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| RAF inhibitors induce dimer formation between KSR and RAF, and activate KSR by CRAF. (A) GDC0879 but not PLX4720 induces BRAF/CRAF dimers. Cells overexpressing myc-CRAF and BRAF were treated with drug for 1 h and CRAF immunoprecipitates were immunoblotted for BRAF and CRAF (epitope tagged with myc). (B) GDC0879 but not PLX4720 enhances KSR/BRAF complexes. KSR immunoprecipitates were prepared from cells overexpressing FLAG-KSR and BRAF after treatment with the indicated drug for 1 h and immunoblotted using antibodies to BRAF. (C) Both GDC0879 and PLX4720 induce KSR/CRAF complexes.KSR immunoprecipitates were prepared from cells overexpressing FLAG-KSR and myc-CRAF after treatment with the indicated drug for 1 h and immunoblotted for CRAF using myc antibodies. (D and E) Requirement of KSR for drug-induced ERK activation. Lysates fromwild-type and KSR deficient fibroblasts, transfected with RASV12, were treated with the indicated doses of either GDC-0879 (D) or PLX4720 (E) for 1 h. Lysates were immunoblotted for phospho-ERK1 and 2, ERK2, and RASV12. (F) KSR and CRAF cooperate to activate MEK. Cells expressing the indicated constructs were treated with a 50 uM PLX4720 for 2 h before cell lysates were prepared and analyzed for pMEK by immunoblotting. CRAF(TM) refers to the T421M gatekeeper mutant that cannot bind to the drug(4). (G) KSR in vitro kinase reactions. Cells were cotransfected with WT or ATP binding deficient KSR and CRAF and immunoprecipitates prepared after cells were treated with an activating dose of PLX (10 uM) for 1 h. KSR immunoprecipitates were prepared, pretreated with 50 uM PLX4720 to inhibit coprecipitating RAF activity, and then tested for kinase activity using purified MEK. MEK phosphorylation was detected using a pMEK specific antibody. |
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Rating |
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| Source |
Proc Natl Acad Sci USA , 2011, 108, 6067-6072. PLX-4720 purchased from Selleck |
| Method |
Western blotting |
| Cell Lines |
Cells overexpressing myc-CRAF and BRAF |
| Concentrations |
10/50 µM |
| Incubation Time |
1/2 h |
| Results |
GDC0879 but not PLX4720 induced BRAF/CRAF dimer formation (Fig. A). However, both drugs induced complexes between KSR1 and CRAF and enhanced interactions between KSR1 and BRAF (Fig. B and C), which suggested that KSR1/RAF complexes induced by the drug might explain the effects ofthe type I BRAF specific inhibitors. As reported previously, treatment of wild-type cells with either drug strongly induced ERK activation at low to intermediate doses but inhibited ERK activation at higher doses (Fig. D and E). Similar results were obtained with cells expressing constitutively active RAS (Fig. D and E) or after serum treatment. Strikingly, in KSR deficient cells, ERK activation was significantly attenuated after PLX4720 or GDC0879 treatment (Fig. D and E), which demonstrates that the ability of PLX4720 and GDC0879 to activate ERK requires the presence of KSR1. We found that, when KSR1 was overexpressed withCRAF, MEK activation was induced by PLX4720 or GDC0879 treatment (Fig. F). this result suggested that induction of the CRAF/KSR1 complex might be important in the activation of MEK. In vitro kinase activity toward MEK was detected but only after drug treatment (Fig. G), which suggests KSR1/CRAF complex formation kinase activity toward MEK. |
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| PBLs from CLL5 were treated with vehicles (0.1% ethanol and 0.005% DMSO), 106 M dexamethasone, 50 nM BIBF 1120, or both for 24 h. Cells were also treated with 0.1% ethanol or 106 M dexamethasone in the presence of either nonphosphorylated (control) EGQYEEIP or phosphorylated EGQY*EEIP H2O soluble peptides (200 nM) for 24 h. |
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Rating |
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| Source |
Cell Death Differ , 2010, 17, 1381-1391. BIBF1120 (Vargatef) purchased from Selleck |
| Method |
Cell viability assay |
| Cell Lines |
primary CLL cells |
| Concentrations |
50 nM |
| Incubation Time |
24 h |
| Results |
We also used the compound BIBF 1120, which has approximately one order of magnitude higher selectivity for Lck than other Src family kinases. BIBF (50 nM) produced similar results, further showing the importance for Lck inhibition (Figure 8i). Thus, we conclude that inhibition of Lck significantly enhances sensitivity to dexamethasone in a model of lymphoid malignancy that is relatively insensitive to glucocorticoid treatment. |
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| A, ANOVA of IC50 values of the dual PI3K/mTOR inhibitor NVP-BEZ235 in 23 melanoma cell lines showing no significant difference in sensitivity to this compound in B-Raf mutant and Braf wild-type cells. B, decreases in pAkt and pP70S6K in a dose and time-dependent fashion in 2 melanoma cell lines treated with NVP-BEZ235. pP70S6K levels are undetectable at all concentrations and time points studied, whereas levels of pAkt start rising again after 4 hours of drug exposure in a dose-dependent fashion. C, clonogenic assays in 2 melanoma cell lines (YUVON and YUSIK) treated with NVP-BEZ235 at different concentrations. NVPBEZ235 was effective in inhibiting colony formation at low nanomolar concentrations. |
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Rating |
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| Source |
Clin Cancer Res, 2010, 16, 6029-6039. BEZ235 (NVP-BEZ235) purchased from Selleck |
| Method |
Synergism studies, Immunoblot assays, Clonogenic assays |
| Cell Lines |
Melanoma cell |
| Concentrations |
0.1-100 μM |
| Incubation Time |
1-24 h |
| Results |
One of the proposed mechanisms of resistance to PI3KIs is mutation in the Ras-Raf pathway, which are found in more than half of melanomas. By ANOVA, no association was found between the IC50 values of NVP-BEZ235 and the presence or absence of B-Raf mutations (Fig. A). The targets of NVP-BEZ235, pAkt and pP70S6K, were both decreased with exposure to the drug in a time- and dose-dependent fashion, as shown in Figure B for YUVON and YUSIK cell lines. Clonogenicity was studied in YUVON and YUSIK cells with exposure to the dual PI3K/mTOR inhibitor. As shown in Figure C, NVP-BEZ235 effectively inhibits clonogenicity at low nanomolar concentrations. |
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| Inhibition of the MAPK signaling pathway results in downregulation of Plk-1 protein expression. (a) WB analysis for Plk-1 protein after treatment of human melanoma cell lines M14 and WM-115 with MEK 1/2 inhibitor PD98059 (10 mM), JNK inhibitor (16 mM), p38 inhibitor SB203580 (20 mM), and multikinase inhibitor sorafenib (10 mM) for 48 h showing significant reduction in the expression of Plk-1 protein after 48 hours. (b) Annexin V/PI staining of cells treated with MAPK inhibitors and induction of apoptosis. JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK 1/2, mitogen-activated protein kinase kinase 1/2; Plk-1, polo-like kinase 1; WB, western blot. |
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Rating |
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| Source |
J Invest Dermatol , 2011, 131, 1886–1895. Sorafenib (Nexavar) purchased from Selleck |
| Method |
Western blot |
| Cell Lines |
human melanoma cell lines M14 and WM-115 |
| Concentrations |
10 µM |
| Incubation Time |
48 h |
| Results |
To see whether inhibition of this pathway has an impact on the expression of Plk-1 in human melanoma, we used specific inhibitors of mitogen-activated protein kinase kinase (MEK) 1/2 (PD98059), multikinase inhibitor (sorafenib), c-Jun N-terminal kinase (JNK) (JNK inhibitor II), and the p38 MAPK (SB203580). Treatment of human melanoma cell lines WM-115 and M14 with these inhibitors was accompanied with significant reduction of Plk-1 protein levels (Figure a). This phenomenon was accompanied by induction of apoptosis(Figure b). |
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| ERK activation is increased in Il6ra2/2 mice compared with WT mice. A, Total and phosphorylated (p) Stat3 and ERK were assessed in small punch biopsy wounds collected after 30 min (Stat3) or 1 d (ERK) by Western blotting. Densitometry results for the blots are provided to the right. pp , 0.05. B, Total and p-ERKs were assessed in small wounds generated in WTand Il6ra2/2 mice treated topically with vehicle (DMSO) or with the MEK inhibitor U0126.Wounds were harvested after 1 d, and western blotting was performed on lysates. C, Wound contraction was assessed macroscopically in large wounds of WT (left panel) and Il6ra2/2 mice treated daily with vehicle (DMSO) or U0126. pp , 0.05. |
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Rating |
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| Source |
J. Immunol, 2010, 184, 7219-7228. U0126-EtOH purchased from Selleck |
| Method |
Western blotting |
| Cell Lines |
Il6ra-/-mice |
| Concentrations |
0.4 μg/μL |
| Incubation Time |
0-10 d |
| Results |
Stat3 phosphorylation was reduced to a similar extent in Il6-/-, Il6ra-/-, and Il6-/-/Il6ra-/-mice (Fig. A) compared with WT mice. phosphorylation of the MAPK ERK1/2 was undetectable in Il6-/-mice yet increased in Il6ra-/-relative to WT mice 1 d after wounding (Fig. A). Topical application of U0126 reduced ERK phosphorylation in wounds of both WT and Il6ra-/- mice compared with vehicle-treated controls (Fig. B). The difference in wound contraction rates was significantly delayed in Il6ra-/-mice treated with U0126 compared with vehicle control-treated mice but did not reach significance in WT mice (Fig. C). |
