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. 2022 Apr 6;13:1853. doi: 10.1038/s41467-022-29479-y

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© The Author(s) 2022

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a HeLa cells expressing endogenous DELE1^HA were treated with cycloheximide (CHX) and analyzed by immunoblotting. ACTB, β-actin. b The endogenous DELE1 locus in HAP1 cells was engineered with a C-terminal triple ALFA tag using CRISPR-Cas9. In-frame fusion verified by Sanger sequencing. c HAP1 DELE1^ALFA cells were lentivirally transduced to express a single guide non-targeting control (sgNTC) or an sgRNA targeting DELE1 (sgDELE1) together with Cas9-P2A-RFP at a multiplicity of infection < 1, such that each population comprises sgRNA-containing cells (RPF+) and non-transduced wild-type cells. DELE1^ALFA was stained with Atto488, values specify mean Atto488 intensity in transduced (RFP+; red box) and non-transduced (RFP-) cells. d, e HAP1 cells of the indicated genotypes were treated with CCCP and the ability of endogenous HRI to co-precipitate the respective S-DELE1 proteins was analyzed by immunoblotting. WT, wild-type. f Haploid genetic screen for regulators of DELE1. Per gene (dots), the ratio of the frequency of mutations in DELE1^ALFA-high versus DELE1^ALFA-low cells (y-axis) is plotted against the combined number of unique mutations identified in both populations (x-axis). Genes significantly enriched for mutations in either population are dark gray or colored (two-sided Fisher’s exact test, FDR-corrected P-value (P_adj) < 0.05). g HAP1 DELE1^ALFA cells were exposed to sgRNAs as indicated. Knockout efficiency was analyzed by immunoblotting. Asterisk: nonspecific band. h DELE1 protein levels (cells as in (g)) analyzed by flow cytometry. Mean ± s.d. of n = 3 independent biological samples (arb. u., arbitrary units). Statistical significance compared to sgNTC assessed using ordinary one-way ANOVA with Dunnett’s multiple comparisons correction. ****P < 0.0001, **P = 0.0094. i Mitochondrial DELE1 protein levels of clonal HAP1 DELE1^ALFA PITRM1 or CLUH knockout cells analyzed by immunoblotting. j Clonal HAP1 DELE1^ALFA PITRM1 and CLUH knockout cells reconstituted with the respective cDNAs or vector control analyzed by immunoblotting. k DELE1 protein levels (cells as in (j)) analyzed by flow cytometry. Mean ± s.d. of n = 3 independent biological samples. Statistical significance compared to WT + vector assessed with ordinary one-way ANOVA and Dunnett’s multiple comparisons correction; ns, non-significant. ****P < 0.0001, **P = 0.0020, ns ≥ 0.9783.