the IC₅₀ values from two independent experiments. The relative fold was obtained by normalizing the IC₅₀ value for RIP3 as 1. (b) Molecular docking of dabrafenib binding to B-Raf. The B-Raf protein was shown as a cartoon style, the original ligand in 3SKC was colored cyan and dabrafenib was colored marine blue. (c) Molecular docking of dabrafenib binding to RIP3. RIP3 was shown in cartoon and dabrafenib was shown in the stick model. (d) The surface plasmon resonance assay showed that dabrafenib did not bind to the GST tag protein (upper) but bound to the recombinant RIP3 protein (middle). The RIP1 inhibitor necrostatin-1 did not bind to RIP3 (lower). The representative data were shown from two independent experiments

Protection of dabrafenib from acetaminophen-induced cell viability reduction in human hepatocytes. (a) Human liver QSG-7701 and HL-7702 cells were treated with 20 mM acetaminophen in the presence or absence of 10 μM dabrafenib for the indicated times and the cell viability was examined. Insets, the levels of RIP1 and RIP3 proteins in the cells were detected by western blotting. (b) The cells exposed to 20 mM acetaminophen for the indicated times in the presence or absence of 10 μM dabrafenib were observed under an inverted microscope at × 20 magnification. Scale bar, 50 μm. (c) The effects of dabrafenib and necrostatin-1 on QSG-7701 and HL-7702 cells treated with 20 mM acetaminophen for 10 or 21 h were examined. (d and e) RIP3 (d) or RIP1 (e) was silenced with the specific siRNA in QSG-7701 cells and HL-7702 cells. The protein levels were examined by western blotting. The cell viability of the indicated cells exposed to 20 mM acetaminophen at the indicated times was examined. *P=0.004; **P=0.005; ***P=0.03; ****P=0.05. All the image data were representative of three independent experiments

Dabrafenib inhibits RIP3-mediated necroptosis. (a) Cell viability was examined in HT29 cells with or without TNFα (20 ng/ml) plus Smac mimetic (100 nM) and the caspase inhibitor z-VAD (20 μM; TSZ) in the presence of the indicated compounds (dabrafenib, 10 μM; all the others, 100 μM) for 24 h. (b) HT29 cells exposed to TSZ for 24 h in the presence or absence of 10 μM dabrafenib were observed under an inverted microscope at 20 × magnification. Scale bar, 50 μm. The data were representative of three independent experiments. (c) The cell viability of N and A cells (upper panel) or the RIP3-silenced N cells (lower panel) exposed to TSZ or TSZ plus dabrafenib for 24 h was determined. The protein levels of RIP3 in the cells were examined by western blotting. *P=0.002; **P=0.001. shNC and shRIP3, N cells transfected with the control shRNA and RIP3 shRNA, respectively. (d) The effects of the indicated compounds on cell viability were examined in HT29 cells or U937 cells treated with TSZ for 24 h. (e) The cell viability of HT29 cells treated with SZ plus 1 μg/ml Fas ligand or 200 ng/ml TRAIL was examined in the presence or absence of 10 μM dabrafenib. *P=0.003; **P=0.0003