Figure 5. Regulation of ROS and recycling pathway by TPx I.

A549 cells were transfected with vector (PCR3.1) or TPXI. (A) The mRNA levels of TPx I were measured by RT–PCR, showing the overexpression of TPXI in transfected A549 cells (lane 2) compared with vector-transfected controls (lane 1). (B) Control vector- and TPx I-transfected cells were grown with or without 20 μM C₆-cer in the presence and absence of 1 nM TNF-α, and the levels of ROS were measured using DCFH-DA. (C) The same treatments as in (B) were applied in the presence of 1 μCi/ml [³H]palmitate for 2 h, and long-chain ceramide formation was measured by TLC. (D) Radioactivity was measured in ceramide bands from the TLC plate shown in (C). The results shown are means±S.D. and are representative of three independent experiments performed in duplicate. * and #, P<0.05 compared with untreated or ceramide (alone)-treated controls respectively.