FIGURE 8.

CERK is required for the activation of JNK and cPLA₂α and for LD biogenesis. CHO-K1 cells were transfected with three siRNA-CERK duplexes as described under “Experimental Procedures” and maintained in 7.5% FBS-containing medium during 72 h and then in medium without FBS for 24 h. During these last 24 h, cells were prelabeled with [³H]palmitate or with [³H]AA to monitor synthesis of Cer-1-P and cPLA₂α activity, respectively (B and C). A, Western blots showing that siRNA-CERK1 and siRNA-CERK3 knocked down the expression of CERK, precluded phosphorylation of JNK and cPLA₂α induced by a 6-h treatment with FBS, and decreased ADRP content. B, [³H]palmitate-prelabeled cells were stimulated with FBS during 15 min. Lipid extracts were separated by TLC, and areas co-migrating with Cer-1-P standard were scraped onto vials and counted. C, [³H]AA-prelabeled cells were stimulated with FBS during 15 min, and radioactivity in the medium was counted. D, cells were stimulated with FBS during 6 h, fixed, and stained with Nile red, and LD were quantified by flow cytometry. Results in C and D are means ± S.E. of three experiments. Results in B are means ± range of two experiments. *, significantly different (p < 0.01) from controls. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.