Figure 1. Neurons are resistant to Torin1-mediated induction of autophagy.

(A) Targeting strategy using CRISPR/Cas9 to knock-in mEGFP immediately 5’ to exon1 in the MAP1LC3B gene. (B) Schematic of the cassette used to integrate NGN1 and NGN2 at the CLYBL safe harbor locus under the control of a Tet-ON system. Puro, puromycin-resistance gene; pA, poly-A tail; P₁, P₂, promotors; iRFP, near-infrared fluorescent protein; rTTA, reverse tetracycline-controlled transactivator; NGN1 and NGN2, neurogenin-1 and -2; T2A, self-cleaving peptide; TRE, tetracycline response element. (C) Protocols used to differentiate iPSCs into iNeurons using doxycycline-mediated, forced expression of differentiation factors, or into iAstrocytes using dual-SMAD inhibition followed by terminal differentiation by culturing in CNTF. (D) Protocol for differentiating iPSCs into iMuscle using a piggybac/transposase system to integrate doxycycline-inducible MYOD and OCT4 shRNA. (E) Representative 100X images of mEGFP-LC3-positive vesicles in the cell types indicated after treatment with DMSO vehicle or 250nM Torin1 for 4h. Dotted lines indicate cell borders, within which cell area in μm² was calculated using Fiji. (F) Scatterplots of blinded manual quantifications of mEGFP-LC3-positive vesicles imaged as in (E), normalized to cell area in μm². Data are from three independent experiments. n.s., not significant; *p<0.05; ****p<0.0001; one-way ANOVA with Šídák’s multiple comparisons test. (G) Density plots of data from (F), which visualize the distribution of quantification data over a continuous interval with kernel smoothing to reduce noise and facilitate visualization of the distribution shape of data and peaks in which data are concentrated. n.s., not significant; ****p<0.0001, two-sample Kolmogorov-Smirnov test. See also Figure S1, Figure S2, Figure S5, and the STAR Methods.