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. Author manuscript; available in PMC: 2020 Jul 25.
Published in final edited form as: Cell Rep. 2020 Jun 23;31(12):107780. doi: 10.1016/j.celrep.2020.107780

Figure 4. Reduced Ciliation in the Tsc2-sh Neurons Is Prevented by mTORC1 and Hsp90 Inhibition during an Age- and Time-Sensitive Window.

Figure 4.

(A and B) Overview of rapamycin time-course experiment using the ciliaHCA with endpoint at DIV13 (A) and DIV20 (B).

(C and D) Quantification of cilia in ctrl-sh and in Tsc2-sh-vehicle-treated or -rapamycin-treated neurons (20 nM) with assay endpoint at DIV13 (C) and DIV20 (D) using the ciliaHCA. Data were quantified relative to each respective vehicle-treated group (n = 8–18 biological replica/condition; two-way ANOVA with Dunnett’s multiple-comparison test, *p < 0.05, **p < 0.01, ns, non-significant) and are represented as mean ± SEM.

(E) Effect of 17-AGG dose-curve between DIV9 and DIV13 on ciliation using the ciliaHCA. Data represent GFP+ACIII+ in the Tsc2-sh neurons as percent of vehicle-treated control neurons.

(F) Effect of 17-AGG dose-curve between DIV9 and DIV13 on pS6 using the mTORC1HCA. Data represent GFP+pS6+ in the Tsc2-sh neurons as percent of vehicle-treated Tsc2-sh neurons. Quantification relative to vehicle-treated Tsc2-sh neurons (n = 8–16 biological replica/condition, E; n = 15–64 biological replica/condition, F, one-way ANOVA with Dunnett’s multiple-comparison test, *p < 0.05, ****p < 0.0001). Data are mean ± SEM.

(G) Representative images of cilia in vehicle-treated ctrl-sh and in Tsc2-sh neurons vehicle-treated or treated with 50-nM 17-AGG between DIV9–13. Neurons and cilia were identified by GFP and ACIII staining. Scale bar is 25 μm.

(H) Western blot of protein lysates from ctrl-sh and Tsc2-sh neurons treated with vehicle or with 17-AGG dose-curve between DIV9 and DIV13.

(I–K) Quantification of pS6 (I), total IGF-IRβ (J), and p-IGF-IRβ (K). Western blot data were normalized using GAPDH as loading control. Data are average fold changes of vehicle-treated ctrl-sh neurons. Quantification is relative to vehicle-treated Tsc2-sh neurons (n = 4 experiment/condition; one-way ANOVA with Dunnett’s multiple-comparison test, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001). Error bars indicate ± SEM.

See also Figure S4.