Fig. 3
KIT migrates to EL through endocytosis in a manner dependent on their kinase activity. a Kasumi-1 or HMC-1.1 cells were cultured for 24 h in the presence of KIT kinase inhibitors (imatinib, open circles; PKC412, closed circles). The graphs show the levels of [3H] thymidine deoxyribonucleotide (TdR) incorporation into cells (counts per minute, c.p.m., growth index) at the indicated inhibitor concentrations. Results are means ± s.d. (n = 3). b Kasumi-1 or HMC-1.1 cells were treated for 4 h with 1 μM PKC412 or 1 μM imatinib. Lysates were immunoblotted for KIT, phospho-KIT Y703 (pKITY703), AKT, pAKT, STAT5, pSTAT5, ERK, and pERK. c-f Kasumi-1 cells were treated for 12 h with 1 μM PKC412 (PKC) or 1 μM imatinib (IMA). c Cells were immunostained with anti-KIT (green) and anti-calnexin (ER marker, red). Confocal immunofluorescence images are shown. Insets show magnified images of the PM region. Bars, 10 μm. d Cell surface KIT levels determined by flow cytometry are shown. Non-permeabilized cells were stained with anti-KIT extracellular domain antibody. Green histogram, with KIT inhibitor treatment; white histogram, no KIT inhibitor; gray histogram, no anti-KIT antibody control. e Cells were immunostained with anti-KIT (green) and anti-TFR (endosome marker, red). Confocal immunofluorescence images are shown. Bars, 10 μm. f Pearson’s R correlation coefficients were calculated by analyzing the intensity of KIT vs. TFR. Results are means ± s.d. (n = 22~29). ***P < 0.001. Note that these inhibitors lowered KIT in vesicular structures and increased KIT in the PM