Extended Data Fig. 4: Generation of a genetic toolkit for functional analysis of ER-phagy receptors in iNeurons.
From: Combinatorial selective ER-phagy remodels the ER during neurogenesis
a, MiSeq analysis of single ER-phagy receptor mutants in hESCs. The green highlights the target of the CRISPR gRNA. The sequence of the major MiSeq output is indicated for each allele. b, Immunoblot validation of targets knockout clones at day 12 of differentiation. Confirmation immunoblots for protein deletion in these different genetic backgrounds were performed for each proteomics experiment. Cell extracts were subjected to immunoblotting with the indicated antibodies, employing a Rhodamine-labelled α-tubulin as loading controls. *, position of the ATG12-ATG5 conjugate. c, MiSeq analysis of combinatorial ER-phagy receptor mutants in hESCs, as performed for the single knockouts in a. d, Immunoblot validation of targets in combinatorial knockout clones at day 12. Cell extracts were subjected to immunoblotting as in b. Confirmation immunoblots for protein deletion in these different genetic backgrounds were performed for each proteomics experiment. e, Karyotype analysis of QKO and PKO hESCs revealed no detectable alterations in chromosome number. f, Ratiometric flow cytometry analysis of Keima-RAMP4 flux was measured in WT, ATG12−/−, or the indicated ER-phagy receptor knockout ES cells (day 0 of differentiation). The ratio of acidic to neutral Keima fluorescence was normalized to samples treated with BAFA (100 nM) for 4 h prior to analysis, and where indicated, cells were cultured with VPS34i prior to analysis. Each measurement (represented by a point) reflects a biological triplicate sample. g, As in panel f, but at day 4 of differentiation to iNeurons. For f, and g, Data are presented as mean values +/−SD. n.s., not significant; Brown–Forsythe and Welch One-way ANOVA and Dunnett’s T3 multiple comparisons test. h, Day 12 iNeurons treated Propidium iodine staining and were analysed via flow cytometry. The same gating strategy for live cells was applied to all genotypes, as was done in all Keima flux experiments. Mean values +/-SEM of the percent of live cells (not stained with PI) is displayed. N=3 biological replicates; n.s. not significant; two sided Mann–Whitney test. i, Examples of enlarged axonal structures from WT, ATG12−/− and PKO day 30 iNeurons containing dense tubular ER, as visualized by TEM. Some of these examples were also shown in Fig. 2 to compare only WT and ATG12−/−. Three independent examples are shown. Scale bar, 500nm. Source numerical data, flow cytometry gating strategy, and unprocessed blots are available in source data.
