Ceramide-up-regulated Txnip interacts with thioredoxin and reduces its activity. (A) Jurkat T cells were treated with 20 μM C2-ceramide or 25 μM etoposide for 6 hours, and 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 colocalization of Txnip and thioredoxin was visualized (Merge; yellow). (B) In vector control– and hTxnip siRNA-transfected Jurkat T cells, Txnip protein expression was detected using Western blot analysis. The GAPDH expression was used as an internal control. The relative ratio of Txnip-GAPDH is shown. The thioredoxin activity was detected using an insulin reduction assay after 20 μM C2-ceramide treatment for 12 hours. Results are shown as means plus or minus SD of triplicate cultures. The vector control group without ceramide treatment was normalized as 100%. (***P < .001).
