Fig. 1. Expression of DGKα in human melanoma cells and melanocytes. (A) 70W, AKI, G361, MMAc, SK-mel-23 and SK-mel-118 human melanoma cells were harvested and the cell lysates (15 μg of protein) were analyzed by Western blotting using anti-DGKα antibody. Lysis buffer alone (–) was used as a control. Size marker is indicated on the left in kDa. (B) AKI human melanoma cells and NHEM were harvested and the cell lysates (15 μg of protein) were analyzed by Western blotting using anti-DGKα antibody (upper panel) or anti-actin antibody (lower panel). Size markers are indicated on the left in kDa. (C) Total RNAs of AKI human melanoma cells and NHEM were isolated and the aliquots (500 ng) were subjected to RT-PCR. Upper and lower panels in each set show the amplified 282- and 983-bp cDNA fragments of DGKα (30 cycles) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 25 cycles), respectively, in agarose gel electrophoresis.

Fig. 2. Effects of overexpression of DGKs α, β and γ on apoptosis in AKI melanoma cells. AKI melanoma cells were transfected with expression plasmids encoding GFP alone, GFP-DGKα-WT, GFP-DGKα-KD, GFP-DGKβ-WT or GFP-DGKγ-WT as indicated. After 24 h, cells were further incubated with or without 50 ng/ml of TNF-α for 24 h. (A and B) The cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Cell apoptosis was detected by TUNEL assay. Cells were examined using an inverted confocal laser scanning microscope (Zeiss LSM 510). A representative of three repeated experiments is shown (A). Bar = 100 μm. (B) Quantification of apoptosis of AKI cells. Random fields (ten fields per experiment) including (A) were analyzed, and TUNEL-positive cells were counted. For each experiment, > 1000 cells were counted. The results are presented as the percentage of the TUNEL-positive cell and represent the means ± S.D. of the values obtained in three separate experiments. Statistical significance was determined using the Student's t test. (C) After incubation with TNF-α, the cells were harvested, and cell lysates (10 μg of protein) were analyzed by Western blotting with anti-GFP antibody. Size markers are indicated on the left in kDa.

Fig. 3. Effects of DGKα-knockdown on apoptosis in AKI melanoma cells. AKI melanoma cells were transfected with or without siRNA targeting either DGKα or GFP (control) as indicated. After 24 h, cells were further incubated with or without 50 ng/ml of TNF-α. (A) After incubation with TNF-α for 24 h, the cells were harvested, and cell lysates (10 μg of protein) were analyzed by Western blotting with anti-DGKα antibody (upper panel) or anti-actin antibody (lower panel). The relative intensity of each band is shown below upper panel. A representative of three repeated experiments is shown. (B and C) After incubation with TNF-α for 24 h, the cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Cell apoptosis was detected by TUNEL assay. Cells were examined using an inverted confocal laser scanning microscope (Zeiss LSM 510). A representative of three repeated experiments is shown (B). Bar = 100 μm. (C) Quantification of apoptosis of AKI cells. Random fields (ten fields per experiment) including (B) were analyzed, and TUNEL-positive cells were counted. For each experiment, > 1000 cells were counted. The results are presented as the percentage of the TUNEL-positive cell and represent the means ± S.D. of the values obtained in three separate experiments. Statistical significance was determined using the Student's t test. (D) After incubation with TNF-α for 48 h, the cells were treated with RNase A, stained with propidium iodide (PI) and analyzed by FACS. Apoptotic cells fall in the sub-G1 gate. A representative of three repeated experiments is shown.

Fig. 4. Effects of DGKα-knockdown on apoptosis in SK-mel-118 melanoma cells. SK-mel-118 melanoma cells were transfected with siRNA targeting either DGKα or GFP (control) as indicated. After 24 h, cells were further incubated with or without 50 ng/ml of TNF-α for 24 h. (A) The cells were harvested, and cell lysates (10 μg of protein) were analyzed by Western blotting with anti-DGKα antibody (upper panel) or anti-actin antibody (lower panel). The relative intensity of each band is shown below upper panel. A representative of three repeated experiments is shown. (B and C) After incubation with TNF-α, the cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Cell apoptosis was detected by TUNEL assay. Cells were examined using an inverted confocal laser scanning microscope (Zeiss LSM 510). A representative of three repeated experiments is shown (B). Bar = 100 μm. (C) Quantification of apoptosis of SK-mel-118 cells. Random fields (ten fields per experiment) including (B) were analyzed, and TUNEL-positive cells were counted. For each experiment, > 500 cells were counted. The results are presented as the percentage of the TUNEL-positive cell and represent the means ± S.D. of the values obtained in three separate experiments. Statistical significance was determined using the Student's t test.

Fig. 5. Effects of overexpression and knockdown of DGKα on Akt, ERK and NF-κB activities in AKI melanoma cells. (A and B) AKI melanoma cells were transfected with siRNA targeting either DGKα or GFP (control) as indicated. After 24 h, cells were further incubated with or without 50 ng/ml of TNF-α. After incubation with TNF-α for 24 h, the cells were harvested, and cell lysates (10 μg of protein) were analyzed by Western blotting with anti-phospho-Akt antibody (p-Akt), anti-Akt antibody (Akt), anti-phospho-ERK antibody (p-ERK) or anti-ERK antibody (ERK) as indicated. (C) AKI melanoma cells were co-transfected with pNF-κB-Luc Vector, pSV-β-Galactosidase Control Vector, and with either pEGFP, pEGFP-DGKα-WT or pEGFP-DGKα-KD as indicated. After 24 h, cells were further incubated with or without 50 ng/ml of TNF-α for 12 h. Luciferase activity was measured and was normalized to β-galactosidase activity. The results are presented as the percentage of the value of GFP alone (TNF-α (–)) and represent the means ± S.D. of the values obtained in three separate experiments. Statistical significance was determined using the Student's t test. (D) AKI melanoma cells were co-transfected with pNF-κB-Luc Vector, pSV-β-Galactosidase Control Vector, and with siRNA targeting either DGKα or GFP (control) as indicated. After 24 h, cells were further incubated with or without 50 ng/ml of TNF-α for 12 h. Luciferase activity was measured and was normalized to β-galactosidase activity. The results are presented as the percentage of the value of control siRNA (TNF-α (–)) and represent the means ± S.D. of the values obtained in three separate experiments. Statistical significance was determined using the Student's t test.

Fig. 6. Effects of overexpression and knockdown of DGKα on nuclear localization of NF-κB in AKI melanoma cells. (A and B) AKI melanoma cells were transfected with expression plasmids encoding GFP alone, GFP-DGKα-WT or GFP-DGKα-KD as indicated. After 24 h, cells were further incubated with or without 50 ng/ml of TNF-α for 12 h. A, The cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100. NF-κB was labeled with anti-NF-κB p65 antibody and Alexa Fluor 594-conjugated secondary antibody. Cells were examined using an inverted confocal laser scanning microscope (Zeiss LSM 510). A representative of three repeated experiments is shown (A). Bar = 10 μm. (B) The nucleus/cytoplasm (N/C) ratio of NF-κB (fluorescence intensity) was quantified using Image J software. For each experiment, > 20 cells were analyzed. The results represent the means ± S.D. of the values obtained in three separate experiments. Statistical significance was determined using the Student's t test. (C and D) AKI melanoma cells were transfected with siRNA targeting DGKα or GFP (control) as indicated. After 24 h, cells were further incubated with or without 50 ng/ml of TNF-α for 12 h. The cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100. NF-κB was labeled with anti-NF-κB p65 antibody and Alexa Fluor 594-conjugated secondary antibody. Cells were examined using an inverted confocal laser scanning microscope (Zeiss LSM 510). A representative of three repeated experiments is shown (C). Bar = 10 μm. (D) The N/C ratio of NF-κB (fluorescence intensity) was quantified using Image J software. For each experiment, > 20 cells were analyzed. The results represent the means ± S.D. of the values obtained in three separate experiments. Statistical significance was determined using the Student's t test. (E) AKI melanoma cells were transfected with siRNA targeting DGKα or GFP (control) as indicated. After 24 h, cells were further incubated with 50 ng/ml of TNF-α for 0.5, 2 and 12 h as indicated. Cytosol (C), membrane/organelle (M), nuclear (N) and cytoskeletal (CS) fractions were isolated using the ProteoExtract subcellular proteome extraction kit. Each fraction (10 μl) was analyzed by Western blotting with anti-NF-κB p65 antibody. The blots are quantified and relative intensities of the bands are shown below each panel. A representative result of two repeated experiments is shown.

Fig. 7. Effects of NF-κB inhibitor on apoptosis and NF-κB activity enhanced by DGKα overexpression in AKI melanoma cells. (A) AKI melanoma cells were co-transfected with pNF-κB-Luc Vector, pSV-β-Galactosidase Control Vector, and either pEGFP or pEGFP-DGKα-WT as indicated. After 23 h, cells were further incubated with or without 5 μM of MG-132 for 1 h. Then, cells were incubated with 50 ng/ml of TNF-α for 12 h. Luciferase activity was measured and was normalized to β-galactosidase activity. The results are presented as the percentage of the value of GFP alone (MG-132 (–)) and represent the means ± S.D. of the values obtained in three separate experiments. (B and C) AKI melanoma cells were transfected with expression plasmids encoding GFP alone or GFP-DGKα-WT as indicated. After 23 h, cells were further incubated with or without 5 μM of MG-132 for 1 h. Then, cells were incubated with 50 ng/ml of TNF-α for 24 h. The cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Cell apoptosis was detected by TUNEL assay. Cells were examined using an inverted confocal laser scanning microscope (Zeiss LSM 510). A representative of three repeated experiments is shown (B). Bar = 100 μm. (C) Random fields (ten fields per experiment) including (B) were analyzed, and TUNEL-positive cells were counted. For each experiment, > 500 cells were counted. The results are presented as the percentage of the TUNEL-positive cell and represent the means ± S.D. of the values obtained in three separate experiments.

Expression of Type I DGKs in human melanoma cells

70W, AKI, G361, MMAc, SK-mel-23 and SK-mel-118 human melanoma cells were harvested, and the cell lysates (15 μg of protein) were analyzed by Western blotting using anti-DGKs antibodies. Moreover, total RNAs were isolated and the aliquots (500 ng) were subjected to RT-PCR. +: specific bands were detected; N.D.: not detected.