1. De Novo Ceramide Synthesis Participates in the Ultraviolet B Irradiation-Induced Apoptosis in Undifferentiated Cultured Human Keratinocytes 
Yoshikazu Uchida, Anna Di Nardo, Vanessa Collins, Peter M. Elias, Walter M. Holleran 
Journal of Investigative Dermatology 
Volume 120, Issue 4, Pages (April 2003)
DOI: /j x
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
UVB irradiation inhibits DNA synthesis of CHK. CHK were incubated for 24h after a single dose of UVB (0–100mJ per cm2) (panel A), or for 2–24h after a single UVB dose (60mJ per cm2) (panel B). Data are expressed as a percentage (mean±SEM) of sham-irradiated control cells. The inhibitory effect of a 60mJ per cm2 dose was evident by 2h after treatment (panel B), and doses ≥60mJ per cm2 induced complete inhibition of CHK proliferation at 24h (panel A); *p<0.001 for all time points (2–24 h) vs sham-irradiated control cells; n=12.
Journal of Investigative Dermatology  , DOI: ( /j x)
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3. Figure 2
UVB irradiation-induced apoptosis in CHK. CHK were incubated for 24 or 48h following single UVB exposure (60mJ per cm2). Apoptosis was assessed by TUNEL staining (panel A); by trypan blue dye exclusion assay (panel B); and by caspase-3 assay (panel C). TUNEL- and trypan blue-positive cells are reported as a percentage of total cells; caspase activity is expressed as pmol per min per mg protein. In each case, data are reported as mean±SEM (panels A,B, n=6; panel C, n=3). TUNEL-positive and trypan blue-positive cells were evident by 24h, and further increased at 48h (panels A,B), whereas caspase-3 activity was increased at 16h, and remained elevated at 24h (panel C); *p<0.001 and **p<0.01 vs sham-irradiated control cells, respectively.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
UVB irradiation increased ceramide content of CHK (60mJ per cm2). Data for ceramide (closed circles), glucosylceramide (open rhombus), and sphingomyelin (open circles) content are expressed as a percentage (mean±SEM) of sham-irradiated controls. UVB induced a significant increase in total ceramide content at 8 and 16h. Increased glucosylceramide content was evident at 8h, whereas sphingomyelin content remained unchanged throughout the treatment period; *p<0.02, **p<0.01, and ***p<0.001 each vs sham-irradiated control values; n=4.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
UVB irradiation increases de novo ceramide and glucosylceramide, but not sphingomyelin, synthesis. CHK were incubated with [14C]palmitate for 8h following UVB irradiation (60mJ per cm2). Fumonisin B1 (FB1) was added immediately after UVB or sham treatment. Data are expressed as a percentage (mean±SEM) of sham-irradiated controls. The UVB-induced increases in ceramide and glucosylceramide synthesis (*p<0.005) were significantly inhibited by fumonisin B1; **p<0.01 vs UVB treated cells without fumonisin B1; n=4.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Both fumonisin B1 and ISP-1 decreased UVB-induced apoptosis in CHK. CHK were irradiated (60mJ per cm2) and incubated with or without inhibitors for 48h. Cell viability/apoptosis were assessed by trypan blue dye exclusion (cf. Fig 2). Data are expressed as a percentage (mean±SEM) of sham control values (note expanded axis). Both fumonisin B1 (50μM) and ISP 1 (≥0.05μM) significantly attenuated the deleterious effect(s) of UVB on CHK viability; *p<0.01 vs UVB-treated cells without inhibitor(s); n≥3.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Co-administration of fumonisin B1 and caspase-3 inhibitor further decreased UVB-induced apoptosis in CHK. CHK were irradiated (60mJ per cm2) and incubated with or without fumonisin B1 (50μM) for 42 h; the caspase inhibitor, Ac-DEVD-CMK, was added at the doses indicated (i.e., 62.5 or 125μM) 2h after irradiation. Cell viability was assessed by trypan blue exclusion (as above), and reported as a percentage (mean±SEM) of non-UVB-treated sham control values (note expanded axis). Fumonisin B1 again significantly attenuates the UVB-induced decrease in cell viability. Although the effect of the caspase-3 inhibitor alone did not reach statistical significance, the combination of fumonisin B1 and Ac-DEVD-CMK showed further attenuation of the UVB-induced cell death; *p<0.01 vs UVB-treated cells without inhibitors (i.e., 0/0 value).
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
Proposed pathway of ceramide-induced apoptosis in CHK following UVB irradiation.
Journal of Investigative Dermatology  , DOI: ( /j x)
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