Figure 4

c-Jun was directly involved in LC3 transcription in response to ceramide treatment. (A) CNE2 cells were treated with 20 μM ceramide for 24 h in the absence or presence of c-Jun siRNA. Then LC3, c-Jun or phospho-c-Jun protein expression were analyzed with immunoblotting. LC3 mRNA was examined by RT-PCR analysis. Pepstatin A is an inhibitor of acid proteases (aspartyl peptidases). (B) Sequential analysis of the core region of LC3 promoter was analyzed by TESS. CNE2 cells were treated with or without ceramide 20 μM for 12 h, then ChIP analysis was to detect the binding of c-Jun to LC3 promoter in vivo according to the manufacturer's instructionand. (C) CNE2 cells were transfected with LC3 (-1993/+7)-luc or LC3(-1993/+7)-MUT-luc; empty vector was co-transfected as negative control; PCMV-RL was co-transfected as internal control. 16 h after transfection, the cells were treated with or without ceramide 20 μM for 12 h. Luciferase activities were detected as described in Material and Methods. (p < 0.05)