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. 2024 Mar 1;26(3):378–392. doi: 10.1038/s41556-024-01356-4

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© The Author(s) 2024, corrected publication 2025

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a, Proteomic analysis of two PKO clones (A2 and E4) in parallel with ATG12^−/− and WT iNeurons (day 20). The upper panel provides a schematic of the TMT multiplex approach employed. n = 4 biological replicates. Middle panel displays Log₂FC for ER protein and selected ER protein categories. Lower panel displays Log₂FC for the indicated organelles. b, Heatmap for Log2FC values for the indicated proteins from the experiment in panel a. c, Immunoblot validating deletion of FAM134B, FAM134C, TEX264, and CCPG1 in both clone A2 and E4 for the PKO mutant. d, Modulation of iNeuron proteome in response to inhibition of MTOR with Torin1 (100 nM,15 h). Upper panel shows a schematic of the experimental set-up employing TMT based proteomics to quantity alterations in the proteome if WT or ATG12^−/−, PKO iNeurons. Lower panel: Correlation plots comparing the effect of Torin1 on organelles of ATG12^−/− cells relative to WT cells and PKO cells relative to WT cells. e, MiSeq analysis of a CALCOCO1^−/− H9 hESC clone, showing the position of the gRNA used for CRISPR-Cas9 deletion, and the position of out of frame deletions in the two alleles of CALCOCO1. f, Schematic showing the TMT total proteome strategy for analysis of the effect of CALCOCO1 deletion on organelle abundance in hESCs and day 12 iNeurons (n=3 biological replicates). ATG12^−/− cells were included as a positive control. g, Heatmap of Log₂FC values for selected proteins from the TMT experiment outlined in panel f, which also demonstrates loss of the CALCOCO1 protein in the CALCOCO1^−/− iNeurons. h, Violin plots (Log₂FC) for the indicated organelles (top panel) and selected classes of ER proteins (lower panel) for ATG12^−/− and CALCOCO1^−/− iNeurons (day 12). Loss of CALCOCO1 does not affect the abundance of any of the organelles tested. i, Comparison of Log₂FC (mutant/WT) distributions of ER proteins (top) or Golgi proteins (bottom) to distributions of randomized selections of the same number of proteins (100 iterations). p-values for comparisons were calculated with a Kolmogorov–Smirnov Test (two-sided). Unprocessed blots are available in source data.

Source data