Fig. 2: Autophagy-dependent clearance of ER in axons during iNeuron differentiation.

a, WT or ATG12^−/− day-20 iNeurons immunostained with ER-tubule marker α-RTN4 (white) and with DAPI (nuclei, blue). Scale bars, 50 μm (full images) and 10 μm (zooms). b, Enlarged ER-positive structures in ATG12^−/− day-20 iNeuron axons revealed by immunostaining with α-calnexin, ER (white); α-MAP2, dendrites (green); α-NEFH, axons (magenta); and DAPI, nuclei (blue). Scale bars, 10 μm (full image) and 5 μm (zooms). c, As in b, day-20 iNeurons were immunostained with α-NEFH and α-calnexin to identify aberrant ER structures; here we compare the zoomed-in region of axons in ATG12^−/− iNeurons to a similar region for WT iNeurons. Scale bar, 5 μm. d, Min-to-max box-and-whiskers plot for the number of axonal ER accumulations per nucleus, where the box represents the 25th to 75th percentiles, whiskers extend from min to max values, the line represents the median and + the mean. Points represent mean values from four independent differentiations (n = 4). *P < 0.05, two-sided Mann–Whitney test. e, Min-to-max box-and-whiskers plot for the area of ER accumulations in axons, where the box represents the 25th to 75th percentiles, whiskers extend from min to max values, the line represents the median and + the mean. Four points for each condition give the resulting mean areas from four independent differentiations. *P < 0.05; two-sided Mann–Whitney test. f,g, Scanning transmission EM of thin sections from WT and ATG12^−/− iNeuron cultures (day 20, one differentiation). Panel f presents low-magnification images through multiple axons. Panel g presents high-magnification images of WT example 1 and example 2 and one ATG12 region, all outlined in f, as well as one additional zoom example 2 from another ATG12^−/− iNeuron field of view. Scale bars, 500 nm.