JY and MS1418 cells (2.3 × 10⁶) were grown in a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 15% FBS. The cells were starved for 12 h in medium containing 0.5% FBS and then irradiated with UVA (80 kJ/m²), UVB (8 kJ/m²), or UVC (60 J/m²). After lysis of cells, protein concentration and acid SMase activity in supernatant fractions were measured as described under “Materials and Methods.” The figure shows the relative fluorescence units in JY and MS1418 cells normalized to mg of cellular protein. The value for control cells was subtracted from each sample. Each bar indicates the mean and S.D. from four assay samples.

JY or MS1418 cells (3 × 10⁶) were starved for 12 h in medium containing 0.5% FBS and then irradiated with UVA, UVB, or UVC as described in Fig. 1. Extraction of lipids and analysis of ceramides were performed as described under “Materials and Methods.” The ceramide level was normalized to cell number and is shown as % of control (value of 100). The figure shows that ceramide levels increase in UVA-, UVB-, or UVC-irradiated JY cells but not in MS1418 cells following irradiation. Each point represents the mean from triplicate incubations.

JY and MS1418 cells (3 × 10⁶) were grown for 24 h in a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 15% FBS and then irradiated with UVA, UVB, or UVC at the doses indicated. Following irradiation, the cells were harvested at the time points indicated. Fragmented DNA in supernatant fractions was extracted and separated after lysis and centrifugation, and quantitative analysis of apoptotic cells was determined by DNA laddering analysis (A and B) and Hoechst staining (C) as described under “Materials and Methods.” The upper (A) and lower (C) panels show that the dose-dependent apoptosis induced by UVA occurs in JY cells but not in MS1418 cells. On the other hand, a similar level of apoptosis induced by UVB and UVC occurs in both cell types. B, the middle left panel shows that apoptosis induced by UVA, UVB, or UVC is time-dependent in JY cells. The lower right panel shows that only apoptosis induced by UVB or UVC is time-dependent, and UVA-induced apoptosis is less significant in MS1418 cells.

JY and MS1418 cells (3 × 10⁶) were cultured in a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 15% FBS and then were treated for 14 h with C²-ceramide, C₂-dihydroceramide, SM, or acid SMase at the doses indicated. Fragmented DNA was extracted and separated as described under “Materials and Methods.” A, the upper two panels show that C²-ceramide but not C₂-dihydroceramide can induce apoptosis in both JY and MS1418 cells. B, the lower two panels show that SM only induces apoptosis in normal JY cells (lower left panel) and not in SMase-deficient MS1418 cells (lower right panel). On the other hand, addition of exogenous SMase induces apoptosis in both cell types (lower two panels).

JY or MS1418 cells (3 × 10⁶) were starved for 24 h in a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 0.5% FBS. Cells were irradiated with UVA (80 kJ/m²), UVB (8 kJ/m²), or UVC (60 J/m²) and then harvested by centrifugation at time points as indicated. After lysis and sonication, samples containing equal amounts of protein were separated by 8% SDS-PAGE and analyzed by Western immunoblotting using anti-phospho-JNKs antibody as described under “Materials and Methods.” Nonirradiated cells were used as a negative control. A, The figure shows that phosphorylation of JNKs was induced in UVA-, UVB-, or UVC-irradiated JY cells but almost not in MS1418 cells. B, intensity of bands was quantitated with Image-QuanNT™ software. Each value indicates the mean and S.D. from three independent experiments and is expressed as percentage of increase in phosphorylation compared with control.

JY or MS1418 cells (3 × 10⁶) were starved for 24 h in a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 0.5% FBS. Cells were irradiated with UVA (80 kJ/m²), UVB (8 kJ/m²), or UVC (60 J/m²) and then harvested by centrifugation at time points as indicated. After lysis and sonication, samples containing equal amounts of protein were separated by 8% SDS-PAGE and analyzed by Western immunoblotting using anti-phospho-ERKs antibody as described under “Materials and Methods.” Nonirradiated cells were used as a negative control. A shows that UVA–, UVB-, or UVC-induced phosphorylation of ERKs was induced in MS1418 cells but was markedly less in JY cells. B, intensity of bands was quantitated with Image-QuanNT™ software. Each value indicates the mean and S.D. from three independent experiments and is expressed as percentage of increase in phosphorylation compared with control.

JY or MS1418 cells (3 × 10⁶) were starved for 24 h in a mixture of RPMI 1640 and DMEM (1:1, v/v) containing 0.5% FBS. Cells were irradiated with UVA (80 kJ/m²), UVB (8 kJ/m²), or UVC (60 J/m²) and then harvested by centrifugation at time points as indicated. After lysis and sonication, samples containing equal amounts of protein were separated by 8% SDS-PAGE and analyzed by Western immunoblotting using anti-phospho-p38 kinase antibody as described under “Materials and Methods.” Nonirradiated cells were used as a negative control. A shows that phosphorylation of p38 kinase was induced in UVA, UVB, or UVC-irradiated JY and MS1418 cells, and the phosphorylation by UVA but not UVB or UVC was less in JY cells. B, intensity of bands was quantitated with Image-QuanNT™ software. Each value indicates the mean and S.D. from three independent experiments and is expressed as percentage of increase in phosphorylation compared with control.

JY or MS1418 cells (3 × 10⁶) were pretreated for 1.5 h with Me₂SO as control, or with PD98059 or SB202190 and then irradiated with UVA (80 kJ/m²). Twelve hours after irradiation, cells were harvested by centrifugation. Fragmented DNA was extracted and separated as described under “Materials and Methods.” Nonirradiated cells were used as a negative control, and cells treated only with UVA irradiation were the positive control. The figure shows that PD98059 and SB202190 do not inhibit UVA-induced apoptosis in JY cells (left panel), and UVA-induced apoptosis occurs after pretreatment of MS1418 with PD98059 or SB202190 (right panel).

Primary embryonic fibroblasts were prepared from wild-type and jnk1−/− and jnk2−/− knockout mice. The cells were starved for 12 h in serum-free DMEM and then irradiated with UVA (80 kJ/m²). A, the cells were harvested at indicated times following irradiation. Then acid SMase activities in supernatant fractions were determined as described in Fig. 1, except that the fluorescence intensity was measured with a Lab-systems Fluoroskan (Franklin, MA) with filter combination 527 nm (excitation) and 620 nm (emission). The UVA-induced SMase activity was normalized to nonirradiated control (value of 100) and is shown as percent. The data are representative of three independent experiments, and each bar represents the mean and S.D. from triplicate samples. B, 14 h following irradiation, detached and attached cells were harvested by scraping and centrifuging. Fragmented DNA in supernatant fractions was extracted and separated as described under “Materials and Methods.” Nonirradiated cells were used as negative control. The figures show that induction of acid SMase activity by UVA occurs (A) but UVA-induced apoptosis is completely blocked (B) in jnk1−/− and jnk2−/− cells.