Figure 7. Oncogenic role and intracellular trafficking of mutant Kit in rat and human cells.

(a) Mutant Kit is constitutively active in HMC-1 and RBL-2H3 cells. (Left) Schematic structures of Kit in HMC-1 and RBL-2H3 cells. (Right) Immunoblots of anti-Kit immunoprecipitates from HMC-1 or RBL-2H3 cells treated with 1 μM PKC412 (Kit kinase inhibitor) for 4 or 12 h, respectively. (b,c) The effect of PKC412 on proliferation. (b) [³H]-thymidine incorporation in HMC-1 cells treated with PKC412 for 24 h. Results (c.p.m.) are means±s.d. (n=3). (c) Growth of RBL-2H3 cells treated with (filled circles) or without (open circles) 1 μM PKC412. Results are means±s.d. (n=3). (d) Glycosylation of Kit(D816V) and Kit(D817Y) performed as for Fig. 2a. (e,f) Subcellular localization of Kit. (e) Methanol-fixed HMC-1 or (f) PFA-fixed RBL-2H3 cells were double-stained with anti-Kit (green) and anti-cathepsin D (endolysosome marker; red), anti-LAMP1 (endolysosome marker; red), anti-calnexin (ER marker; red), or anti-GM130 (Golgi marker; blue). Magnified images of the boxed area are shown. Representative images of Kit-positive endolysosomes containing cathepsin D are shown. Bars, 5 μm. The graphs show the correlation coefficient (Pearson’s R) between Kit and organelle markers. Results are means±s.d. from 15 to 18 cells. Data were subjected to one-way ANOVA with Dunnett’s multiple comparison post-hoc test. **P<0.01, ***P<0.001. (g,h) Endocytosis of mutant Kit in HMC-1 and RBL-2H3 cells. (g) HMC-1 (left) or RBL-2H3 cells (right) treated with 1 μM PKC412 for 4 or 12 h, respectively. Cells were stained with anti-Kit (green) and anti-calnexin (ER marker; red). Insets show boxed areas at higher magnification. Bars, 10 μm. (h) HMC-1 cells treated with 1 μg ml⁻¹ filipin or 0.45 M sucrose for 3 h to block endocytosis. Cells were stained with anti-Kit (cyan) and anti-p85 (red). Bars, 10 μm. Immunoblots for Kit, p85 and TfR are shown.