Research Paper Volume 14, Issue 19 pp 7662—7691

Figure 4. Lotus germ extract activated the JNK and DAPK1-Beclin1 pathways. (A) Lotus germ extract phosphorylated JNK at an early time point. Aging NB1RGB cells were treated with 50 μg/mL lotus germ extract for the indicated times and subjected to immunoblotting using the indicated antibodies. (B) Lotus germ extract inhibited the Beclin1 and BcL-2 interaction via Beclin1 phosphorylation. Aging NB1RGB cells were treated with DMSO (−) or 50 μg/mL lotus germ extract (+) for 24 h, and proteins were crosslinked with DSP prior to protein extraction. A coimmunoprecipitation assay was performed with cell lysates using the indicated antibodies, followed by western blotting. (C) Lotus germ extract induced DAPK1 expression. Aging NB1RGB cells were treated with DMSO (−) or 50 μg/mL lotus germ extract (+) for 24 h and subjected to real-time quantitative PCR. (D–F) DAPK1 plays an important role in the effects of lotus germ extract. Aging NB1RGB cells were treated with DMSO (−) or 10 μM TC-DAPK6 and/or 50 μg/mL lotus germ extract for 24 h and subjected to immunoblotting using the indicated antibodies (D), coimmunoprecipitation assay (E) or JC-1 activity measurementṣ (F). (G–J) The overexpression of DAPK1 stimulates the mitochondrial activity in aging fibroblast cells. Aging fibroblast cells transfected with pcDNA3.1 or pcDNA3.1-DAPK1 for 48 h were subjected to immunoblotting using the indicated antibodies (G), SA-β-gal activity measurement (H), TMRM activity measurement (I), or collagen production (J). Data are presented as the mean ± SD of three simultaneously performed experiments (C, F, H–J). Each P value was calculated using two-way ANOVA; n.s.: not significant, ^*P < 0.05, ^**P < 0.01.