Beclin1 is known to play a crucial role in autophagosome initiation and maturation. 20 Up-regulated LC3-II and down-regulated P62 represent a high-level autophagy flux or active autophagy process. degeneration of the K?llikers organ prior to apoptosis during the early postnatal development. and were the target genes to assess and was used as the endogenous reference. The primers were listed on Table 8-Dehydrocholesterol 1. The coefficient of variation (CV) values of the target genes and endogenous reference were calculated. The expression levels of mRNAs were calculated by the 2-DDCT method. Table 1. Primers for quantitative real time-PCR. 8-Dehydrocholesterol P7), and decreased again at P14 (P 0.01). The expression level of P62 was gradually increased from P1 to P10 (Figure 5 A,?,C;C; *P 0.05, **P 0.01), but decreased significantly at P14 (P 0.05 P14 P10). Beclin 1 expression showed no significant difference in p3 to p7, but p10 to p14 showed a decreasing trend (Figure 5D; *P 0.05). These changes indicated that the autophagy flux was declined over time. Open in a separate window Figure 5. Western blot analysis of autophagy and apoptosis related proteins in the K?llikers organ. A) Blots of LC3-II, P62, Beclin1 and -actin at different developmental stages. B-D) Half quantitative analysis of LC3-II, P62 and Beclin1 expression levels. E) Blots of Caspase3, cleaved-Caspase3, Bcl2, and GAPDH at different developmental stages. F-G) Half F2RL1 quantitative analysis of Caspase-3, cleaved- Caspase3 and Bcl2. Repeated measures ANOVA was used. *P 0.05; **P 0.01. mRNA levels of autophagy associated genes in the K?llikers organ during postnatal development The mRNA expression levels of were evaluated in the K?llikers organ at different development stages (Figure 6). Consistent with the overall protein expression pattern, mRNA expression of was declined at P3, (Figure 6A; *P 0.05 P3 vs P1), remained at low level from P3 to P10, and sharply declined again at P14 (P 0.05). The expression level of increased significantly from P1 to P7 (Figure 6B; *P 0.05, **P 0.01), then significantly declined at P10 (P 0.05). There was no difference between P10 and P14. was increased from P1 to P5 (Figure 6C; *P 0.05, **P 0.01), then declined from P7 to P10 (P 0.05). There was also no difference between P10 and P14. Open in a separate window Figure 6. Real-time PCR analysis of the mRNA expression of and in the K?llikers organ. The K?llikers organ samples from 12 cochleae at different developmental stages were assessed. Data from triplicate samples were calculated by the 2-CT method, normalized to P1 levels and presented as mean SEM. Repeated measures ANOVA was used. *P 0.05; **P 0.01. Expression of apoptosis-related genes in the K?llikers organ during postnatal development The apoptosis-related proteins were parallelly analyzed, including Bcl-2, Caspase-3 and cleaved-Caspase-3 (Figure 5E). The caspase-3 expression gradually increased from P3 to P7 (Figure 5F; P 0.05), then sharply declined from P7 to P14 (P 0.01); cleaved-Caspase-3 remains steady from P1 to P7, increased sharply at P10 (Figure 5I, P 0.05), then decreased sharply at P14 (Figure 5I; P 0.01). The Bcl-2 expression showed a similar pattern as cleaved-Caspase-3 (Figure 5G; *P 0.05, **P 0.01). Consistent with the protein expression, the and mRNA 8-Dehydrocholesterol expression peaked at P7, a little earlier than the protein expression change (Figure 6 D,?,EE). Discussion In mammals, the development of the auditory system undergo a tremendous sequence of morphological and gene expression changes, with the K?llikers organ as one such example.14 Its degeneration in time-course program may decide auditory function. Here we explored the morphological 8-Dehydrocholesterol and gene expression changes of the K?llikers organ from E16.5 to P14. Our work supported that autophagy may participate in the degeneration process during postnatal development. We first observed abundant autophagic vacuoles containing organelles and proteins in supporting cells of.
Beclin1 is known to play a crucial role in autophagosome initiation and maturation
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