Fig 2: Deubiquitination at AIF K244 disrupts mitochondrial structure and compromises OXPHOS. A,B) Representative electron microscopy images of the indicated cells (KYSE30 background). Scale bars, 2 µm in the top panel and 200 nm in the bottom panel. C–F) Whole‐cell OCR as measured using C,E) Seahorse assays and D,F) statistical analyses of basal respiration, maximal respiration and ATP production in the indicated cells (KYSE30 background). The data are the means ± s.e.m.; n = 5. Two‐tailed t tests, ***p < 0.001, **p < 0.01, *p < 0.05. G,H) Statistical analyses of the basal glycolysis rate in the indicated cells (KYSE30 background). The data are the means ± s.e.m.; n = 5. Two‐tailed t tests, ***p < 0.001. I,J) Whole‐cell OCR as measured using Seahorse assays in the indicated cells (KYSE150 background). The data are the means ± s.e.m.; n = 5. K) IB for respiratory chain proteins in the indicated cells (KYSE150 background). L) IB to assess interactions between AIF and CHCHD4 in the indicated cells (KYSE150 background).

Fig 3: OTUD1 activates the caspase‐dependent apoptotic pathway by recruiting the CUL4A‐DDB1 complex to facilitate ubiquitination‐mediated degradation of MCL1. A) IB to assess MCL1 ubiquitination in KYSE30 cells expressing EV, OTUD1, or OTUD1C320A. B) Quantitative analyses of MCL1 expression in KYSE30 cells expressing EV, OTUD1, or OTUD1C320A and subjected to a CHX pulse‐chase assay. Quantification of MCL1 expression relative to β‐actin expression is shown. The data are the means ± s.e.m.; n = 3. C) IB to assess MCL1 ubiquitination in KYSE150 cells expressing shC, shD1#1, or shD1#2 shRNAs. D) Quantitative analyses of MCL1 expression in KYSE150 cells expressing shC, shD1#1, or shD1#2 shRNAs and subjected to a CHX pulse‐chase assay. Quantification of MCL1 expression relative to β‐actin expression is shown. The data are the means ± s.e.m.; n = 3. E–G) IB for the indicated proteins in co‐IP assays. H) Relative viability of AIF‐ablated KYSE30 cells expressing EV, OTUD1, or OTUD1C320A and treated with DDP with or without Z‐VAD (50 × 10−6 m). The data are the means ± s.d.; n = 5. I) Cell death fraction of AIF‐ablated KYSE30 cells expressing EV, OTUD1, or OTUD1C320A and treated with or without Z‐VAD (50 × 10−6 m). The data are the means ± s.e.m.; n = 3. Two‐tailed t tests, ***p < 0.001. J)IB for the indicated proteins in AIF‐ablated KYSE30 cells expressing EV, OTUD1, or OTUD1C320A or in K) AIF‐ablated KYSE150 cells expressing shC, shD1#1, or shD1#2 shRNAs and treated with or without DDP (5 µg mL−1)/Z‐VAD (50 × 10−6 m).

Fig 4: OTUD1 deubiquitinates and stabilizes DCAF10. A,B) Relative protein expression levels of DCAF10 in a pulse‐chase assay performed in the indicated A) KYSE30 or B) KYSE150 cells. Quantification of DCAF10 expression relative to β‐actin expression is shown. The data are the means ± s.e.m.; n = 3. C,D) IB to assess MCL1 ubiquitination in the indicated C) KYSE150 or D) KYSE30 cells (D1:OTUD1; shDC: shDCAF10; shCU: shCUL4A; shDD: shDDB1). E) IB for OTUD1, DCAF10, MCL1, and AIF expression in ESCC cell lines. F) IB to assess Myc‐DCAF10 ubiquitination in 293T cells cotransfected with Myc‐DCAF10, HA‐Ub mutants and V5‐OTUD1. K48 and K63 indicate that all lysines except K48 or K63 were mutated to arginines. G) Tumor weights of xenografts from the indicated cells (KYSE150 background). The data are the means ± s.e.m.; n = 7. Two‐tailed t tests, ***p < 0.001, ns: not significant. H) Tumor weights of xenografts from the indicated cells (KYSE150 background) and treated with DDP, Z‐VAD, or S63845. The data are the means ± s.e.m.; n = 7. Two‐tailed t tests, ***p < 0.001, **p < 0.01, ns: not significant. I) A schematic model showing the overlapping and distinct downstream events of OTUD1 overexpression and loss. OTUD1 overexpression not only activates both parthanatos and caspase‐dependent apoptosis pathways, but also compromises OXPHOS to further promotes cell death. In contrast, OTUD1 loss inhibits DDP‐induced apoptosis by suppression of parthanatos and caspase‐dependent apoptotic pathway.

Fig 5: Deubiquitination at AIF K255 leads to an increase in DNA‐binding ability. A) IB to assess AIF ubiquitination in 293T cells cotransfected with Flag‐AIF, HA‐Ub, and V5‐OTUD1 (wild‐type or C320A). B) IB to assess AIF ubiquitination in an in vitro ubiquitination assay. C) IB to assess AIF ubiquitination in KYSE30 cells expressing EV, OTUD1 or OTUD1C320A. D) IB detection for the ubiquitination of AIF in KYSE150 cells expressing shC, shD1#1, or shD1#2 shRNAs. E) Ubiquitinated peptides and lysine sites in the MS analysis. F) IB to assess Myc‐AIF ubiquitination in 293T cells cotransfected with Myc‐AIF mutants, HA‐Ub mutants and Flag‐OTUD1. K27, K63, K244, and K255 indicate that all lysines, except K27, K63, K244, or K255, respectively, were mutated to arginines. G) ChIP assays to detect the association between the indicated AIF mutants and genomic DNA of GAPDH and RPL30 in KYSE30 cells. The data are the means ± s.e.m.; n = 3. H) IB detection for the soluble and DNA‐binding AIF mutants in KYSE150 cells. I) IB detection for AIF level in the mitochondria of indicated cells before or after DDP treatment. Quantification of AIF expression relative to TOMM20 expression is shown. The data are the means ± s.e.m.; n = 3. J) IB to assess interactions between AIF and PAR/MIF in the indicated cells.