Figure 2. Overproduction of Lass6 induces ceramide synthesis in mammalian cells.

(A) Lass6 enhances ceramide synthesis in cultured cells. HEK 293T cells were transfected with pcDNA3 (vector) or pcDNA3-HA-Lass6 (Lass6) and incubated for 4 h in medium containing 1 μCi of [³H]dihydrosphingosine in the presence or absence of FB₁ (20 μM). Lipids were extracted and separated by TLC. (B) Sensitivity to FB₁ of Lass6 activity in vitro. Total cell lysates were prepared from HA–Lass6-overproducing HEK 293T cells or from cells transfected with control vector, and preincubated for 1 h on ice with (+) or without (−) FB₁ (20 μM). A dihydroceramide synthase assay was performed by incubating 25 μM C_16:0-CoA, 5 μM dihydrosphingosine (including [³H]dihydrosphingosine) and cell lysates from control (40 μg of protein) or HA–Lass6-overproducing (20 μg of protein) cells for 15 min at 37 °C. Results shown are means±S.D. from three independent experiments. (C) Lass6 is localized in the ER. CHO cells transfected with pcDNA3-HA-Lass6 were immunostained with anti-HA (left panel) and anti-KDEL (right panel) antibodies. Bar, 10 μm.