Research Paper Volume 14, Issue 19 pp 7662—7691

Lotus germ extract rejuvenates aging fibroblasts via restoration of disrupted proteostasis by the induction of autophagy

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.