Journal: Molecular cell

Article Title: The Proto-oncogene c-Kit Inhibits Tumor Growth by Behaving as a Dependence Receptor.

doi: 10.1016/j.molcel.2018.08.040

Figure Lengend Snippet: Figure 3. c-Kit-Induced Apoptosis Is Inhibited by SCF and Is Mediated by Intracellular Caspase-like Cleavage Driving a Caspase Activation Amplification Loop (A and B) The ligand SCF inhibits c-Kit-induced cell death. A549 and IMR32 stable cell lines inducible for kinase-mut c-Kit were treated with Dox and recombinant SCF proteins derived from E. coli (R&D Systems, 1 mg/mL) or from 293T cells (Cell Signaling Technology, 25 ng/mL). Apoptotic cell death was then evaluated in the A549 stable cell line by PI staining 48 hr after treatment and quantified with the Incucyte Zoom instrument (A) and in the IMR32 stable cell line by caspase-3 activation and quantified with a fluorometric detection kit (B). (C–F) c-Kit is cleaved by caspase-3. The in vitro-translated intracellular region of WT or caspase cleavage site-mutated (D816V) c-Kit was incubated with 0.3 mM of purified active caspase-3, and digested bands were analyzed by autoradiography (C). Although WT c-Kit cleavage revealed the presence of two major bands at 31 kDa (corresponding to a c-Kit fragment from the transmembrane domain to D816) and 18 kDa (c-Kit fragment to D816 to stop codon), these bands dis- appeared when using the D816V mutant. c-Kit cleavage was then confirmed in IMR32 cells transfected with the kinase-mut (D816) or kinase- and caspase- mutated (V816) intracellular region of c-Kit (D). After treatment for 2 hr with the proteasome inhibitor MG132, western blot analysis using a C-terminal hemag- glutinin (HA) tag revealed the presence of a band at 18 kDa that disappeared in the V816 construct. Cleavage at the D816 site of full-length c-Kit was also confirmed by using IMR32 stable inducible cell lines (E) in which c-Kit or its mutants was induced by Dox. After a 2-hr treatment with the proteasome inhibitor bortezomib, western blot analysis using an antibody targeting the C-terminal region of c-Kit revealed the presence of a band at 18 kDa in WT and kinase-mut (D792N) c-kit but not in c-Kit stable cells mutated at the D816 site. The western blot presented here is representative of three independent experiments. In addition, endogenous c-Kit could also be cleaved (F). In the M2Ge melanoma cell line, an 18-kDa band was detected by c-Kit antibody targeting the C-terminal region in the presence of bortezomib. (G and H) c-Kit interacts with and activates caspase-9. 293T cells were transfected with kinase-mut c-Kit and caspase-9-HA plasmids. After lysis, caspase-9 was pulled down by HA antibody, and c-Kit was detected in the immunoprecipitated fraction using a-c-Kit antibody (G). IMR32 stable cells, inducible for kinase-mut c-Kit, were treated with Dox for 24 hr, and, after lysis, kinase-mut c-Kit was pulled down by FLAG antibody. Active caspase-9 that bound to pulled down c-Kit was quantified with a caspase-9 activity detection kit (H). Data are representative of at least three replicates as mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Recombinant SCF protein was obtained from R&D systems (E.coli-derived) or Cell Signaling (293T-derived).

Techniques: Activation Assay, Stable Transfection, Recombinant, Derivative Assay, Staining, In Vitro, Incubation, Autoradiography, Mutagenesis, Transfection, Western Blot, Construct, Lysis, Immunoprecipitation, Activity Assay