Fig 1: 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.
Fig 2: OTUD1 knockdown promoted NSCLC cell proliferation, migration and invasion. (a) NSCLC cells were transfected with vector with sh-NC, sh-OTUD1-1# or sh-OTUD1-2#, then the expression level of OTUD1 was measured by western blot assay to verify the efficiency. (b) NSCLC cells were transfected with vector with sh-NC, sh-OTUD1-2#. Cell viability was then measured by CCK-8 assay (p < 0.01). (c) NSCLC cells were transfected with vector with sh-NC, sh-OTUD1-2#. Then the migration ability of PC9 and A549 cells was measured by transwell assay (p < 0.01). (d) NSCLC cells were transfected with vector with sh-NC, sh-OTUD1-2#. Then the invasion ability of PC9 and A549 cells was measured by transwell assay (p < 0.01)
Fig 3: OTUD1 interacts with AIF and promotes its nuclear translocation. A) Silver staining for Flag-OTUD1-associated proteins after immunoprecipitation using anti-Flag magnetic beads. B) Number of unique peptide hits for OTUD1 and AIF. C) IB for the indicated proteins in in vivo co-IP assays using anti-Flag and anti-V5 magnetic beads. D) IB for the indicated proteins in in vitro co-IP assays using anti-Flag magnetic beads. E) IB for the indicated proteins in a co-IP assay using anti-AIF antibody. F,G) Confocal microscopy assessment of PLA spots (red) in the indicated cells. Each spot indicates a single interaction between proteins. Nuclei were stained with DAPI (blue). Scale bars, 30 µm. The data are the means ± s.e.m.; n = 3. Two-tailed t tests, ***p < 0.001, **p < 0.01, *p < 0.05. H,I) Confocal microscopy assessment of AIF (red) localization in the indicated cells. Nuclei were stained with Hoechst 33 342 (blue). Percentages indicate the relative intensity of the AIF signal inside the nucleus compared to the AIF signal of the whole cell. Scale bars, 30 µm. The data are the means ± s.e.m.; n = 15. Two-tailed t tests, ***p < 0.001.
Fig 4: OTUD1 activates the caspase-independent apoptotic pathway. A,B) Representative images and fraction of EdU-positive cells. Scale bars, 60 µm. The data are the means ± s.e.m.; n = 3. Two-tailed t tests, ***p < 0.001, **p < 0.01, ns: not significant. C) Representative image and tumor weights of xenografts from KYSE30 cells expressing EV, OTUD1, or OTUD1C320A and treated with saline or DDP. The data are the means ± s.e.m.; n = 7. Two-tailed t tests, ***p < 0.001, **p < 0.01. D) Representative image and tumor weights of xenografts from KYSE150 cells expressing shC, shD1#1, or shD1#2 shRNAs and treated with saline or DDP. The data are the means ± s.e.m.; n = 7. Two-tailed t tests, ***p < 0.001, ns: not significant. E,F) Statistical analyses of the cell death fraction of the indicated cells as determined using flow cytometric analysis. The data are the means ± s.e.m.; n = 3. Two-tailed t tests, ***p < 0.001, **p < 0.01, ns: not significant. G) Representative image and statistical analyses of JC-1 staining of KYSE30 cells expressing EV, OTUD1 or OTUD1C320A and treated with DDP. Scale bars, 30 µm. Fluorescence intensity (fold) indicates the relative intensity of Red/Green. The data are the means ± s.e.m.; n = 5. Two-tailed t tests, **p < 0.01, ns: not significant. H) In vitro growth of KYSE30 cells expressing EV or OTUD1 and treated with or without Z-VAD (50 × 10-6 m). The data are the means ± s.d.; n = 5. Two-tailed t tests, ***p < 0.001. I) Cell death fractions of the indicated KYSE30 cells expressing EV or OTUD1 and treated with DDP together with or without Z-VAD (50 × 10-6 m). The data are the means ± s.e.m.; n = 3. Two-tailed t tests, **p < 0.01, *p < 0.05. J) Representative image and statistical analyses of JC-1 staining of KYSE30 cells expressing EV or OTUD1 and treated with DDP or Z-VAD (50 × 10-6 m). Scale bars, 30 µm. Fluorescence intensity (fold) indicates the relative intensity of Red/Green. The data are the means ± s.e.m.; n = 5. Two-tailed t tests, **p < 0.01.
Fig 5: OTUD1 is involved in the regulation of RNA virus-induced production of type I IFNs and proinflammatory factors.(A) Relative mRNA expression of IFNß in HEK293T cells transfected with control shRNAs (shCON) or OTUD1 shRNAs (shOTUD1) and then infected by SeV for 10 hr. The data were shown as fold change normalized to that of uninfected cells in the shCON group. (B) Otud1+/+ or Otud1-/- MEFs were infected with SeV, or VSV, or HSV. 10 hr after infection, IFNß mRNAs were analyzed by quantitative real time PCR (q-PCR). The data were shown as fold change normalized to that in uninfected Otud1+/+ MEFs. (C) Q-PCR analysis of IFNß mRNA expression in Otud1+/+ or Otud1-/- MEFs stimulated by ISD (2 µg/ml) or cGAMP (1 µg/ml) for 10 hr. The data were shown as fold change normalized to that in unstimulated Otud1+/+ MEFs. (D) IFIT1, ISG15 and ISG54 mRNA levels in Otud1+/+ or Otud1-/- MEFs infected with SeV for 10 hr were analyzed by q-PCR. The data were shown as (B). (E) Otud1+/+ or Otud1-/- MEFs were infected with VSV for 10 hr. The relative mRNA amounts of TNFa and IL-6 were determined by q-PCR. The data were shown as (B). (F) Otud1+/+ or Otud1-/- MEFs were infected with SeV, or VSV, or HSV for 10 hr and then viral RNAs were analyzed by q-PCR. The data were shown as (B). (G and H) Otud1+/+ or Otud1-/- MEFs were infected with VSV (G) or HSV (H) for 10 hr. Viral titers in supernatants were tested by TCID50 assay. (I) Determination of VSV titers in supernatants of Otud1+/+ or Otud1-/- MEFs infected with VSV for the indicated time by TCID50 assay. NS: not significant (P>0.05). *P<0.05, **P<0.01 and ***P<0.001. Error bars represent the mean and s.d. of three independent experiments.
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