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. 2016 Dec 22;26(1):158–172. doi: 10.1093/hmg/ddw376

Figure 6.

Figure 6

Autophagy activators in the mTOR-dependent or mTOR-independent pathways both ameliorate cystic phenotypes in pkd1a-/- embryos. (A,B) Rapamycin, carbamazepine, and minoxidil all enhanced LC3II expression while differentially affecting phospho-S6 levels. Zebrafish embryos were treated with DMSO (D), rapamycin (R, 400 nM), carbamazepine (C, 20 μM), or minoxidil (M, 400 nM) at 4 dpf for 16 h, and Western blot analysis was performed using whole embryo lysates. The gel is representative of three independent experiments (A), and LC3II levels were normalized by actin expression and presented as means ± s.d. (B). (C) Autophagy activators suppressed cystogenesis in the pkd1a-/- embryos. Embryos derived from heterozygous pkd1a inter-crosses were incubated with vehicle (D) or autophagy activators (R: 400 nM,; C: 20 μM; or M: 400 nM) at 2.5 dpf for 16 h. Percentage of embryos with kidney cysts was analysed by HE staining of JB-4 sections. Note: rapamycin, but not other drugs, led to cyst formation in 14% of wild type embryos. (D) Autophagy activators preserved kidney excretion function in the pkd1a-/- embryos. Embryos at 2.5 dpf were treated with vehicle (D) or autophagy activators (R, C, or M) for 16 h, and kidney fluid flow was analysed via dye injection. (E) Autophagy activators promoted the clearance of ubiquitinated protein aggregates. Human PKD1-/- cells were incubated with MG132 and 13 h later MG132 was removed and replaced with vehicle (D) or autophagy activators (R, C, or M) for 11 h. Insoluble fractions of the cell lysates were subjected to Western blot analysis using an Ub antibody. Shown is a representative blot of three independent experiments. (F) Low doses of rapamycin significantly alleviated cyst formation in the pkd1a-/- embryos when combined with low doses of carbamazepine. Embryos at 2.5 dpf were treated with 40 nM rapamycin (R), 2 μM carbamazepine (C), or 40 nM rapamycin plus 2 µM carbamazepine (R + C). Data are presented as means ± s.d. from three independent experiments, and 7 to24 embryos per group were scored in each experiment (C,D,F). *: P < 0.05. **: P < 0.01. NS: not statistically significant (P > 0.05).