1. Hydrolytic Pathway Protects against Ceramide-Induced Apoptosis in Keratinocytes Exposed to UVB 
Yoshikazu Uchida, Evi Houben, Kyungho Park, Sounthala Douangpanya, Yong-Moon Lee, Bill X. Wu, Yusuf A. Hannun, Norman S. Radin, Peter M. Elias, Walter M. Holleran 
Journal of Investigative Dermatology 
Volume 130, Issue 10, Pages (October 2010)
DOI: /jid
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2. Figure 1
Apoptosis is only evident in parallel with sustained elevation of ceramide (Cer) in high UVB (H-UVB)-treated cultured human keratinocytes (CHK), whereas inhibition of ceramidase (Cdase) potentiates UVB-induced apoptosis. CHK were treated with low UVB (L-UVB) (30mJcm−2) or H-UVB (60mJcm−2). (a) Apoptosis was assessed by TUNEL staining and poly(ADP-ribose) polymerase cleavage (western immunoblot analysis). Cell lysates were combined from five to seven individual treatment groups. MW, molecular markers. (b) Cer synthesis was assessed by [3H]palmitate incorporation into Cer fraction. (c) Cer content. Similar results were obtained when the experiment was repeated (twice) using different cell preparations (a-c). (b and c) Mean±SD from three independent treatments. Bar=50μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Blockade of ceramidase (CDase) activities sensitizes cultured human keratinocytes (CHK) to UVB-induced apoptosis. (a) Low-UVB (L-UVB)-treated CHK were incubated for 8hours with or without the CDase inhibitor, N-oleoylethanolamine (NOE) (20μM in DMSO). (b and c) Apoptosis in CHK transfected with small interfering RNA (siRNA) for acidic aCDase (aCDase) and/or neutral CDase (nCDase) was assessed by lactate dehydrogenase release assay and/or poly(ADP-ribose) polymerase cleavage 24hours after UVB irradiation. (b) *P<0.01 versus. sham- or L-UVB-irradiated cells or sham-irradiated-random-siRNA-transfected cells; **P<0.01 versus sham-irradiated-aCDase and/or nCDase-siRNA-transfected cells. Samples that were not originally run next to each other were juxtaposed in the Figure. (d) Cer production was determined 24hours after UVB irradiation. n=4–6, mean±SD. Similar results were obtained when the experiment was repeated (a–d).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Alterations of mRNA and protein levels of neutral ceramidase (nCDase) or acidic ceramidase (aCDase) after UVB. (a) Expression levels for ceramidase (CDase) mRNAs in cultured human keratinocytes (CHK) were determined by quantitative reverse transcriptase-PCR; n=3; *P<0.01 versus respective sham-irradiated cells. (b) Western immunoblots: Cell lysates were combined from six individual treatment groups for sham, low UVB (L-UVB) and high UVB (H-UVB). The experiments were repeated twice. The duplicate lanes for aCDase were from two separate experiments. The third experiment (not shown) yielded similar results. Two repeat experiments for nCDase also gave results similar to those shown here. (c) The intensity of aCDase or nCDase was normalized to β-actin expression, and then compared with sham-irradiated controls. Relative protein expression levels of aCDase or nCDase were determined vs. those of sham-irradiated control cells (set to 100%). Mean±SD from three independent experiments; *P<0.02 versus respective sham-irradiated cells.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Inhibition of sphingosine kinase 1 (SPHK1) (but not SPHK2) potentiates cultured human keratinocyte sensitivity to UVB-induced apoptosis. CHK were treated with low UVB (L-UVB) or high UVB (H-UVB), and then incubated for 8hours. Cells were incubated with SPHK inhibitor dimethylsphingosine (DMS) (2.5μM), or with vehicle alone (DMSO) (a), whereas SPHK1-, SPHK2-, or random small interfering RNA (siRNA) were introduced into cells before UVB treatment (b). Cell viability was assessed by dehydrogenase activity after 24hours treatment; n=3; *P<0.01, versus sham-irradiated cells; **P<0.01 versus UVB-irradiated, vehicle control cells; ***P<0.01 versus sham-irradiated-, UVB-irradiated random-siRNA-transfected cells. Mean±SD.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Proposed mechanism for ceramide (Cer) production and hydrolysis that determines cell survival or apoptosis in keratinocytes after UVB irradiation. Ceramidase (CDase) is an essential step for initiating protective mechanisms, that is, generation of nonapoptotic metabolites, sphingosine-1-phosphate (S1P), as well as regeneration of glucosylceramide (GlcCer), but unlikely sphingomyelin (SM), to protect against Cer-induced apoptosis.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions