Figure 4A , B , Fluorescence microscopy images ( A ) and quantification ( B ) of mitophagy of HeLa wild - type ( WT ) , PentaKO + empty vector , PentaKO + NDP52 WT or NDP52 C18 , 153 , 163 , 321S ( Mut ) cells , stably expressing YFP - Parkin and mt - mKeima were pre - treated with or without MitoQ for 22 h and treated with 4 µM / 10 µM AO for the indicated times . Data information Data are mean ± s . e . m . ( B or displayed as cell popular violin plots ( B ) . P values were calculated by one - way ANOVA followed by Sidak test on three independent experiments ( B * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § , p < 0 . 05 , § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 μm ( AC , HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the absence or presence of 10 μM MG132 ( MG ) and 400 nM Baf , followed by a mitochondrial fractionation and immunoblotting for NDP52 in non - reducing conditions . The mitochondrial protein UQCRC2 was used as a loading control . Data information : # , non - specific band or a post - translational modification . D , HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima were treated with 1 mM DFP for 24 h ( D ) , or pre - treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h ( D Cells were then analysed by MitoSOX staining ( D ) Data information : # , Data are mean ± s . e . m . D ) P values were calculated by one - way ANOVA followed by Sidak test on three independent experiments D ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § , p < 0 . 05 , § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 μm D ) . E , HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima were treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h E ) in the presence or absence of 400 nM Baf ( E ) . immunoblotting for NDP52 ( E ) . Data information : # , non - specific band or a post - translational modification .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, B, Fluorescence microscopy images (A) and quantification (B) of mitophagy of HeLa wild-type (WT), PentaKO + empty vector, PentaKO + NDP52 WT or NDP52 C18,153,163,321S (Mut) cells, stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without MitoQ for 22 h and treated with 4 µM / 10 µM AO for the indicated times. Data information Data are mean ± s.e.m. (B or displayed as cell popular violin plots (B). P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments (B *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm (AC, HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the absence or presence of 10 μM MG132 (MG) and 400 nM Baf, followed by a mitochondrial fractionation and immunoblotting for NDP52 in non-reducing conditions. The mitochondrial protein UQCRC2 was used as a loading control. Data information: #, non-specific band or a post-translational modification.D, HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 1 mM DFP for 24 h (D), or pre-treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h (D Cells were then analysed by MitoSOX staining (D) Data information: #, Data are mean ± s.e.m. D) P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments D). *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm D).E, HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h E) in the presence or absence of 400 nM Baf (E). immunoblotting for NDP52 (E). Data information: #, non-specific band or a post-translational modification.


Figure 5A , Fluorescence microscopy images and quantification of mitophagy in HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM rotenone ( R ) , 4 µM A , 10 µM O , or the combination of RO or AO for 3h . Data information Data are mean ± s . e . m . ( A or displayed as cell popular violin plots ( A , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated ( A , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm ( AB , Fluorescence microscopy images and quantification of MitoSOX staining in HeLa PentaKO + NDP52 WT cells in the same conditions as ( A ) . Data information Data are mean ± s . e . m . B , or displayed as cell popular violin plots B , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated B , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm B , HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima ( C , were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h , followed by mitophagy measurement ( C ) Data information Data are mean ± s . e . m . C , or displayed as cell popular violin plots C , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated C , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μmHeLa PentaKO + NDP52 WT cells ( D ) were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h , followed by MitoSOX staining ( D ) Data information Data are mean ± s . e . m . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm , DHeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima E ) were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h , followed by immunoblotting for NDP52 in non - reducing conditions ( E ) . Data information : # , non - specific band or a post - translational modification . F , HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin and mt - mKeima were pre - treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by mitophagy measurements ( F ) Data information Data are mean ± s . e . m . F ) or displayed as cell popular violin plots F ) . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated F ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm F ) . G , HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin and mt - mKeima were pre - treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by immunoblotting for NDP52 in non - reducing conditions ( G ) . Data information : # , non - specific band or a post - translational modification .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, Fluorescence microscopy images and quantification of mitophagy in HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM rotenone (R), 4 µM A, 10 µM O, or the combination of RO or AO for 3h. Data information Data are mean ± s.e.m. (A or displayed as cell popular violin plots (A, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated (A, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm (AB, Fluorescence microscopy images and quantification of MitoSOX staining in HeLa PentaKO + NDP52 WT cells in the same conditions as (A). Data information Data are mean ± s.e.m. B, or displayed as cell popular violin plots B, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated B, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm B,HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima (C, were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by mitophagy measurement (C) Data information Data are mean ± s.e.m. C, or displayed as cell popular violin plots C, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated C, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μmHeLa PentaKO + NDP52 WT cells (D) were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by MitoSOX staining (D) Data information Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm , DHeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima E) were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by immunoblotting for NDP52 in non-reducing conditions (E). Data information: #, non-specific band or a post-translational modification.F, HeLa PentaKO + NDP52 WT stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by mitophagy measurements (F) Data information Data are mean ± s.e.m. F) or displayed as cell popular violin plots F). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated F). *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm F).G, HeLa PentaKO + NDP52 WT stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by immunoblotting for NDP52 in non-reducing conditions (G). Data information: #, non-specific band or a post-translational modification.


Figure 6HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf , followed by immunofluorescence analyses ( A ) The number of foci of the indicated proteins colocalised with NDP52 , or foci of NDP52 colocalised with Parkin , was quantified . Data information : Data are mean ± s . e . m . ( A , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments ( A * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - significant ) . Scale bars : 20 μm ( A , HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf , followed by immunoblotting for LC3 and NDP52 in mitochondrial fraction ( B ) or whole cell lysate ( C ) in reducing conditions Data information : Data are mean ± s . e . m . B , C , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments B , C * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - significant ) . HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf , followed by co - immunoprecipitation assay to analyse the interaction of NDP52 with FIP200 , ULK1 and ATG13 ( D ) . Data information : Data are mean ± s . e . m . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments D , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - significant ) . E , MEFs stably expressing YFP - Parkin , mt - mKeima and empty , human NDP52 ( hNDP52 ) WT or hNDP52 Mut were pre - treated with or without 500 nM MitoQ for 21 h and treated with AO for 3 h followed by mitophagy measurement . Data information : Data are mean ± s . e . m . E ) or displayed as cell popular violin plots ( E ) . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments E ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 μm E ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunofluorescence analyses (A) The number of foci of the indicated proteins colocalised with NDP52, or foci of NDP52 colocalised with Parkin, was quantified. Data information: Data are mean ± s.e.m. (A, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments (A *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant). Scale bars: 20 μm (A,HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunoblotting for LC3 and NDP52 in mitochondrial fraction (B) or whole cell lysate (C) in reducing conditions Data information: Data are mean ± s.e.m. B, C, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments B, C *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by co-immunoprecipitation assay to analyse the interaction of NDP52 with FIP200, ULK1 and ATG13 (D). Data information: Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments D, *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).E, MEFs stably expressing YFP-Parkin, mt-mKeima and empty, human NDP52 (hNDP52) WT or hNDP52 Mut were pre-treated with or without 500 nM MitoQ for 21 h and treated with AO for 3 h followed by mitophagy measurement. Data information: Data are mean ± s.e.m. E) or displayed as cell popular violin plots (E). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments E). *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm E).


Figure 1 ( B ) Serum CRAMP determination by ELISA ( n = 8 ) . Data information : Data in B were representative and were the mean ± SD from three independent experiments . P values were calculated by unpaired two - tailed t - test for comparison of two groups ( E ) Localization and expression of CRAMP ( red ) , E - cadherin ( green ) , F4 / 80 ( green ) and Ly6G ( green ) in duodenum by immunofluorescent staining . Representative photomicrographs of individual and merged staining were shown . Nuclei were stained with DAPI ( blue ) . Scale bar : 50 μm . Data information : Data were representative from three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Serum CRAMP determination by ELISA (n = 8). Data information: Data in B were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups(E) Localization and expression of CRAMP (red), E-cadherin (green), F4/80 (green) and Ly6G (green) in duodenum by immunofluorescent staining. Representative photomicrographs of individual and merged staining were shown. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data information: Data were representative from three independent experiments.


Figure 2 ( C ) Left , representative images of duodenal damage by H & E staining . Scale bar : 200 μm . Right , graph depicted the ratio of the morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . . ( D ) Western blot and densitometry analyses of duodenal tight junction proteins ( claudin - 1 , occludin , ZO - 1 and ZO - 2 ; n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . . ( E ) Western blot and densitometry analysis of duodenal zonulin ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..(D) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..(E) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


Figure 3 ( D ) Left , representative images of duodenal damage by H & E staining . Scale bar : 200 μm . Right , graph depicted the ratio of the morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . ( E ) Western blot and densitometry analyses of duodenal tight junction proteins ( claudin - 1 , occludin , ZO - 1 and ZO - 2 ; n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . ( F ) Western blot and densitometry analysis of duodenal zonulin ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

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Figure 3(D) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(E) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(F) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


Figure 4 ( A ) Western blot and densitometry analyses of gliadin - triggered inflammatory signaling [ p - EGFR ( Tyr1068 ) , p - Akt , Akt , MyD88 , TRAF6 , p - p65 and p65 ] in epithelial cells ex vivo ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

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Figure 4(A) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65 and p65] in epithelial cells ex vivo (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


Figure 5 ( E - G ) The percentages of ( E ) Foxp3 + Treg , ( F ) T - bet + Th1 and ( G ) RORγt + Th17 among CD3 + CD4 + population in lamina propria were shown by flow cytometry ( n = 8 ) . Data information : Data were representative and were the median ± interquartile range from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

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Figure 5(E-G) The percentages of (E) Foxp3+ Treg, (F) T-bet+ Th1 and (G) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


Figure 6 ( D ) Duodenal colonizing P . aeruginosa and A . muciniphila as determined by RT - qPCR using bacterial - specific species gene primers ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by unpaired two - tailed t - test for comparison of two groups ( E ) The production of LasB ( a protease of P . aeruginosa ) was examined in duodenum ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by unpaired two - tailed t - test for comparison of two groups ( H ) Duodenal colonizing PA14 and PAO1 as determined by RT - qPCR using P . aeruginosa - specific species gene primer ( n = 9 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple

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Figure 6(D) Duodenal colonizing P. aeruginosa and A. muciniphila as determined by RT-qPCR using bacterial-specific species gene primers (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups(E) The production of LasB (a protease of P. aeruginosa) was examined in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups(H) Duodenal colonizing PA14 and PAO1 as determined by RT-qPCR using P. aeruginosa-specific species gene primer (n = 9). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons


Figure 7 ( C ) The levels of CRAMP in duodenum ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . ( D ) Representative images of intestinal damage by H & E staining . Scale bar : 200 μm . The graph depicted the ratio of the morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . ( E ) Western blot and densitometry analyses of tight junction proteins ( claudin - 1 and occludin ) and zonulin in duodenum ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . ( I ) The percentages of three subsets of CD45 + CD11c + DCs ( n = 8 ) . ( J - L ) The percentages of ( J ) Foxp3 + Treg , ( K ) T - bet + Th1 and ( L ) RORγt + Th17 among CD3 + CD4 + population in lamina propria were shown by flow cytometry ( n = 8 ) . Data information : Data were representative and were the median ± interquartile range from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

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Figure 7(C) The levels of CRAMP in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(D) Representative images of intestinal damage by H&E staining. Scale bar: 200 μm. The graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(E) Western blot and densitometry analyses of tight junction proteins (claudin-1 and occludin) and zonulin in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.(I) The percentages of three subsets of CD45+CD11c+ DCs (n = 8). (J-L) The percentages of (J) Foxp3+ Treg, (K) T-bet+ Th1 and (L) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


Figure 1A , B Representative size - exclusion chromatography profiles of the MICU1 - MICU2 complex and chimaera in 2 mM EGTA ( A ) and 2 mM CaCl2 ( B ) conditions .

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Figure 1A, B Representative size-exclusion chromatography profiles of the MICU1-MICU2 complex and chimaera in 2 mM EGTA (A) and 2 mM CaCl2 (B) conditions.


Figure 4A The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA . B The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2 + . C Comparison of binding of MICU1 and EMRE peptide in both conditions . D MICU1 - MICU2 heterodimer interacts with the EMRE peptide at a 1 : 1 ratio . E Carbachol ( CCh ) - induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt - EMRE and scramble peptides . Solid lines , mean ; shaded regions , ± SEM , n = 3 . F Resting mitochondrial Ca2 + levels in mt - EMRE and scramble peptides treated cells , evaluated via ratiometric imaging of the mitochondrial targeted mt - riG6m . Mean ± s . d . , student t - test , * * p < 0 . 01 , n = 3 . Data information : Error bars for some points are too small to be visible . G Mean mitochondrial Ca2 + peaks response to 100 μM CCh . Mean ± s . d . , student t - test , * * * p ≤ 0 . 001 , n = 3 . Data information : Error bars for some points are too small to be visible . H Representative [ Ca2 + ] c clearance traces of U2OS cells treated with PBS and mt - EMRE peptide . I Quantification of mitochondria Ca2 + uptake rate in response to 100 μM histamine . Mean ± s . d . , student t - test , n . s . , not significant , n = 4 . Data information : Error bars for some points are too small to be visible .

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Figure 4A The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA. B The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2+. C Comparison of binding of MICU1 and EMRE peptide in both conditions.D MICU1-MICU2 heterodimer interacts with the EMRE peptide at a 1:1 ratio.E Carbachol (CCh)-induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt-EMRE and scramble peptides. Solid lines, mean; shaded regions, ± SEM, n = 3.F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3. Data information: Error bars for some points are too small to be visible.G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3. Data information: Error bars for some points are too small to be visible.H Representative [Ca2+]c clearance traces of U2OS cells treated with PBS and mt-EMRE peptide.I Quantification of mitochondria Ca2+ uptake rate in response to 100 μM histamine. Mean ± s.d., student t-test, n.s., not significant, n = 4. Data information: Error bars for some points are too small to be visible.


Figure 1 ( B ) Western blot analyses of whole - cell lysates of wild - type ( CC - 124 ) , bld10 , bld10 : : B10 - N - HA , bld10 : : B10 - M - HA , and bld10 : : B10 - C - HA cells using antibodies against the C - terminal region of Bld10p ( Anti - Bld10 - C antibody , upper blot ) and the HA antigen ( Anti - HA antibody , lower blot ) . The apparent molecular weights of tagged Bld10ps are slightly larger than that of native Bld10p due to their HA tags . ( C ) Percentages of flagellated cells in wild - type ( CC - 124 ) , bld10 , bld10 : : B10 - N - HA , bld10 : : B10 - M - HA , bld10 : : B10 - C - HA , bld12 , bld10bld12 , bld10bld12 : : ∆ N3 , bld10bld12 : : ∆ C2 , bld10 : : ∆ N3 , and bld10 : : ∆ C2 cells . Cells with two ( red ) , one ( green ) , and zero ( blue ) flagella were counted . n , number of cells counted .

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Figure 1(B) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld10, bld10::B10-N-HA, bld10::B10-M-HA, and bld10::B10-C-HA cells using antibodies against the C-terminal region of Bld10p (Anti-Bld10-C antibody, upper blot) and the HA antigen (Anti-HA antibody, lower blot). The apparent molecular weights of tagged Bld10ps are slightly larger than that of native Bld10p due to their HA tags.(C) Percentages of flagellated cells in wild-type (CC-124), bld10, bld10::B10-N-HA, bld10::B10-M-HA, bld10::B10-C-HA, bld12, bld10bld12, bld10bld12::∆N3, bld10bld12::∆C2, bld10::∆N3, and bld10::∆C2 cells. Cells with two (red), one (green), and zero (blue) flagella were counted. n, number of cells counted.


Figure 2 ( D ) Immunoelectron micrographs of wild type centrioles treated with anti - Bld10p ( B10 - C ) after embedding in the resin and sectioning . Localization of 16 gold particles in 13 centriole images are plotted on the triangular map ( the right panel ) . Data information : Scale bar , 100 nm .

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Figure 2(D) Immunoelectron micrographs of wild type centrioles treated with anti-Bld10p (B10-C) after embedding in the resin and sectioning. Localization of 16 gold particles in 13 centriole images are plotted on the triangular map (the right panel). Data information: Scale bar, 100 nm.


Figure 3 ( C ) A schematic diagram showing the positions of the observed filamentous structures ( L1 , L2 , and L3 ) . The distance between each filamentous structure and the inner side of the A - tubule is indicated in parentheses ( mean ± SEM ) . The numbers of observed L1 , L2 , and L3 are 25 , 19 , and 13 , respectively . ( D ) Slices from the reconstructed cryo - electron tomograms of probasal bodies . Lower panels show the regions with L1 , L2 and L3 , boxed from the upper panels . Red dots denote the connection points between the spokes and filamentous structures . Data information : Scale bar , 100 nm .

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Figure 3(C) A schematic diagram showing the positions of the observed filamentous structures (L1, L2, and L3). The distance between each filamentous structure and the inner side of the A-tubule is indicated in parentheses (mean ± SEM). The numbers of observed L1, L2, and L3 are 25, 19, and 13, respectively. (D) Slices from the reconstructed cryo-electron tomograms of probasal bodies. Lower panels show the regions with L1, L2 and L3, boxed from the upper panels. Red dots denote the connection points between the spokes and filamentous structures. Data information: Scale bar, 100 nm.


Figure 4 ( A ) Western blot analyses of whole - cell lysates of wild - type ( CC - 124 ) , bld12 , bld10 , bld10bld12 , and bld10bld12 : : ∆ N3 cells using an antibody against the C - terminal one - third of Bld10p ( Anti - Bld10 - C antibody ) . The band corresponding to truncated Bld10p ( Bld10p ∆ N3 ) overlaps with a non - specific band at 75 kDa . ( B ) Western blot analyses of whole - cell lysates of wild - type ( CC - 124 ) , bld12 , bld10 , bld10bld12 , and bld10bld12 : : ∆ C2 cells using an antibody raised against the N - terminal 120 amino - acid sequence of Bld10p ( Anti - Bld10 - N antibody ) . ( C ) Cross - sectional electron micrographs of centrioles in bld12 , bld10 : : ∆ N3 , bld10bld12 : : ∆ N3 , bld10 : : ∆ C2 , and bld10bld12 : : ∆ C2 cells . Scale bar , 100 nm . ( D ) Distribution of the number of triplets in centrioles of bld12 , bld10 : : ∆ N3 , bld10bld12 : : ∆ N3 , bld10 : : ∆ C2 , and bld10bld12 : : ∆ C2 cells . Red lines denote the normal number of microtubules in the centriole ( nine ) . n , number of centrioles observed .

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Figure 4(A) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld12, bld10, bld10bld12, and bld10bld12::∆N3 cells using an antibody against the C-terminal one-third of Bld10p (Anti-Bld10-C antibody). The band corresponding to truncated Bld10p (Bld10p ∆N3) overlaps with a non-specific band at 75 kDa. (B) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld12, bld10, bld10bld12, and bld10bld12::∆C2 cells using an antibody raised against the N-terminal 120 amino-acid sequence of Bld10p (Anti-Bld10-N antibody).(C) Cross-sectional electron micrographs of centrioles in bld12, bld10::∆N3, bld10bld12::∆N3, bld10::∆C2, and bld10bld12::∆C2 cells. Scale bar, 100 nm. (D) Distribution of the number of triplets in centrioles of bld12, bld10::∆N3, bld10bld12::∆N3, bld10::∆C2, and bld10bld12::∆C2 cells. Red lines denote the normal number of microtubules in the centriole (nine). n, number of centrioles observed.


Figure 5 ( A and B ) Cross - sectional electron micrographs of the proximal ( A ) and distal ( B ) regions of centrioles containing eight or nine triplets in bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 cells . The arrangement of triplets in images of axonemes were circularized using CentrioleJ program The colored circles represent the circles inscribed to the innermost positions of the A - tubules in the centriole . Scale bar , 100 nm . ( C and D ) Distances between adjacent A - tubules at the proximal ( C ) and distal ( D ) regions of centrioles containing seven , eight , nine , and ten triplets in wild - type ( cw92 ) , bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 cells . The distances between the contact points of A - tubules with the inscribed circle were measured . These distances were ~ 10 % shorter in cells expressing truncated Bld10ps than in wild - type and bld12 cells . * * * , P < 0 . 0001 . n , number of centrioles measured . Data information : Data were analyzed by unpaired t - test . The graphs show mean ± SEM . ( E ) Lengths of A - C linkers in wild - type ( cw92 ) , bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 centrioles . Centrioles containing nine triplets and eight triplets were separately analyzed . n , number of A - Clinkers measured . ( F ) Angles between adjacent triplets in centrioles analyzed in panel € . The angle ( θ ) measured was defined as indicated in the schematic diagram ; the angle between the line connecting the centers of B - tubules of the two adjacent triplets ( black line ) and the line connecting the centers of A - and C - tubules of the triplet of the two ( brown line ) . * , P < 0 . 01 . n , number of triplet angles measured . Data information : Data were analyzed by unpaired t - test . The graphs show mean ± SEM .

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Figure 5(A and B) Cross-sectional electron micrographs of the proximal (A) and distal (B) regions of centrioles containing eight or nine triplets in bld12, bld10bld12::∆N3, and bld10bld12::∆C2 cells. The arrangement of triplets in images of axonemes were circularized using CentrioleJ program The colored circles represent the circles inscribed to the innermost positions of the A-tubules in the centriole. Scale bar, 100 nm. (C and D) Distances between adjacent A-tubules at the proximal (C) and distal (D) regions of centrioles containing seven, eight, nine, and ten triplets in wild-type (cw92), bld12, bld10bld12::∆N3, and bld10bld12::∆C2 cells. The distances between the contact points of A-tubules with the inscribed circle were measured. These distances were ~10% shorter in cells expressing truncated Bld10ps than in wild-type and bld12 cells. ***, P<0.0001. n, number of centrioles measured. Data information: Data were analyzed by unpaired t-test. The graphs show mean ± SEM.(E) Lengths of A-C linkers in wild-type (cw92), bld12, bld10bld12::∆N3, and bld10bld12::∆C2 centrioles. Centrioles containing nine triplets and eight triplets were separately analyzed. n, number of A-Clinkers measured. (F) Angles between adjacent triplets in centrioles analyzed in panel €. The angle (θ) measured was defined as indicated in the schematic diagram; the angle between the line connecting the centers of B-tubules of the two adjacent triplets (black line) and the line connecting the centers of A- and C-tubules of the triplet of the two (brown line). *, P<0.01. n, number of triplet angles measured. Data information: Data were analyzed by unpaired t-test. The graphs show mean ± SEM.


Figure 1 ( A ) LC - MS / MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA . Signal intensities of the modifications are scaled to that for m1A , the ribonucleoside with the highest MS

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Figure 1(A) LC-MS/MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA. Signal intensities of the modifications are scaled to that for m1A, the ribonucleoside with the highest MS signal


Figure 2 ( A ) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC - MS / MS in total tRNA extracted from parasites across the IDC time course . The average fold - change values ( relative to an arbitrary average control ) were subjected to hierarchical clustering analysis ( log2 - transformed data ) . The scale of the heat map has saturated the fold - change values of some modifications . Refer to Appendix Table S6 . ( B ) Three groups of modifications show differential regulation during the course of IDC . The majority ( 22 ) increase synchronously across the IDC ( blue ) , while 4 show asynchronous or irregular behavior ( purple ) and 2 ( orange ) are uniquely up - regulated in the ring stage . Log2Fold - change values plotted in the three graphs represent mean ± SEM ( N = 3 ) . Differences between the highest value ( denoted as peak ) and the lowest value ( denoted as trough ) were subjected to a two - tailed Student ' s t - test : NS , not

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC-MS/MS in total tRNA extracted from parasites across the IDC time course. The average fold-change values (relative to an arbitrary average control) were subjected to hierarchical clustering analysis (log2-transformed data). The scale of the heat map has saturated the fold-change values of some modifications. Refer to Appendix Table S6. (B) Three groups of modifications show differential regulation during the course of IDC. The majority (22) increase synchronously across the IDC (blue), while 4 show asynchronous or irregular behavior (purple) and 2 (orange) are uniquely up-regulated in the ring stage. Log2Fold-change values plotted in the three graphs represent mean ± SEM (N = 3). Differences between the highest value (denoted as peak) and the lowest value (denoted as trough) were subjected to a two-tailed Student's t-test: NS, not significant


Figure 3iTRAQ proteomics analysis ( Appendix Fig . S2 ) resulted in 2 , 100 proteins consistently quantified at every point across the IDC . The heat map shows hierarchical clustering analysis of fold - changes ( log2 - transformed ) in the levels of the proteins relative to an arbitrary average across the IDC . The 100 most up - regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P . falciparum 3D7 . The significantly ( P < 0 . 05 ) enriched pathways among the up - regulated proteins at each time point are summarized in tables for each time

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Figure 3iTRAQ proteomics analysis (Appendix Fig. S2) resulted in 2,100 proteins consistently quantified at every point across the IDC. The heat map shows hierarchical clustering analysis of fold-changes (log2-transformed) in the levels of the proteins relative to an arbitrary average across the IDC. The 100 most up-regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P. falciparum 3D7. The significantly (P < 0.05) enriched pathways among the up-regulated proteins at each time point are summarized in tables for each time point


Figure 5LC - MS / MS analysis was performed on T1 - digested tRNA and deconvolution yielded precise masses of the unmodified ( 3055 . 425 Da ) and modified ( 3143 . 43 Da ) oligos for Glu - tRNAGAA ( BCollision - induced dissociation of the parent molecular ions for m / z 1046 . 477 - 3 from Glu - tRNAGA produced expected - c , - w and - y ions for the sequences CU [ mcm5s2U ] UCACCCLC - MS / MS analysis was performed on T1 - digested tRNA and deconvolution yielded precise masses of th unmodified ( 1835 . 20 Da ) and modified ( 1923 . 262 Da ) oligos for Gln - tRNACAA ( E ) Collision - induced dissociation of the parent molecular ions fo m / z 1923 . 262 from Gln - tRNACAA produced expected - c , - w and - y ions for the sequence CC [ mcm5s2U ] CCAARatios of modified oligonucleotides from the anticodon stem - loop o ( H ) GluGA isoacceptors expressed as percentages of their sum totalRatios of modified oligonucleotides from the anticodon stem - loop o ( G ) GlnCARatios of modified oligonucleotides from the anticodon stem - loop o ( I ) GlyGGA isoacceptors expressed as percentages of their sum

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 5LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of the unmodified (3055.425 Da) and modified (3143.43 Da) oligos for Glu-tRNAGAA (BCollision-induced dissociation of the parent molecular ions for m/z 1046.477-3 from Glu-tRNAGA produced expected -c, -w and -y ions for the sequences CU[mcm5s2U]UCACCCLC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of th unmodified (1835.20 Da) and modified (1923.262 Da) oligos for Gln-tRNACAA (E)Collision-induced dissociation of the parent molecular ions fo m/z 1923.262 from Gln-tRNACAA produced expected -c, -w and -y ions for the sequence CC[mcm5s2U]CCAARatios of modified oligonucleotides from the anticodon stem-loop o (H)GluGA isoacceptors expressed as percentages of their sum totalRatios of modified oligonucleotides from the anticodon stem-loop o (G)GlnCARatios of modified oligonucleotides from the anticodon stem-loop o (I)GlyGGA isoacceptors expressed as percentages of their sum total


Figure 6 ( A , B ) Comparison of arbitrary translation efficiency ( TE ) values of ( A ) late trophozoite stage ( TP 40 ) and ( B ) schizont stage ( TP 46 ) proteins across all experimental time points . The arbitrary TE is defined here as the normalized protein abundance per mRNA abundance to mimic the translational output per mRNA copy in a biological system . The top 5 % up - regulated proteins ( n = 100 ) at ( A ) late trophozoite stage ( TP 40 ) and ( B ) schizont stage ( TP 46 ) were used for this analysis . Differences among all groups were first tested by one - way ANOVA analysis , followed by Dunnett ' s test as multiple comparison post - test to find significant differences between pairs of ( A ) TP 40 or ( B ) TP 46 and other time points . P < 0 . 01 is denoted as * * . P < 0 . 0001 is denoted as * * * * . Scatterplots of mRNA and protein fold - changes ( log - transformed ) at time points ( C ) 40 h and ( D ) 46 h in the erythrocytic cycle . Quadrants are designated with roman numerals . Data points falling into quadrant I are designated as high translationally efficient ( HTE ) proteins while those falling into quadrant IV are designated low translationally efficient ( LTE )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A, B) Comparison of arbitrary translation efficiency (TE) values of (A) late trophozoite stage (TP 40) and (B) schizont stage (TP 46) proteins across all experimental time points. The arbitrary TE is defined here as the normalized protein abundance per mRNA abundance to mimic the translational output per mRNA copy in a biological system. The top 5% up-regulated proteins (n = 100) at (A) late trophozoite stage (TP 40) and (B) schizont stage (TP 46) were used for this analysis. Differences among all groups were first tested by one-way ANOVA analysis, followed by Dunnett's test as multiple comparison post-test to find significant differences between pairs of (A) TP 40 or (B) TP 46 and other time points. P<0.01 is denoted as **. P<0.0001 is denoted as ****. Scatterplots of mRNA and protein fold-changes (log-transformed) at time points (C) 40 h and (D) 46 h in the erythrocytic cycle. Quadrants are designated with roman numerals. Data points falling into quadrant I are designated as high translationally efficient (HTE) proteins while those falling into quadrant IV are designated low translationally efficient (LTE) proteins


Figure 1 ( C ) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study . The relevant markers are listed to the left of the micrographs . Scale bar : 5 µm . ( D ) Pie charts showing the proportion of specific phenotype mutants ( SPMs ) that have one or more distinct aberrant phenotypes , and non - phenotype - specific mutants for each of the compartments screened .

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Figure 1(C) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study. The relevant markers are listed to the left of the micrographs. Scale bar: 5 µm. (D) Pie charts showing the proportion of specific phenotype mutants (SPMs) that have one or more distinct aberrant phenotypes, and non-phenotype-specific mutants for each of the compartments screened.


Figure 2Representative images of wild - type and mutant cells organized by marker and phenotype ( labels on the left of each panel ) . For each phenotype , three cells labelled for the training set ( labelled single cells ) and cells identified by the 2NN classifier ( identified single cells ) are shown . The table to the right of the images shows ( from left to right ) : [ 1 ] the occurrence of each phenotype in a wild - type population ( % in WT ) ; [ 2 ] the number of specific phenotype mutants ( all ) and stringent specific phenotype mutants ( str ) for each of the 17 mutant morphologies ; [ 3 ] the most significantly enriched GO Slim biological process and ; [ 4 ] the most significantly enriched protein complex . # denotes term below statistical significance .

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Figure 2Representative images of wild-type and mutant cells organized by marker and phenotype (labels on the left of each panel). For each phenotype, three cells labelled for the training set (labelled single cells) and cells identified by the 2NN classifier (identified single cells) are shown. The table to the right of the images shows (from left to right): [1] the occurrence of each phenotype in a wild-type population (% in WT); [2] the number of specific phenotype mutants (all) and stringent specific phenotype mutants (str) for each of the 17 mutant morphologies; [3] the most significantly enriched GO Slim biological process and; [4] the most significantly enriched protein complex. # denotes term below statistical significance.


Figure 4Endocytic patch formation dynamics in the ydl176wΔ ( ipf1Δ ) strain . Patch dynamics were examined using time - lapse fluorescence microscopy of wild - type ( WT ) and ipf1Δ deletion strains carrying reporters for the coat ( Sla1 - GFP ; green ) , and actin ( Sac6 - tdTomato ; red ) modules . Left : Representative kymographs for the WT and ipf1Δ strains . Right : Box plot illustrating the distribution of lifetimes of Sla1 - GFP and Sac6 - tdTomato patches . Significance was determined using unpaired t - tests . Scale bar : 10 seconds .

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Figure 4Endocytic patch formation dynamics in the ydl176wΔ (ipf1Δ) strain. Patch dynamics were examined using time-lapse fluorescence microscopy of wild-type (WT) and ipf1Δ deletion strains carrying reporters for the coat (Sla1-GFP; green), and actin (Sac6-tdTomato; red) modules. Left: Representative kymographs for the WT and ipf1Δ strains. Right: Box plot illustrating the distribution of lifetimes of Sla1-GFP and Sac6-tdTomato patches. Significance was determined using unpaired t-tests. Scale bar: 10 seconds.


Figure 6Penetrance as a function of replicative age . Top : Bar graph showing the fraction of outliers in populations of increasing replicative age ( # of divisions ) for wild - type ( WT ) , and 5 mutant strains ( rrd2Δ , rpl20bΔ , cka2Δ , vac8Δ and vac17Δ ) . Data are presented as mean of 3 biological replicates + / - SD . Bottom : Box plot with the distribution of cell sizes for the same populations of cells . Whiskers extend to the 5th and 95th percentile . Micrographs of young ( top row of images ) and older ( bottom row of images ) wild - type ( WT ) cells expressing Vph1 - EGFP ( green vacuole ) and stained with CF640R WGA ( magenta bud scars ) . Scale bar : 5 µm . Combined effect of replicative age and a vacuole inheritance defect on penetrance . Micrographs of wild - type and vac17Δ cells expressing Vph1 - EGFP ( green vacuole ) and Hta2 - mCherry ( red nucleus ) , stained with CF640R WGA ( magenta bud scars ) . Cells with increasing bud - scar staining ( replicative age ) are shown from left to right . Scale bar : 5 µm . Relationship between stress response and penetrance . Single - cell UPRE - RFP levels were measured in ~ 60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology . Cells were binned into equal - sized bins , from low to high stress response , assessed as outlier or inlier , and clustered based on their penetrance profile ( composed from the fraction of outliers in each stress - response bin ) . Each line plot represents a penetrance profile . * denotes the cluster with a profile most similar to wild - type . Insert pie charts show the proportion of mutant strains in each cluster .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Penetrance as a function of replicative age. Top: Bar graph showing the fraction of outliers in populations of increasing replicative age (# of divisions) for wild-type (WT), and 5 mutant strains (rrd2Δ, rpl20bΔ, cka2Δ, vac8Δ and vac17Δ). Data are presented as mean of 3 biological replicates +/- SD. Bottom: Box plot with the distribution of cell sizes for the same populations of cells. Whiskers extend to the 5th and 95th percentile.Micrographs of young (top row of images) and older (bottom row of images) wild-type (WT) cells expressing Vph1-EGFP (green vacuole) and stained with CF640R WGA (magenta bud scars). Scale bar: 5 µm.Combined effect of replicative age and a vacuole inheritance defect on penetrance. Micrographs of wild-type and vac17Δ cells expressing Vph1-EGFP (green vacuole) and Hta2-mCherry (red nucleus), stained with CF640R WGA (magenta bud scars). Cells with increasing bud-scar staining (replicative age) are shown from left to right. Scale bar: 5 µm.Relationship between stress response and penetrance. Single-cell UPRE-RFP levels were measured in ~60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology. Cells were binned into equal-sized bins, from low to high stress response, assessed as outlier or inlier, and clustered based on their penetrance profile (composed from the fraction of outliers in each stress-response bin). Each line plot represents a penetrance profile. * denotes the cluster with a profile most similar to wild-type. Insert pie charts show the proportion of mutant strains in each cluster.


Figure 1 ( A ) C - circle assay for tumor samples . The presence of C - circles was tested in 22 osteosarcomas by Rolling Circle Amplification ( RCA ) assay using Φ29 DNA polymerase . Φ29 DNA polymerase negative controls and Φ29 DNA polymerase - based reactions starting respectively with 75 , 37 . 5 , and 18 . 75 ng of DNA were applied to dot blots and C - circles were detected by hybridization with a 32P - ( CCCTAA ) 4 telomeric probe . U2OS and HeLa cells correspond to positive and negative control samples , respectively . ( B ) Representative images of telomere FISH ( red ) and PML IF ( green ) colocalizations ( APBs ) in osteosarcoma tumor samples . DNA is counterstained blue with DAPI . APBs are indicated by white arrows . Scale bars are 5 μm . ( C ) Representative image of TeSLA Southern Blot of two representative samples of ALT - positive ( left ) and telomerase - positive ( right ) osteosarcomas . Nine TeSLA PCRs ( 30 pg each reaction ) were done for each DNA sample . DIG - labelled MW ladder has been added a posteriori . ( D ) Plot of cumulative TeSLA fragment sizes ( in kb ) in ALT - positive ( n = 16 ) and ALT - negative ( n = 6 ) osteosarcomas . Points and error bars ( ± SEM ) represent cumulative percentage of TeSLA fragments from all the TeSLA PCRs ( n = 9 per tumor sample ) from all the patients in both groups ( n = 16 in ALT - positive group , and n = 6 in ALT - negative group ) . The Kolmogorov - Smirnov test was applied to identify statistical differences in TeSLA fragments distributions ( p = 0 . 0016 ) . ( E ) Individual TeSLa fragment lengths in ALT - positive ( n = 16 ) and ALT - negative ( n = 6 ) osteosarcomas . Each dot represents a TeSLA fragment , bars represent the median value for individual patient ' s samples .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) C-circle assay for tumor samples. The presence of C-circles was tested in 22 osteosarcomas by Rolling Circle Amplification (RCA) assay using Φ29 DNA polymerase. Φ29 DNA polymerase negative controls and Φ29 DNA polymerase-based reactions starting respectively with 75, 37.5, and 18.75 ng of DNA were applied to dot blots and C-circles were detected by hybridization with a 32P-(CCCTAA)4 telomeric probe. U2OS and HeLa cells correspond to positive and negative control samples, respectively.(B) Representative images of telomere FISH (red) and PML IF (green) colocalizations (APBs) in osteosarcoma tumor samples. DNA is counterstained blue with DAPI. APBs are indicated by white arrows. Scale bars are 5 μm.(C) Representative image of TeSLA Southern Blot of two representative samples of ALT-positive (left) and telomerase-positive (right) osteosarcomas. Nine TeSLA PCRs (30 pg each reaction) were done for each DNA sample. DIG-labelled MW ladder has been added a posteriori. (D) Plot of cumulative TeSLA fragment sizes (in kb) in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Points and error bars (± SEM) represent cumulative percentage of TeSLA fragments from all the TeSLA PCRs (n=9 per tumor sample) from all the patients in both groups (n=16 in ALT-positive group, and n=6 in ALT-negative group). The Kolmogorov-Smirnov test was applied to identify statistical differences in TeSLA fragments distributions (p=0.0016). (E) Individual TeSLa fragment lengths in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Each dot represents a TeSLA fragment, bars represent the median value for individual patient's samples.


Figure 2 ( A ) Oncoprint graph showing the distribution of mutations ( green rectangles ) and copy number variations ( red rectangle , amplification ; blue rectangle , copy loss ) in all samples . Tumors are distributed according to ALT - positivity ( ALT + ) or - negativity ( ALT - ) and ATRX status ( ATRX - mutated or ATRX - wt ) . ( B ) Tissue sections of two representative high - grade osteosarcomas with HES ( hematoxylin eosin saffron ) staining ( left panel ) , anti - ATRX ( HPA064684 ) immunochemistry ( middle panel ) , and anti - DAXX ( HPA008736 ) immunochemistry ( right panel ) , showing ATRX protein expression in two ALT - positive samples . In the top panel , intratumoral ATRX expression is negative ( positive control osteoclasts are shown ) . In the bottom panel , intratumoral ATRX expression is high . Scale bars are 250 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Oncoprint graph showing the distribution of mutations (green rectangles) and copy number variations (red rectangle, amplification; blue rectangle, copy loss) in all samples. Tumors are distributed according to ALT-positivity (ALT+) or -negativity (ALT-) and ATRX status (ATRX-mutated or ATRX-wt).(B) Tissue sections of two representative high-grade osteosarcomas with HES (hematoxylin eosin saffron) staining (left panel), anti-ATRX (HPA064684) immunochemistry (middle panel), and anti-DAXX (HPA008736) immunochemistry (right panel), showing ATRX protein expression in two ALT-positive samples. In the top panel, intratumoral ATRX expression is negative (positive control osteoclasts are shown). In the bottom panel, intratumoral ATRX expression is high. Scale bars are 250 μm.


Figure 3 ( A ) Copy - number - alteration ( CNA ) profiles of osteosarcomas . Representation of the GISTIC analysis with false discovery rate ( FDR ( q - value ) ) for all cases ( n = 22 ; left panel ) , ALT - positive / ATRX - wt cases ( n = 11 ; middle panel ) , and ALT - positive / ATRX - mutated cases ( n = 5 ; right panel ) . ( B ) Gain / amplification of TOP3A gene region ( 17p11 ) is a genomic signature of ATRX - wt osteosarcomas . For each ATRX - mutated ( top part ) and ATRX - wt ( bottom part ) case , from the left to the right , the chromosome 17 and regional genomic profiles were established , with CGH Analytics software . A focus was made within the genomic interval ( 17 . 25 - 19 . 11 Mb ) of the short arm of chromosome 17 ( hg38 human genome mapping ; Build 38 from NCBI , December 2013 version ) including TOP3 . Color profiles corresponding to the different tumors are defined at the top of each group . Most ATRX - wt cases show gain or amplification of this region ( bottom part ) , whereas no ATRX - mutated cases display this alteration ( top part ) . ( C ) Chromosome 17 copy number variation analysis showing amplification / gain of the TOP3A region that do not extend into the TP53 gene in ALT ATRX - wt tumors ( left panel ) , and amplification of the TOP3A region for which TP53 region is at the edge of the amplification / gain in ALT - positive ATRX - wt tumors ( right panel ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Copy-number-alteration (CNA) profiles of osteosarcomas. Representation of the GISTIC analysis with false discovery rate (FDR (q-value)) for all cases (n=22; left panel), ALT-positive/ATRX-wt cases (n=11; middle panel), and ALT-positive/ATRX-mutated cases (n=5; right panel).(B) Gain/amplification of TOP3A gene region (17p11) is a genomic signature of ATRX-wt osteosarcomas. For each ATRX-mutated (top part) and ATRX-wt (bottom part) case, from the left to the right, the chromosome 17 and regional genomic profiles were established, with CGH Analytics software. A focus was made within the genomic interval (17.25-19.11 Mb) of the short arm of chromosome 17 (hg38 human genome mapping; Build 38 from NCBI, December 2013 version) including TOP3. Color profiles corresponding to the different tumors are defined at the top of each group. Most ATRX-wt cases show gain or amplification of this region (bottom part), whereas no ATRX-mutated cases display this alteration (top part).(C) Chromosome 17 copy number variation analysis showing amplification/gain of the TOP3A region that do not extend into the TP53 gene in ALT ATRX-wt tumors (left panel), and amplification of the TOP3A region for which TP53 region is at the edge of the amplification/gain in ALT-positive ATRX-wt tumors (right panel).


Figure 4 ( A ) Volcano plot of the supervised analysis of mRNA expression profiles of osteosarcomas according to ALT - positive vs . ALT - negative samples , showing upregulated ( in red ) and downregulated ( in green ) genes . Twelve tumors had sufficient RNA quality to be analyzed : seven ALT - positive ATRX - wt , two ALT - positive / ATRX - mutated and three ALT - negative . ( B ) TOP3A gene expression according to ALT and ATRX status : ALT + / ATRX - wt ( n = 7 ) , ALT + / ATRX - mut ( n = 2 ) and ALT - ( n = 3 ) . Box - and - whisker plot were defined with default parameters by median value ( central band at the 50th percentile ) , interquartile ranges ( IQR , box limited by 25th and 75th percentile ) and whisker boundaries defined by minimum and maximum value . An ANOVA statistical test , and Tukey ' s range test were used to compare modalities .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Volcano plot of the supervised analysis of mRNA expression profiles of osteosarcomas according to ALT-positive vs. ALT-negative samples, showing upregulated (in red) and downregulated (in green) genes. Twelve tumors had sufficient RNA quality to be analyzed: seven ALT-positive ATRX-wt, two ALT-positive/ATRX-mutated and three ALT-negative.(B) TOP3A gene expression according to ALT and ATRX status: ALT+/ATRX-wt (n=7), ALT+/ATRX-mut (n=2) and ALT- (n=3). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined by minimum and maximum value. An ANOVA statistical test, and Tukey's range test were used to compare modalities.


Figure 5 ( A ) Western blot showing ATRX protein expression in nine osteosarcoma cell lines and HTC116 as positive control . ( B ) Western blot showing TOP3A protein expression in nine osteosarcoma cell lines and NY siTOP3A as control . ( D ) C - circle assay showing inverse effect of ATRX ectopic expression on C - circle levels in ATRX - mutated and ATRX - wt cell lines . Error bars represent the mean ± SEM from n = 2 experiments , n . s . = non - significant , * p < 0 . 05 , Mann - Whitney test . ( E ) C - circle assay showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma cells Error bars represent the mean ± SEM from n = 2 experiments , n . s . = non - significant , * p < 0 . 05 , Mann - Whitney test . Western blot and quantification of TOP3A expression is shown for U2OS cells overexpressing TOP3A .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Western blot showing ATRX protein expression in nine osteosarcoma cell lines and HTC116 as positive control.(B) Western blot showing TOP3A protein expression in nine osteosarcoma cell lines and NY siTOP3A as control.(D) C-circle assay showing inverse effect of ATRX ectopic expression on C-circle levels in ATRX-mutated and ATRX-wt cell lines. Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test.(E) C-circle assay showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma cells Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test. Western blot and quantification of TOP3A expression is shown for U2OS cells overexpressing TOP3A.


Figure 6 ( B ) Representative images of telomeric DNA ( green ) and PML protein ( red ) colocalizations ( APBs ) in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD . Scale bars are 5 μm . ( C ) Quantification of APB frequency in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD ; Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , n . s . , non - significant ; Mann - Whitney test . ( D ) Effect of TOP3A KD on Telomere Dysfunction - Induced Foci ( TIFs ) in osteosarcoma and in vitro - immortalized cell lines ; Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , * p < 0 . 05 , * * p < 0 . 005 ; Mann - Whitney test . ( E ) Distributions of TeSLA fragments in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD ( left panel ; each dot represents a TeSLA fragment ) , and representative TeSLA Southern Blot image for NY ( right panel ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Representative images of telomeric DNA (green) and PML protein (red) colocalizations (APBs) in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD. Scale bars are 5 μm. (C) Quantification of APB frequency in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s., non-significant; Mann-Whitney test. (D) Effect of TOP3A KD on Telomere Dysfunction-Induced Foci (TIFs) in osteosarcoma and in vitro-immortalized cell lines; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, *p < 0.05, **p < 0.005; Mann-Whitney test.(E) Distributions of TeSLA fragments in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD (left panel; each dot represents a TeSLA fragment), and representative TeSLA Southern Blot image for NY (right panel).


Figure 7 ( A ) FISH - IF of telomeres ( in red ) and BLM ( in green ) in TOP3A - depleted cells BLM foci at telomeres are indicated by white arrows . Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , n . s . = non - significant , * * p < 0 . 01 , Mann - Whitney test . Scale bars are 5 μm . ( B ) ATSA assay assessing ALT - mediated telomeric DNA synthesis ( telomere in red , EdU in purple ) in PML ( in green ) according to TOP3A KD . EdU signal at APB ( telomere / PML ) are indicated by white arrows . Approximately 150 cells were divided into five groups ( 0 , 1 - 2 , 3 - 4 , 5 - 6n and ≥ 7 ) based on the number of EdU + APBs . Scale bars are 5 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) FISH-IF of telomeres (in red) and BLM (in green) in TOP3A-depleted cells BLM foci at telomeres are indicated by white arrows. Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s. = non-significant, **p < 0.01, Mann-Whitney test. Scale bars are 5 μm.(B) ATSA assay assessing ALT-mediated telomeric DNA synthesis (telomere in red, EdU in purple) in PML (in green) according to TOP3A KD. EdU signal at APB (telomere/PML) are indicated by white arrows. Approximately 150 cells were divided into five groups (0,1-2, 3-4, 5-6n and ≥7) based on the number of EdU+APBs. Scale bars are 5 μm.


Fig 1 ( A - D ) Primary cultures from D . melanogaster embryos expressing GFP - Atg8a under the control of Dmef2 - Gal4 , stained with Phalloidin ( actin ) in red . Inset are magnifications of areas denoted by dotted line . ( A ) In untreated control cultures , GFP - Atg8a localizes throughout the muscle cytoplasm and nuclei . ( B ) Addition of rapamycin ( Rap ) for 12 hrs results in the formation of small GFP - Atg8a labeled punctae . ( C ) Addition of chloroquine ( CQ ) for 12 hrs results in the accumulation of large GFP - Atg8a labeled vesicles that localize around the nucleus and between the myofibers . ( D ) Addition of both CQ and Rap for 12hrs , triggers the formation of greater numbers of enlarged vesicles in the muscles than either drug alone . ( E ) Knockdown of the core autophagy gene , Atg1 , prevents the formation of GFP - Atg8a vesicles in CQ + Rap treated cultures . ( F ) Quantification of the mean ratio of GFP - Atg8a punctae area to total muscle area per well ( n = 4 ) . Error bars indicate SEM with * * p < . 01 ; * * * p < . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 1(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the muscle cytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the muscles than either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.


Fig 3 ( A - D ) Primary cultures from D . melanogaster embryos expressing GFP - Atg8a under the control of Dmef2 - Gal4 , stained with Phalloidin ( actin ) in red . Inset are magnifications of areas denoted by dotted line . ( A ) In untreated control cultures , GFP - Atg8a localizes throughout the musclecytoplasm and nuclei . ( B ) Addition of rapamycin ( Rap ) for 12 hrs results in the formation of small GFP - Atg8a labeled punctae . ( C ) Addition of chloroquine ( CQ ) for 12 hrs results in the accumulation of large GFP - Atg8a labeled vesicles that localize around the nucleus and between the myofibers . ( D ) Addition of both CQ and Rap for 12hrs , triggers the formation of greater numbers of enlarged vesicles in the musclesthan either drug alone . ( E ) Knockdown of the core autophagy gene , Atg1 , prevents the formation of GFP - Atg8a vesicles in CQ + Rap treated cultures . ( F ) Quantification of the mean ratio of GFP - Atg8a punctae area to total muscle area per well ( n = 4 ) . Error bars indicate SEM with * * p < . 01 ; * * * p < . 001 . ( E ) Control white RNAi muscles starved and treated with CQ accumulate GFP - Atg8a punctae , but very few colocalize with ubiquitin ( Ubi ) . ( F ) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP - Atg8a punctae . The remainder colocalize with ubiquitin . ( G ) Quantification of the mean GFP - Atg8a punctae area and Mander ' s overlap coefficient for GFP - Atg8a and Ubi for genotypes shown in E and F . Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals , with p - values for Rpn1 RNAi relative to white RNAi control ( * * * p < . 001 ) . ( H , I ) Autophagosome number is reduced , but autophagic flux is not significantly altered in starved Rpn1 vs . white RNAi muscles expressing GFP - mCherry - Atg8 . ( J ) Flux measurement is shown on the right as the ratio of mCherry punctae to GFP - mCherry ( n = 6 ) . SEM is indicated . ( K ) Anti - Atg8a Western blot of third instar muscle lysate from Dmef2 - Gal4 ; UAS - RNAi animals . In fed white RNAi control , endogenous Atg8a appears as a single band ( Atg8a - I ) . 6 hrs starvation induces lipid modification of Atg8a and the appearance of a second band ( Atg8a - II ) . Knockdown of Atg1 or Rpn1 inhibits Atg8a modification , indicating reduced autophagy levels .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 3(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the musclecytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the musclesthan either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.(E) Control white RNAi muscles starved and treated with CQ accumulate GFP-Atg8a punctae, but very few colocalize with ubiquitin (Ubi). (F) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP-Atg8a punctae. The remainder colocalize with ubiquitin. (G) Quantification of the mean GFP-Atg8a punctae area and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in E and F. Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals, with p-values for Rpn1 RNAi relative to white RNAi control (***p<.001).(H, I) Autophagosome number is reduced, but autophagic flux is not significantly altered in starved Rpn1 vs. white RNAi muscles expressing GFP-mCherry-Atg8. (J) Flux measurement is shown on the right as the ratio of mCherry punctae to GFP-mCherry (n = 6). SEM is indicated.(K) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4; UAS-RNAi animals. In fed white RNAi control, endogenous Atg8a appears as a single band (Atg8a-I). 6 hrs starvation induces lipid modification of Atg8a and the appearance of a second band (Atg8a-II). Knockdown of Atg1 or Rpn1 inhibits Atg8a modification, indicating reduced autophagy levels.


Fig 4 ( A - D ) Inhibition of the proteasome by overexpression of a temperature sensitive proteasome mutant ( DTS ) . Dmef2 - Gal4 , UAS - GFP - Atg8a control and UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a and animals were shifted to the restrictive temperature then starved and CQ treated 6 hrs prior to dissection . 18 hrs post - shift ( PS ) , both control ( A ) and DTS muscles ( B ) accumulate GFP - Atg8a punctae , but the latter also accumulate ubiquitin ( Ubi ) aggregates . 48 hrs PS , control muscles ( C ) maintain strong autophagy induction , but DTS mutant muscles ( D ) have reduced GFP - Atg8a punctae accumulation , similar to the Rpn1 knockdown phenotype . ( E ) Quantification of the mean GFP - Atg8a punctae and Mander ' s overlap coefficient for GFP - Atg8a and Ubi for genotypes shown in A - D ( SEM is indicated for n = 8 ventral longitudinal muscles from individual animals with p - values of DTS relative to control ) . Dark green = Dmef2 - Gal4 , UAS - GFP - Atg8a ; Light green = UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a . ( F ) Western blot analysis of endogenous Atg8a from third instar muscle lysate . 6 hrs starvation induces lipidated Atg8a - II in Dmef2 - Gal4 control at both 18 hrs and 48 hrs PS , but not in DTS at 48 hrs PS .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 4(A-D) Inhibition of the proteasome by overexpression of a temperature sensitive proteasome mutant (DTS). Dmef2-Gal4, UAS-GFP-Atg8a control and UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and animals were shifted to the restrictive temperature then starved and CQ treated 6 hrs prior to dissection. 18 hrs post-shift (PS), both control (A) and DTS muscles (B) accumulate GFP-Atg8a punctae, but the latter also accumulate ubiquitin (Ubi) aggregates. 48 hrs PS, control muscles (C) maintain strong autophagy induction, but DTS mutant muscles (D) have reduced GFP-Atg8a punctae accumulation, similar to the Rpn1 knockdown phenotype. (E) Quantification of the mean GFP-Atg8a punctae and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in A-D (SEM is indicated for n = 8 ventral longitudinal muscles from individual animals with p-values of DTS relative to control). Dark green = Dmef2-Gal4, UAS-GFP-Atg8a; Light green = UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a.(F) Western blot analysis of endogenous Atg8a from third instar muscle lysate. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-Gal4 control at both 18 hrs and 48 hrs PS, but not in DTS at 48 hrs PS.