Journal: International Journal of Molecular Sciences

Article Title: Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy

doi: 10.3390/ijms25084493

Figure Lengend Snippet: Torin1-induced autophagy in ATG16L1 ΔWDR cells is lagged. ( A ) Abridged general view of full length ATG16L1 (ATG16L1 FL ) and ATG16L1 ΔWDR . AFIM: ATG5 interaction motif, CCD: coiled-coil domain, WDR: WD40 repeats domain. ( B ) WT-HeLa cells and ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 ΔWDR were treated with 1 μM of Torin1 for 0, 1, 3, 6, 12 h. p62, LC3, and β-actin were analyzed by immunoblotting. ( C , D ) Quantification of LC3-II/β-actin ratios and p62/β-actin ratios in ( B ), n = 3. * p < 0.05, ** p < 0.01. ( E , F ) ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 ΔWDR were treated with 1 μM of Torin1 for 24 h. p62, LC3, and β-actin were analyzed by immunoblotting and quantified, n = 3. ** p < 0.01 and *** p < 0.001.

Article Snippet: WT-HeLa and ATG16L1 −/− -HeLa cells were purchased from Abclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Stable Transfection, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy

doi: 10.3390/ijms25084493

Figure Lengend Snippet: Starvation-induced autophagy in ATG16L1 ΔWDR cells is lagged. ( A – F ) WT-HeLa cells ( A , B ) and ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 ( C , D ) or GFP-ATG16L1 ΔWDR ( E , F ) were incubated with EBSS for 0, 1, 3, 6, 9, and 12 h. Protein levels of p62 and LC3 were analyzed by immunoblotting and quantified, respectively, n = 3. * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Article Snippet: WT-HeLa and ATG16L1 −/− -HeLa cells were purchased from Abclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Stable Transfection, Expressing, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy

doi: 10.3390/ijms25084493

Figure Lengend Snippet: ATG16L1 ΔWDR cells have lower LLP degradation capacity. ( A , B ) WT-HeLa cells were firstly labeled by AHA for 18 h, then short-lived proteins were chased for 2 h before cells were treated with 0.5 μM of Baf (Bafilomycin A1) or 1 μM of Torin1 with or without Baf and incubated in regular culture medium supplemented with 10× L-methionine for 12 h. Cells were collected and incubated with “Click” reaction mixture. The degradation of LLPs was analyzed by flow cytometry ( A ) and quantified ( B ) by relative fluorescence intensity per cell in ( A ), n = 3. * p < 0.05, *** p < 0.001, and **** p < 0.0001. ( C – H ) ATG16L1KO-HeLa cells ( C , D ), GFP-ATG16L1-HeLa cells ( E , F ), and GFP-ATG16L1 ΔWDR -HeLa ( G , H ) were treated with 1 μM of Torin1 and were incubated in regular culture medium supplemented with 10× L-methionine for 12 h after labeling by AHA. Degradation of LLPs was analyzed by flow cytometry and quantified by relative fluorescence intensity per cell, n = 3. *** p < 0.001, **** p < 0.0001, ns: not significant. ( I , J ) WT-HeLa cells (WT), ATG16L1KO-HeLa (KO), GFP-ATG16L1-HeLa cells (FL), GFP-ATG16L1 ΔWDR -HeLa cells (ΔWDR) were firstly labeled by AHA for 18 h, then short-lived proteins were chased for 2 h. Cells were incubated in regular culture medium supplemented with 10× L-methionine for 12 h. Degradation of basal LLPs was analyzed by flow cytometry and quantified, respectively, n = 3. *** p < 0.001, **** p < 0.0001, ns: not significant. ( K , L ) GFP-ATG16L1 ΔWDR -HeLa cells and WT-HeLa cells were treated with 1 μM of Torin1 and 0.5 μM of Baf (Bafilomycin A1) for 3 h. Protein level of LC3 was analyzed by immunoblotting ( K ) and quantified ( I ), n = 3. * p < 0.001, **** p < 0.0001.

Article Snippet: WT-HeLa and ATG16L1 −/− -HeLa cells were purchased from Abclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Labeling, Incubation, Flow Cytometry, Fluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy

doi: 10.3390/ijms25084493

Figure Lengend Snippet: Deletion of WDR contributes to stalled autophagy flux without affecting fusion steps and lysosome function. ( A ) ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 FL and GFP-ATG16L1 ΔWDR were preincubated with DQ-BSA Green working solution for 2 h followed by treatment with 0.5 μM of Bafilomycin A1 for 6 h and 4 μM of Torin1 for the indicated time. Confocal images were detected, scale bar = 20 μm. Dots per cell were quantified. **** p < 0.0001. ( B ) ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 FL and GFP-ATG16L1 ΔWDR were treated with 4 μM of Torin1 for 1, 6 h and then stained for LC3 and LAMP1, scale bar = 10 μm. The red frames represent the area to be enlarged.Quantification of LC3 total dots (red) and partial colocalization with LAMP1 (yellow). *** p < 0.001, **** p < 0.0001. ( C ) ATG16L1 −/− -HeLa cells expressed GFP-ATG16L1 FL were transiently transfected with GFP-RFP-LC3. After 24 h expression, the cells were then treated with 4 μM of Torin1 for 1, 6, 12, 24 h. LC3 dots were analyzed and quantified, scale bar = 10 μm. The red frames represent the area to be enlarged. **** p < 0.0001. ( D ) ATG16L1 −/− -HeLa cells expressed GFP-ATG16L1 ΔWDR were transiently transfected with GFP-RFP-LC3. After 24 h expression, the cells were then treated with 4 μM of Torin1 for 1, 6, 12, 24 h. LC3 dots were analyzed and quantified, scale bar = 10 μm. The red frames represent the area to be enlarged. **** p < 0.0001.

Article Snippet: WT-HeLa and ATG16L1 −/− -HeLa cells were purchased from Abclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Stable Transfection, Expressing, Staining, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy

doi: 10.3390/ijms25084493

Figure Lengend Snippet: Deletion of WDR reduces the interaction of ATG16L1 with FIP200 and WIPI2b instead of the interaction of ATG16L1 with ATG5. ( A ) ATG16L1 −/− -HeLa cells expressing GFP-ATG16L1/-ATG16L1 ΔWDR were treated with 1 μM of Torin1 for 1 h. Immunoprecipitation of ATG5 was performed in cell lysates; protein levels of ATG16L1 and ATG5 were analyzed. ( B ) ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 and GFP-ATG16L1 ΔWDR were treated with 1 μM of Torin1 for 1 h and stained for LC3. Then, confocal images of LC3 and GFP-ATG16L1 and GFP-ATG16L1 ΔWDR were analyzed, scale bar = 10 μm. The red frames represent the area to be enlarged. ( C , D ) ATG16L1 −/− -HeLa cells expressing GFP-ATG16L1 or GFP-ATG16L1 ΔWDR were treated with or without 1 μM of Torin1 for 1 h (C or the left of D) or 12 h, immunoprecipitation was performed using ATG16L1 antibody ( C ) and FIP200 antibody ( D ). Protein levels of FIP200 and ATG16L1 were analyzed by immunoblotting. ( E ) ATG16L1 −/− -HeLa cells expressed GFP-ATG16L1 or GFP-ATG16L1 ΔWDR were treated with or without EBSS for 3 h. Immunoprecipitation of WIPI2-binding proteins from cell lysates was analyzed.

Article Snippet: WT-HeLa and ATG16L1 −/− -HeLa cells were purchased from Abclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Immunoprecipitation, Stable Transfection, Staining, Western Blot, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy

doi: 10.3390/ijms25084493

Figure Lengend Snippet: Chemical-induced NCA required the WDR domain and K490 of ATG16L1. ( A ) WT-HeLa cells treated with 10 μM of AMDE-1 with or without 2 μM of wortmannin (WM) or 1 μM of bafilomycin A1 (Baf) for 6 h. LC3 was analyzed and quantified, n = 3. * p < 0.05, *** p < 0.001, ns: not significant. ( B ) ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 ΔWDR were treated with indicated 10 μM of AMDE-1 with or without 2 μM of WM for 6 h. Protein level of LC3-II was analyzed and quantified, n = 3. * p < 0.05, ** p < 0.01. ( C ) ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 ΔWDR were treated with 10 μM of AMDE-1 with or without 2 μM of WM for 6 h. Endogenous LC3 was stained and analyzed, scale bar = 20 μm. LC3 puncta per cell were quantified, n = 50. **** p < 0.0001. ( D ) ATG16L1 −/− -HeLa cells stably expressing GFP-ATG16L1 ΔWDR were treated with 10 μM of AMDE-1, 10 μM of Ion (ionomycin), 10 μM of Nic (niclosamide), 30 μM of CCCP with or without WM (2 or 10 μM), respectively, for 6 h. Protein levels of ATG16L1 and LC3 were analyzed. ( E ) WT-HeLa and ATG16L1 K490A -HeLa cells were treated with 2 μM of Torin1, 10 μM of AMDE-1, and 10 μM of niclosamide (Nic) for 6 h. Immunoblot and quantification of ATG16L1 and LC3 were analyzed, n = 3. **** p < 0.0001, ns: not significant. ( F ) FIP200 −/− -HeLa and FIP200 −/− /ATG16L1 −/− -HeLa cells stably expressing ATG16L1 K490A were treated with 10 μM of AMDE-1 and 10 μM of Nic for 6 h. Immunoblot of ATG16L1 and LC3 were analyzed. ( G ) FIP200 −/− -HeLa cells were treated with 2 μM of Torin1, 10 μM of AMDE-1, and 10 μM of niclosamide (Nic) for 6 h. Immunoblot of LC3 was analyzed, n = 3. ( H ) WT-HeLa cells were treated with 2 μM of Torin1, 10 μM of AMDE-1, and 10 μM of niclosamide (Nic) for 6 h. After fractionation, total lysate, cytosol, and membrane fractions were subjected to Western blotting. ATP6V0D1 and ATP6V1D were analyzed and V0–V1 association of membrane fraction were quantified, n = 3. *** p < 0.001, ns: not significant. ( I ) ATG16L1 −/− -HeLa cells expressing GFP-ATG16L1 or GFP-ATG16L1 ΔWDR were treated with 2 μM of Torin1, 10 μM of AMDE-1, and 10 μM of niclosamide (Nic) for 6 h. Immunoprecipitation of ATP6V1D was performed in cell lysates; protein levels of ATG16L1 and ATP6V1D were analyzed.

Article Snippet: WT-HeLa and ATG16L1 −/− -HeLa cells were purchased from Abclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Fractionation, Membrane, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy

doi: 10.3390/ijms25084493

Figure Lengend Snippet: ATG16L1 with an incomplete WDR could not mediate canonical autophagy or NCA/CASM. ( A , B ) WT-HeLa, FIP200 −/− -HeLa, and ATG16L1 −/− - FIP200 −/− -HeLa cells were treated with 1 μM of Torin1 for 6 h. FIP200, ATG16L1, and LC3 were analyzed and quantified, n = 3. **** p < 0.0001, ns: not significant. ( C ) Diagram of mutants with truncation/redundancy of WD40 fragments. ( D , G ) ATG16L1 −/− / FIP200 −/− -HeLa ( D , E ) or ATG16L1 −/− -HeLa ( F , G ) cells were transiently transfected with ATG16L1 mutants for 42 h and then treated with 10 μM of AMDE-1 for 6 h. Protein levels of ATG16L1 and LC3 were analyzed and quantified, n = 3. *** p < 0.001, ns: not significant. ( H ) ATG16L1 −/− / FIP200 −/− -HeLa or ATG16L1 −/− -HeLa cells were transiently transfected with ATG16L1 mutants for 42 h and then treated with 10 μM of AMDE-1 for 6 h. Cells were stained for LC3. The confocal images of mcherry-ATG16L1 mutants and LC3 were observed, scale bar = 10 μm. LC3 puncta per cell in ( H ) were quantified, n = 50. **** p < 0.0001, ns: not significant. ( I ) Immunoprecipitation using ATG16L1 antibody was performed in cell lysates of ATG16L1 −/− -HeLa cells expressing GFP-ATG16L1, ATG16L1 ΔWD2 , and ATG16L1 ΔWDR . Protein levels of FIP200 and ATG16L1 were analyzed. ( J ) ATG16L1 −/− -HeLa cells expressing GFP-ATG16L1 and ATG16L1 ΔWD2 were treated with 20 μM of AMDE-1 for 3 h. Immunoprecipitation of ATP6V1D was performed in cell lysates; protein levels of ATG16L1 and ATP6V1D were analyzed.

Article Snippet: WT-HeLa and ATG16L1 −/− -HeLa cells were purchased from Abclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Transfection, Staining, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks

doi: 10.1038/s41467-019-09641-9

Figure Lengend Snippet: Peli1 is recruited to DNA double-strand break sites. a Mitotic spreads of primary Peli1 wild-type (WT) and Peli1 knockout (KO). MEF cells were arrested in metaphase with 0.1 mg/ml of colchicine, fixed, and visualized by DAPI staining. Red arrow designates chromatid and chromosome breaks. Scale bars, 5 μm. b Quantification of the number of chromatid and chromosome breaks per cell evaluated in 50 cell metaphase spreads. Student’s t -test was used for statistical analyses. c Peli1 WT MEF cells were treated with IR at different doses (0, 1, 2, 4, and 6 Gy). At 1 hr after IR, cells were collected and then subjected to immunoblotting with Peli1, γH2AX, H2AX, and actin antibodies. d Peli1 WT MEFs were either control treated or treated with IR (10 Gy) and allowed to recover for 1 h before processing for Peli1 and γH2AX immunostaining (left). Scale bars, 10 μm. Quantitative analysis for colocalization of Peli1 with >10 foci of γH2AX without IR or with IR. Plotted values represent mean ± SEM of more than 300 individual cells. Student’s t -test was used for statistical analyses. e Peli1 WT MEF cells were subjected to laser micro-irradiation. After 10 min, cells were co-stained with γH2AX or 53BP1 and Peli1 antibodies. The white lines with DAPI staining indicated laser stripes. Scale bars, 10 μm. f mCherry-LacI-FokI expressing plasmid was cotransfected with GFP-Peli1 (upper panels) or YFP-MRE11 (positive control, lower panels) into U2OS-DSB reporter cells. Scale bars, 10 μm. g mCherry-LacI-FokI plasmid was transfected into U2OS-DSB reporter cells. At 48 h post transfection, cells were immunostained with Peli1 or γH2AX antibodies. The number of FokI focus determines the cell cycle phase. Single FokI (upper panel) represents G1 phase while double FokI represents S/G2 phase of cell cycle. Scale bars, 10 μm. h , i U2OS cells were transfected with GFP-Peli1, GFP-NBS1, or GFP-MDC1 expressing plasmid. At 48 h, cells were subjected to laser micro-irradiation. Laser stripes were examined at indicated time point ( h ). The intensity of each laser stripe was determined by averaging values from ten cells at each time point and graphed ( i ). Scale bars, 10 μm

Article Snippet: HeLa H2AX (WT and KO) cells were provided by Dr. Makoto Nakanishi (University of Tokyo, Japan).

Techniques: Knock-Out, Staining, Western Blot, Control, Immunostaining, Irradiation, Expressing, Plasmid Preparation, Positive Control, Transfection

Journal: Nature Communications

Article Title: Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks

doi: 10.1038/s41467-019-09641-9

Figure Lengend Snippet: Deletion of Peli1 FHA domains impaired DNA damage response. a Diagrams of Peli1 WT, deletion, and NLS-fused deletion mutants. b 293T cells were transfected with plasmids encoding GFP-Peli1 WT, each of deletion and NLS-fused mutants. At 48 h post transfection, cells were collected and cell lysates were analyzed by immunoblotting with anti-GFP and anti-tubulin antibodies. c U2OS cells were transfected with GFP-Peli1 WT or each mutant and treated with BrdU (10 μM) for 30 h followed by laser micro-irradiation. Scale bar, 10 μM. d mCherry-LacI-FokI and GFP-fused Peli1 WT or mutants were cotransfected into U2OS-DSB reporter cells (U2OS 2–6–3). At 48 h, cells were fixed and visualized by confocal microscopy. Scale bars, 10 μm. e Targeting strategy to generate PELI1 mutant mice in which coding for exon 4 was replaced with a puromycin cassette gene, resulting in Peli1 E4 truncated mice. f Peli1 WT and E4 truncated MEFs were treated with IR (4 Gy) as indicated. Cells were collected and then subjected to immunoblotting with Peli1, γH2AX, H2AX, and actin antibodies. g Peli1 WT and E4 truncated MEFs were treated with IR (2 Gy) and allowed to recover for 1 h before processing for γH2AX and phospho-ATM (p-Ser1981) immunostaining. Scale bars, 10 μm

Article Snippet: HeLa H2AX (WT and KO) cells were provided by Dr. Makoto Nakanishi (University of Tokyo, Japan).

Techniques: Transfection, Western Blot, Mutagenesis, Irradiation, Confocal Microscopy, Immunostaining

Journal: Nature Communications

Article Title: Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks

doi: 10.1038/s41467-019-09641-9

Figure Lengend Snippet: Peli1 is phosphorylated by ATM that translocate to DNA damage sites. a , b U2OS cells transiently expressing GFP-Peli1 were pre‐treated with ATM (KU55933, ATMi, 10 μM), ATR (VE-831, ATRi, 10 μM), DNA‐PK (KU57788, DNA‐PKi, 10 μM), PARP (PJ34, PARPi, 100 μM), and SUMO (2Dd-08, SUMOi, 50 μM) inhibitors for 1 h. Cells were subjected to laser micro-irradiation. Laser stripes were examined at the indicated time point ( a ). Scale bars, 10 μm. The intensity of each laser stripe was determined by averaging values from ten cells at each time point and graphed ( b ). c ATM WT and KO MEF cells were subjected to laser micro-irradiation. Co-immunostaining with γH2AX and Peli1 antibodies at laser-induced DNA lesions (10 min after laser micro-irradiation). Scale bar, 10 μM. d Schematic showing potential Peli1 phosphorylation sites by ATM/ATR kinases. e 293T cells were cotransfected with GFP-Peli1 WT and S121A/T127A mutant. At 48 h post transfection, cells were treated with IR (10 Gy). At 30 min after IR, cells were collected and immunoprecipitated with an anti-GFP antibody. GFP-Peli1 protein complexes were subjected to immunoblotting with anti-pSQ/TQ, anti-GFP, anti-γH2AX, and anti-actin antibodies. f 293T cells were cotransfected with GFP-Peli1 WT and S121A/T127A mutant with or without HA-Ub. At 36 h post transfection, cells were treated with IR (10 Gy) and 30 min later cells were collected and immunoprecipitated with an anti-GFP antibody. GFP-Peli1 protein complexes were subjected to immunoblotting with indicated antibodies. g , h U2OS cells were transfected with plasmids encoding GFP-Peli1 WT or S121A/T127A mutant. At 48 h, cells were subjected to laser micro-irradiation and laser stripes were examined at indicated time point. Scale bar, 10 μM ( g ). The intensity of each laser stripe was determined by averaging values from ten cells at each time point and graphed ( h ). i U2OS cells were transfected with plasmids encoding GFP-Peli1 WT or S121A/T127A mutant. At 48 h post transfection, cells were collected and analyzed by immunoblotting with anti-GFP and anti-actin antibodies

Article Snippet: HeLa H2AX (WT and KO) cells were provided by Dr. Makoto Nakanishi (University of Tokyo, Japan).

Techniques: Expressing, Irradiation, Immunostaining, Phospho-proteomics, Mutagenesis, Transfection, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks

doi: 10.1038/s41467-019-09641-9

Figure Lengend Snippet: γH2AX-mediated accumulation of Peli1 at DNA damage sites. a , b Mobilization kinetics of GFP-Peli1 to sites of DNA damage. U2OS cells were cotransfected with plasmids encoding GFP-Peli1 and indicated siRNAs. After 48 h, cells were subjected to laser micro-irradiation and laser stripes were examined at indicated time point. Scale bar, 10 μM. U2OS cells were cotransfected with plasmids encoding GFP-Peli1 and indicated siRNAs ( a ). After 48 h, cell lysates were analyzed by immunoblotting with indicated antibodies ( b ). c The intensity of each laser stripe was determined by averaging values from ten cells at each time point and graphed. d H2AX WT and KO HeLa cells were subjected to laser micro-irradiation. Colocalization of Peli1 and γH2AX at laser-induced DNA lesions (10 min after laser micro-irradiation; left panels) and western blotting analysis (right panels) are shown. Scale bar, 10 μM. e , f H2AX WT and KO HeLa cells were transfected with GFP-Peli1. At 48 h, cells were subjected to laser micro-irradiation. Laser stripes were examined at the indicated time point ( e ). The intensity of each laser stripe was determined by averaging values from ten cells at each time point and graphed ( f ). g H2AX WT and KO HeLa cells were transfected with FLAG empty vector (EV), FLAG-H2AX WT, S139A, or S139E mutant. At 48 h post transfection, cells were subjected to laser micro-irradiation. After 10 min post micro-irradiation, cells were fixed and immunostained with indicated antibodies. Scale bar, 10 μM. h H2AX HeLa cells were cotransfected with H2AX-GFP WT, S139A, or S139E mutant. After 48 h, cells were collected and immunoprecipitated with an anti-GFP antibody. These protein complexes were subjected to immunoblotting with indicated antibodies. i 293T cells were cotransfected with FLAG-H2AX WT and GFP empty vector (EV), GFP-Peli1 WT, or GFP-Peli1 E4 truncated. At 48 h post transfection, cells were collected and immunoprecipitated with an anti-FLAG antibody. These protein complexes were subjected to immunoblotting with antibodies shown

Article Snippet: HeLa H2AX (WT and KO) cells were provided by Dr. Makoto Nakanishi (University of Tokyo, Japan).

Techniques: Irradiation, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation

Journal: Nature Communications

Article Title: Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks

doi: 10.1038/s41467-019-09641-9

Figure Lengend Snippet: Peli1-mediated ATM activation and NBS1 interaction upon DNA damage. a Peli1 WT and ΔE4 MEF cells were treated with 4 Gy IR. At indicated time, cells were collected and then subjected to immunoblotting. b Peli1 WT and E4 truncated MEF cells were micro-irradiated, fixed after 10 min, and immunostained with anti-p-ATM antibody (left panels). Mean levels of p-ATM accumulation at sites of laser tracks were quantified using Image J software and plotted as indicated (right panels). Data show mean ± SEM; n = 20 cells. Student’s t -test was used for statistical analyses. Scale bars, 10 μm. c Peli1 E4 truncated MEFs were transfected with a control pBabe or pBabe-Peli1 (Myc-Peli1) retrovirus and treated with IR (4 Gy). At 1 h post-IR, cells were collected and immunoblotted with anti-Peli1, anti-p-ATM, anti-ATM, and anti-actin antibodies. d Peli1 E4-truncated MEFs were transfected with pBabe or pBabe-Peli1 (Myc-Peli1) retrovirus for 24 h and then treated with IR (4 Gy). At 1 h post-IR, cells were fixed and immunostained with anti-p-ATM antibodies. Scale bars, 10 μm. e 293T cells were transfected with TAP (control) or TAP-Peli1 (Flag-tagged Peli1). At 36 h, cells were treated with or without IR (4 Gy). At 30 min post-IR, cells were collected and isolated through S-tag pull-down assay. Bound proteins were immunoblotted with indicated antibodies. f IR-treated or non-treated 293T cell lysates were immunoprecipitated with anti-NBS1 or anti-Peli1 antibody and immunoblotted with antibodies indicated. γH2AX was used as a positive marker of IR in input panel

Article Snippet: HeLa H2AX (WT and KO) cells were provided by Dr. Makoto Nakanishi (University of Tokyo, Japan).

Techniques: Activation Assay, Western Blot, Irradiation, Software, Transfection, Control, Isolation, Pull Down Assay, Immunoprecipitation, Marker

Journal: Nature Communications

Article Title: Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks

doi: 10.1038/s41467-019-09641-9

Figure Lengend Snippet: Peli1-mediated NBS1 ubiquitination stabilizes the Mre11, RAD50, and NBS1 (MRN) complex. a 293T cells were transfected with shLuc or Peli1 and treated with or without IR (10 Gy). At 30 min post-IR, cells were collected and immunoprecipitated with an anti-NBS1 antibody. NBS1 protein complexes were subjected to immunoblotting. b Immunoblotting of MRN complex components in Peli1 WT and E4 truncated MEF cells using antibodies indicated. c Peli1 WT and E4 truncated MEF cells were treated with proteasome inhibitor MG132 (10 μM) for 6 h. Cells were subsequently subjected to immunoblotting. d 293T cells were transfected with Myc-Peli1 expression plasmid. At 48 h post transfection, cells were collected and cell lysates were analyzed by immunoblotting with antibodies indicated. e siRNA-targeting control (CTRL) or endogenous Peli1 (3′-UTR) was transfected into U2OS cells in combination with GFP-NBS1. U2OS cells reintroduced with empty vector (EV), Myc-Peli1 WT, or RING mutant (HA) were subjected to micro-irradiation at 48 h. At 30 min post micro-irradiation, cells were fixed and immunostained with anti-Peli1 and anti-Myc antibodies. The intensity of GFP-NBS1 at laser stripes was determined by averaging values from 10 to 20 cells and graphed. Scale bars, 10 μm. f Peli1 WT and E4 truncated MEF cells were micro-irradiated with a UV laser, fixed after 10 min, and immunostained with anti-NBS1 and anti-γH2AX antibodies (upper panel). Scale bar, 10 μM. Mean levels of NBS1 accumulation at sites of laser tracks were quantified (lower panel). Student’s t -test was used for statistical analyses

Article Snippet: HeLa H2AX (WT and KO) cells were provided by Dr. Makoto Nakanishi (University of Tokyo, Japan).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Control, Mutagenesis, Irradiation

Journal: Nature Communications

Article Title: Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks

doi: 10.1038/s41467-019-09641-9

Figure Lengend Snippet: Model of Peli1-mediated DNA damage signaling and HR repair. Peli1 recruitment to DSB sites is triggered by ATM-mediated phosphorylation and directly binds to phosphorylated histone H2AX (Step I). Once recruited, Peli1 interacts with NBS1 and catalyzes K63-linked ubiquitination of NBS1, leading to further activation of ATM, which reinforces DNA-end resection and HR repair (Step II)

Article Snippet: HeLa H2AX (WT and KO) cells were provided by Dr. Makoto Nakanishi (University of Tokyo, Japan).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Activation Assay