Removal of Stem Cell Factor or Addition of Monoclonal Anti-c-KIT Antibody Induces Apoptosis in Murine Melanocyte Precursors  Masaru Ito, Yoko Kawa, Mitsuhiro Okura, Takako Baba, Yasuo Kubota, Masako Mizoguchi  Journal of Investigative Dermatology  Volume 112, Issue 5, Pages 796-801 (May 1999) DOI: 10.1046/j.1523-1747.1999.00552.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of c-KIT-positive cells and effect of ACK2 addition in NCC cultures. (a) c-KIT-positive cells were immunohistochemically stained with ACK2 after 7 d of culture with SCF. Scale bar: 500 μm (NC, neural crest). (b) c-KIT-positive cells survived and increased in number with SCF. The cells per well were counted under a microscope. SCF+, with SCF (basic standard medium), c-KIT-positive cells increased in number on day 3–8 and peaked on day 8 (n = 6). SCF–, without SCF, the c-KIT-positive cells found on day 3 soon decreased in number to zero (n = 6). (c) Concentration-dependent effects of ACK2 on the apoptosis of NCC. On day 7 various amounts of ACK2 were added to NCC culture. On the next day (day 8), we counted the number of apoptotic cells. Based on these results, we determined that the optimal dose of ACK2 for the detection of apoptosis was 100 μg per ml. *p < 0.05, n = 4 for each concentration except 1 μg per ml (n = 1). Journal of Investigative Dermatology 1999 112, 796-801DOI: (10.1046/j.1523-1747.1999.00552.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Apoptotic cells around the neural tubes. Apoptotic cells were detected as Apop Tag-positive nuclei and were almost equally distributed among dendritically proliferated cells around the neural tubes of mice in each group. Apoptotic cells were more frequently detected in the SCF group (a) than in the ACK2′ group (b). Scale bar: 200 μm. Journal of Investigative Dermatology 1999 112, 796-801DOI: (10.1046/j.1523-1747.1999.00552.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Removal of SCF or addition of ACK2 increased the number of apoptotic cells in the NCC culture (error bars, mean ± SD). The numbers of Apop Tag-positive cells in each explant were counted under a microscope. The mean numbers of Apop Tag-positive cells were 80 ± 14 (mean ± SD) in the SCF group (n = 5), 192 ± 75 in the SCF+/– group (n = 6), 330 ± 83 in the ACK2 group (n = 5), 425 ± 60 in the ACK2′ group (n = 5), and 87 ± 2 in the IgG group (n = 5). There was a significant difference between the numbers in the SCF and SCF+/– groups (p < 0.005). The numbers in the ACK2 and ACK2′ groups were markedly increased and significantly larger compared with those in the SCF and SCF+/– groups (p < 0.05); however, there was no significant difference between the ACK2 and ACK2′ groups. The number in the IgG group was not significantly different from that in the SCF group. Journal of Investigative Dermatology 1999 112, 796-801DOI: (10.1046/j.1523-1747.1999.00552.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 c-KIT is expressed by apoptotic cells. In the ACK2 group, we performed a double staining with Apop Tag and c-KIT. Using Apop Tag, we first labeled apoptotic cells red by AEC (a, the dark color indicates apoptotic cell’s nucleus, examples are indicated by arrows). Subsequently, we labeled c-KIT-positive cells in the same specimens blue/violet by BCIP/NBT (b, the darker color indicates c-KIT positive cell’s membrane, examples are indicated by arrows). The same cells are apoptotic and c-KIT positive. Scale bar: 100 μm. Journal of Investigative Dermatology 1999 112, 796-801DOI: (10.1046/j.1523-1747.1999.00552.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Apoptotic cells have premelanosomes. Electron microscopy showed that the apoptotic cells (→) had condensed nuclei with homogeneous and compacted chromatin, which are characteristic findings of apoptosis (a). Scale bar: 2 μm. On higher magnification, many premelanosomes (→) are seen in these apoptotic cells (b). Scale bar: 0.5 μm. Journal of Investigative Dermatology 1999 112, 796-801DOI: (10.1046/j.1523-1747.1999.00552.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Agarose gel electrophoresis of nuclear DNA extracted from cultured neural crest cells. Agarose gel electrophoresis was used to detect internucleosomal DNA cleavage in whole NCC. Lane 1, ladder marker; lane 2, SCF group (negative control); lane 3, ACK2′ group. DNA from the ACK2′ group (lane 3) exhibited a ladder-like pattern of oligonucleosome-sized DNA fragments characteristic of apoptosis. Journal of Investigative Dermatology 1999 112, 796-801DOI: (10.1046/j.1523-1747.1999.00552.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions