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Blood, Vol. 111, Issue 8, 4365-4374, April 15, 2008
Ceramide induces p38 MAPK and JNK activation through a mechanism involving a thioredoxin-interacting protein-mediated pathway
Blood Chen et al.
111: 4365
Supplemental materials for: Chen et al
Files in this Data Supplement:
- Figure S1. Ceramide-upregulated Txnip interacts with thioredoxin and reduces its activity (JPG, 99.3 KB)
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(A) 10I cells were treated with 40 µM C2-ceramide for 6 hours, the interaction of Txnip with thioredoxin was determined by confocal microscopic observation. After fixation with 1% formaldehyde in PBS, cells were incubated with Txnip and thioredoxin antibodies followed by FITC-labeled (Txnip-FITC, green) and TRITC-labeled (Trx-TRITC, red) secondary antibodies, respectively. The co-localization of Txnip and thioredoxin was visualized (Merge, yellow). (B) Thioredoxin activity was detected using insulin reduction assay after various doses of C2-ceramide treatment. Results are shown as means ± SD of triplicate cultures. The untreated group was normalized as 100% (***, P < .001).
- Figure S2. ASK1 activation is involved in ceramide stimulation (JPG, 86.3 KB)
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10I cells were treated with 20 µM C2-ceramide for different time periods as indicated. The expression of phospho-ASK1 at serine 83 (S83 pASK1) and at threonine 845 (T845 pASK1), and ASK1 were determined using Western blot analysis. The relative ratio of pASK1:ASK1 is shown. The expression of  -tubulin was an internal control.
- Figure S3. Txnip regulates ASK1-medidated apoptosis under ceramide stimulation (JPG, 370 KB)
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(A) Vector control- and hTxnip siRNA-transfected Jurkat T cells were treated with 20 µM C2-ceramide for 6 hours and the expression of T845 pASK1 was measured by flow cytometric analysis. Representative histograms and the percentages of positive cells are shown as means ± SD of triplicate cultures. R1, transfection-negative; R2, transfection-positive (GFP as a marker). (B) Vector control- and ASK1 siRNA-transfected Jurkat T cells were treated with 20 µM C2-ceramide for 24 hours and the cell apoptosis was measured by annexin V staining. Representative histograms and the percentages of apoptotic cells are shown as means ± SD of triplicate cultures. R1, transfection-negative; R2, transfection-positive.
- Figure S4. Ceramide causes transcription-regulated p38 MAPK and JNK activation, and p38 MAPK- and JNK-mediated apoptosis (JPG, 473 KB)
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(A) 10I cells were treated with C2-ceramide for different time periods as indicated. The expression of phospho-p38 MAPK (pp38 MAPK) at threonine 180/tyrosine 182, p38 MAPK, phospho-JNK (pJNK) at threoine 183/tyrosine 185, JNK, Txnip, and thioredoxin were determined using Western blot analysis. The expression of -tubulin was an internal control. The relative ratios of pp38 MAPK:p38 MAPK, pJNK:JNK, Txnip:-tubulin and thioredoxin:-tubulin are shown. (B) For confocal microscopic observation, cells treated with 20 µM C2-ceramide for 12 hours with or without actinomycin D (0.15 µg/mL) pretreatment were fixed and then incubated with pp38 MAPK (left) or pJNK (right) antibodies followed by FITC-labeled (green) secondary antibody staining. Nuclear staining was performed using PI staining (red). (C) The expression of pp38 MAPK (filled bar) or pJNK (open bar) in cells treated with C2-ceramide for 12 hours with or without actinomycin D (0.15 µg/mL) pretreatment were detected using flow cytometric analysis after staining with pp38 MAPK or pJNK specific antibodies. The percentages of positive cells are shown (means ± SD of triplicate cultures). (D) The pp38 MAPK at threonine 180/tyrosine 182, p38 MAPK, pJNK at threoine 183/tyrosine 185, JNK, Txnip, and -tubulin expression were determined after C2-ceramide treatment with or without actinomycin D (0.15 µg/mL) using Western blot analysis. The relative ratios of pp38 MAPK:p38 MAPK, pJNK:JNK and Txnip:-tubulin are shown. (E and F) Vector control- and ASK1 siRNA-transfected Jurkat T cells were treated with 20 µM C2-ceramide for 6 hours and the expression of pp38 MAPK and pJNK were determined using flow cytometric analysis. Representative histograms and the percentages of positive cells are shown and means ± SD were obtained from triplicate cultures. R1, transfection-negative; R2, transfection-positive. (G) 10I cells were pretreated with SB203580 or SP600125 for 1 hour followed by C2-ceramide treatment for 12 hours. Apoptotic cells were detected using PI staining followed by flow cytometric analysis. The percentages of apoptotic cells are shown (means ± SD of triplicate cultures; ***, P < .001). (H and I) Jurkat T cells were transfected with EGFP-vector control or EGFP-p38 MAPK or -JNK siRNA and 24 hours later, cells were treated with or without C2-ceramide (20 µM) for 24 hours. The percentages of apoptotic cells were determined by PE-conjugated annexin V staining using flow cytometric analysis. Representative histograms and the percentages of apoptotic cells are shown and means ± SD were obtained from triplicate cultures. R1, transfection-negative; R2, transfection-positive.
- Figure S5. Ceramide and etoposide induce ER stress-related proteins Bip, CHOP, caspase-4 and PERK activation (JPG, 113 KB)
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Jurkat T cells were treated with 20 µM C2-ceramide or 25 µM etoposide for different time periods as indicated. The expression of Bip, CHOP, cleaved caspase-4, phospho-PERK at threonine 981 and PERK were detected using Western blot analysis. Cells treated with 5 µg/ml of tunicamycin (TM) were used for the positive control. Untreated cells (Un) were collected as protein basal expression control. The  -actin expression was used as an internal control. The relative ratios of Bip:-actin, CHOP:-actin, cleaved caspase-4:-actin, and pPERK:PERK are shown.
- Figure S6. Down-regulation of Txnip reduces ceramide-induced p38 MAPK and JNK phosphorylation and cell apoptosis (JPG, 315 KB)
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(A) Jurkat T cells were transfected with EGFP-vector control or hTxnip siRNA and 24 hours later, cells were treated with or without C2-ceramide (20 µM) for 24 hours. The percentages of apoptotic cells were determined by PE-conjugated annexin V staining. (B and C) For the pp38 MAPK and pJNK detection in ceramide-treated (20 µM C2-ceramide for 6 hours) vector control- and hTxnip siRNA-transfected cells, pp38 MAPK and pJNK antibodies were used followed by rhodamine-conjugated secondary antibody staining and flow cytometric analysis. Representative histograms and the percentages of positive cells are shown and means ± SD were obtained from triplicate cultures. R1, transfection-negative; R2, transfection-positive.
- Table S1. Microarray data (XLS, 6.66 MB)
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