Extended Data Fig. 2: Quality control of in vitro neurogenesis methods.
From: Combinatorial selective ER-phagy remodels the ER during neurogenesis
a, The indicated hESCs were either cultured in the pluripotent state (day 0) or converted to day 12 iNeurons prior to total proteome analysis by multiplex TMT in biological triplicate cultures (n=3). The relative abundance (Log2 FC) of the indicated neurogenesis or pluripotency factors at day 12 relative to day 0 is shown, indicating that all genotypes undergo differentiation to a similar extent as measured by markers of the process. b, Comparable number of viable iNeurons were observed upon quantitative analysis of intact DAPI-positive nuclei compared to all DAPI-positive (intact and fragmented) DNA structures in cultures of the indicated genotypes. Four points shown for each WT or KO condition represent the measured ratios from the same four independent differentiations also analysed for ER structure per nuclei in Fig. 2b. Data are presented as mean values +/-SEM. *, p<0.05, two sided Mann–Whitney test. c,d Quantification of DAPI -positive TUNEL-negative nuclei in day 20 iNeurons from the indicated genotypes (c) and representative images of DAPI-positive nuclei in green and TUNEL-stained DAPI-positive fragmented nuclei, with TUNEL signal in magenta (d). The lower panels are magnified regions (merges and separate image channels) of the area boxed in the respective image above. Four points shown for each WT or KO condition represent the measured values from four independent differentiations. Data are presented as mean values +/-SEM. *, p<0.05, two sided Mann–Whitney test. Scale bar, 100 microns (top), 10 microns (bottom) e, ATF4 abundance was examined by immunoblotting of extracts from day 12 iNeurons from the indicated genotypes with or without treatment with tunicamycin as a positive control for induction of the ER-stress stress response. Tunicamycin was used at 0.1 or 1.0 μg/ml for 6 or 12 h, as indicated. Blots were reprobed with α-tubulin as a loading control. The relative α-ATF4 signal, normalized for tubulin, is shown in the histogram (right panel) with the dashed line representing ATF4 signal in untreated WT cells. n=1. f, XBP1 and XBP1s mRNA from the indicated iNeurons in biological triplicate (n=3) cultures was subjected to reverse transcription-PCR (see Methods) and examined by agarose gel electrophoresis to resolve spliced and unspliced XBP1. 1.0 μg/ml tunicamycin (6h) was employed as a positive control for induction of ER stress and XBP1 splicing. GAPDH was used as a positive control. The XBP1s/XBP1 ratio was quantified as shown in the histogram (right panel). No evidence of increased XBP1 splicing was observed in any genotype under untreated conditions. Data are presented as mean values +/-SEM. *, p<0.05; **, p<0.01; n.s., not significant; Brown–Forsythe and Welch One-way ANOVA and Dunnett’s T3 multiple comparisons test. g, TEX264-GFP, TEX264F273A-GFP, or FAM134C-GFP were expressed in WT or ATG12−/− hESCs and cells imaged at day 4 of differentiation to iNeurons. In some experiments, VPS34i was added to WT cells for 24h prior to imaging. Arrows mark examples of ER-phagy receptor puncta. h, Expression of TEX264-GFP was verified by immunoblotting of iNeuron extracts using α-HSP90 as a loading control. i, Number of TEX264-GFP puncta was quantified in day 4 iNeurons (n = 1 independent differentiation). Min-to-max box-and-whiskers representing detectable number of puncta per cell, with box representing the 25th to 75th percentile, whiskers going from min to max values, line at median and + at mean. j, Day 30 iNeurons expressing TEX264-GFP and mCh-LC3B were immunostained with α-MAP2 to detect dendrites (white) and α-NEFH to mark axons (blue) and projections imaged by confocal microscopy. One differentiation was fixed and imaged this way. Insets show TEX264-GFP/mCh-LC3B-positive puncta (arrows) or mCh-LC3B-positive but TEX264-GFP-negative puncta (arrowheads) in axons. All scale bars, 10 microns. Source numerical data and unprocessed blots are available in source data.
