1. Keloid Fibroblasts Resist Ceramide-Induced Apoptosis by Overexpression of Insulin- Like Growth Factor I Receptor 
Hiroshi Ishihara, Hiroshi Yoshimoto, Masaki Fujioka, Ryuuichi Murakami, Akiyoshi Hirano, Tohru Fujii 
Journal of Investigative Dermatology 
Volume 115, Issue 6, Pages (December 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Upregulation of IGF-I receptor expression in keloid fibroblasts compared with normal fibroblasts. Immunoblotting of IGF-IR α subunits. Total cell lysates were prepared from the three independent strains of normal and keloid fibroblasts, and equal amounts of proteins were subjected to 7.5% SDS-polyacrylamide gel electrophoresis followed by western blotting against IGF-IR α subunits. Lanes 1–3, normal fibroblasts; lanes 4–6, keloid fibroblasts.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Dose-dependent induction of cell death by C2 ceramide in normal and keloid fibroblasts. (a-f) Phase contrast microscopy of dermal fibroblasts cultured with DMEM containing various concentrations of C2 ceramide for 12 h. (a-c) Normal fibroblasts treated with 0–50 μM C2 ceramide; (d-f) keloid fibroblasts treated with 0–50 μM C2 ceramide; g, normal fibroblasts treated with ethanol only; (h) normal fibroblasts treated with 50 μM dihydro-C2-ceramide; (i) fluorescent microscopy (Hoechst 33342) of normal fibroblasts cultured with DMEM containing 30 μM C2 ceramide for 12 h. Typical morphologic changes of apoptotic cells are observed among normal fibroblasts (arrow). Magnifications: (a–h) × 40; (i) × 200.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Determination of apoptotic fibroblasts by the TUNEL method in vitro. (a, b) Normal fibroblasts treated with 30 and 50 μM C2 ceramide; (c, d) keloid fibroblasts treated with 30 and 50 μM C2 ceramide; (e) normal fibroblasts with no treatment; (f) normal fibroblasts treated with ethanol only; (g) normal fibroblasts treated with 50 μM dihydro-C2-ceramide. Typical apoptotic cells were observed in a concentration-dependent manner by C2 ceramide in normal fibroblasts, but only few TUNEL-positive cells were detected in keloid fibroblasts under the same conditions. Magnifications: ×100.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
DNA contents of normal and keloid fibroblasts after treatment with C2 ceramide. Cells were cultured with serum-free DMEM for 24 h and incubated with DMEM containing 25 μM C2 ceramide for 12 h. Following nuclear staining with PI, flow cytometric analysis was performed as described in Materials and Methods. (a, b) Normal fibroblasts; (c, d) keloid fibroblasts; (e, f) negative controls of normal fibroblasts. Note the increased population of apoptotic cells (48.61% ± 8.82%) in normal fibroblasts after 12 h. In contrast, keloid fibroblasts underwent a G2 arrest and resisted apoptosis (7.76% ± 3.48%).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Cell viability of normal and keloid fibroblasts following C2 ceramide treatment – dose-response. Each of three primary culture cell lines of normal and keloid fibroblasts seeded at 5 × 105 cells per well into 96-well plates were cultured with the indicated concentrations of C2 ceramide, and the cell viability was monitored by WST-1 assay as described in Materials and Methods. The dose-dependent effect of C2 ceramide on the cell viability of normal and keloid fibroblasts 12 h after incubation. Note that keloid fibroblasts were still resistant with the 40 μM concentration of C2 ceramide even though the same dose of C2 ceramide strongly abolished the cell viability of normal fibroblasts.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Cell viability of normal and keloid fibroblasts following C2 ceramide treatment – time course. Normal and keloid fibroblasts seeded at 5 × 105 cells per well into 96-well plates were cultured with the indicated concentrations of C2 ceramide, and the cell viability was monitored by WST-1 assay in a similar method to Figure 5. The time course of cell viability in cells treated with 0 μM, 30 μM, and 40 μM C2 ceramide. The viability of normal fibroblasts decreased in a time-dependent manner, whereas the reduction rate of viability was small in keloid fibroblasts.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

8. Figure 7
IGF-I rescues C2-ceramide-induced apoptosis of keloid fibroblasts but not of normal fibroblasts. Normal and keloid fibroblasts were cultured with DMEM containing various concentrations (0–50 μM) of C2 ceramide with or without 100 ng per ml IGF-I for 12 h, and the cell viability was studied by WST-1 assay. Incubation of keloid fibroblasts with IGF-I enhanced the resistance to C2-ceramide-induced apoptosis in keloid fibroblasts but not in normal fibroblasts.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

9. Figure 8
PI3-kinase is involved in IGF-I-mediated resistance to apoptosis in keloid fibroblasts. Keloid fibroblasts were cultured with DMEM containing 40 μM C2 ceramide with or without 100 ng per ml IGF-I, and the cell viability at the indicated time was monitored by WST-1 assay. Preincubation with 100 nM wortmannin for 30 min blocked IGF-I-mediated resistance of keloid fibroblasts against C2-ceramide-induced apoptosis.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions