( b ) LC3 dot counting in CALM knockdown cells . CALM was knocked down in HeLa cells expressing GFP - LC3 and with or without an siRNA - resistant form of wild - type CALM ( rescue ) , as indicated . The cells were starved in HBSS for 4 h , fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell . The knockdown efficiency and the level of the siRNA - resistant form of CALM are shown on the left on the western blotting . The number of LC3 dots per cell ( shown as mean ± s . d . ) is shown on the graph for each condition ( n ≥ 300 cells per condition ; BC , basal conditions ) . The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right ( * P0 . 01 ; NS , not significant , two - tailed t - test ) . Pictures obtained from automated microscope are shown . Scale bars , 20 μm .

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(b) LC3 dot counting in CALM knockdown cells. CALM was knocked down in HeLa cells expressing GFP-LC3 and with or without an siRNA-resistant form of wild-type CALM (rescue), as indicated. The cells were starved in HBSS for 4 h, fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell. The knockdown efficiency and the level of the siRNA-resistant form of CALM are shown on the left on the western blotting. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right (*P0.01; NS, not significant, two-tailed t-test). Pictures obtained from automated microscope are shown. Scale bars, 20 μm.


( a ) p62 vesicle formation in CALM knockdown HeLa cells . Confocal pictures are shown . # CALM - downregulated cells where p62 vesicles accumulate . Data are representative of three independent experiments and shown as mean ± s . e . m . ( n ≥ 500 cells ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) p62 vesicle formation in CALM knockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells where p62 vesicles accumulate. Data are representative of three independent experiments and shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.


( b ) Western blot analysis of p62 , actin and GAPDH in HeLa cells and HEK cells ( basal conditions , BC , or without serum for 1 h ) where CALM was knocked down using shRNA or siRNA , as indicated . Data are mean ± s . d ( n = 3 experiments for HeLa cells and HEK cells ; * P0 . 05 ; two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).


( c ) Percentage of huntingtinQ74 - expressing cells with aggregates in CALM knockdown HeLa cells . Data depict one representative experiments performed in triplicate , out of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Percentage of huntingtinQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


( d ) Tau - positive tangle formation in CALM knockdown cells . HeLa cells transiently expressing DsRed - tau 4R were treated with Baf A1 for 4 h as indicated . Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody ( green ) . Data represent the number of cells with phosphorylated tau - positive tangles as mean ± s . e . m . ( n = 3 experiments ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm .

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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Tau-positive tangle formation in CALM knockdown cells. HeLa cells transiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h as indicated. Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent the number of cells with phosphorylated tau-positive tangles as mean ±s.e.m. (n=3 experiments; *P0.01; two-tailed t-test). Scale bars, 5 μm.


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).


( a ) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal ( BC ) and serum starvation conditions . Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions . Data are from one representative experiment , out of three independent experiments . Data shown as mean ± s . e . m . ( n ≥ 500 cells ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal (BC) and serum starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions. Data are from one representative experiment, out of three independent experiments. Data shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.


( b ) Co - localization between GFP - ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 ( CTX ; 20 min ) in CALM knockdown HeLa cells . Confocal pictures are presented with magnified areas showing the co - localization between ATG16L1 and CTX in greater detail . Scale bars , 5 μm . The Pearson ' s coefficient between ATG16L1 and cholera toxin is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 20 cells ; * P0 . 05 ; two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-localization between GFP-ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 (CTX; 20 min) in CALM knockdown HeLa cells. Confocal pictures are presented with magnified areas showing the co-localization between ATG16L1 and CTX in greater detail. Scale bars, 5 μm. The Pearson's coefficient between ATG16L1 and cholera toxin is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥20 cells; *P0.05; two-tailed t-test).


( c ) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions . Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two - tailed t - test ) . ( AU , arbitrary unit ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). (AU, arbitrary unit). Scale bars, 5 μm.


( d ) Live - cell imaging of ATG16L1 - GFP in CALM knockdown HeLa cells . ATG16L1 pictures from various time points of a 5 - min movie are shown in inverted grey style . Arrows indicate ATG16L1 vesicles . The number of fusion events per 5 min is shown . Data are representative of five movies and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Live-cell imaging of ATG16L1-GFP in CALM knockdown HeLa cells. ATG16L1 pictures from various time points of a 5-min movie are shown in inverted grey style. Arrows indicate ATG16L1 vesicles. The number of fusion events per 5 min is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.


( e ) In vitro fusion assay of post - nuclear supernatant from HeLa cells expressing either GFP - ATG16L1 or mStrawberry - ATG16L1 in control and CALM knockdown conditions . Confocal pictures are shown where ATG16L1 - mStrawberry signal is shown in purple to enable better visualization . Fused vesicles appear in white . The ATP - negative condition , which prevents SNARE - dependent fusion , is also shown as a control for the reaction . Magnified areas are shown to allow visualization of the vesicles . The percentage of fused vesicles is represented . n = numbers of vesicles scored per field ( a minimum of five fields were analysed per condition ) . Data are representative of two independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ) . Scale bars , 5 μm . ( * P0 . 05 ; two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) In vitro fusion assay of post-nuclear supernatant from HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1 in control and CALM knockdown conditions. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control for the reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles is represented. n=numbers of vesicles scored per field (a minimum of five fields were analysed per condition). Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test).


( a ) LC3 levels in CALM knockdown cells . CALM was knocked down in cells expressing or not expressing an siRNA - resistant form of CALM wild type ( wt ) or CALM 219 mutant , as indicated . The cells were lysed and subjected to western blotting . The knockdown efficiency , LC3 - II levels and the level of the siRNA - resistant form of CALM are shown on the western blotting .

(a) LC3 levels in CALM knockdown cells. CALM was knocked down in cells expressing or not expressing an siRNA-resistant form of CALM wild type (wt) or CALM 219 mutant, as indicated. The cells were lysed and subjected to western blotting. The knockdown efficiency, LC3-II levels and the level of the siRNA-resistant form of CALM are shown on the western blotting.


( b ) LC3 dots counting in CALM knockdown cells . CALM was knocked down in cells expressing GFP - LC3 with or without an siRNA - resistant form of CALM wild type ( wt ) or CALM mutant ( 219 mutant ) as indicated . The cells were kept in full medium or starved in Hanks balanced salt solution ( HBSS ) for 4 h , fixed and subjected to microscopy to score the number of LC3 dots per cell . The ratio of the number of LC3 dots per cell ( shown as mean ± s . d . ) between starvation and basal conditions is shown on the graph ( * P0 . 01 ; NS , not significant , two - tailed t - test ; n ≥ 300 cells per condition ; BC , basal conditions ) .

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(b) LC3 dots counting in CALM knockdown cells. CALM was knocked down in cells expressing GFP-LC3 with or without an siRNA-resistant form of CALM wild type (wt) or CALM mutant (219 mutant) as indicated. The cells were kept in full medium or starved in Hanks balanced salt solution (HBSS) for 4 h, fixed and subjected to microscopy to score the number of LC3 dots per cell. The ratio of the number of LC3 dots per cell (shown as mean ±s.d.) between starvation and basal conditions is shown on the graph (*P0.01; NS, not significant, two-tailed t-test; n≥300 cells per condition; BC, basal conditions).


( c ) Co - localization between ATG9A and VAMP3 - HA or GFP - LC3 and VAMP8 - HA in control and CALM knockdown HeLa cells stably expressing VAMP3 - HA or VAMP8 - HA . Confocal pictures are shown with magnified areas showing co - localization between ATG9A and VAMP3 or GFP - LC3 and VAMP8 in control cells and no co - localization in CALM knockdown cells . Quantification of ATG9A - VAMP3 or GFP - LC3 - VAMP8 co - localization is shown on the graph as a Pearson ' s coefficient ( data are mean ± s . d . ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm .

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(c) Co-localization between ATG9A and VAMP3-HA or GFP-LC3 and VAMP8-HA in control and CALM knockdown HeLa cells stably expressing VAMP3-HA or VAMP8-HA. Confocal pictures are shown with magnified areas showing co-localization between ATG9A and VAMP3 or GFP-LC3 and VAMP8 in control cells and no co-localization in CALM knockdown cells. Quantification of ATG9A-VAMP3 or GFP-LC3-VAMP8 co-localization is shown on the graph as a Pearson's coefficient (data are mean ±s.d.; *P0.05; two-tailed t-test). Scale bars, 5 μm.


( a ) Co - localization between ATG12 and VAMP2 - HA in control and CALM knockdown HeLa cells stably expressing VAMP2 - HA . Confocal pictures are shown with magnified areas on the right showing co - localization between ATG12 and VAMP2 in control cells and no co - localization in CALM knockdown cells . Quantification of ATG12 - VAMP2 co - localization is shown on the graph as the Pearson ' s coefficient ( data are mean ± s . d . ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm .

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(a) Co-localization between ATG12 and VAMP2-HA in control and CALM knockdown HeLa cells stably expressing VAMP2-HA. Confocal pictures are shown with magnified areas on the right showing co-localization between ATG12 and VAMP2 in control cells and no co-localization in CALM knockdown cells. Quantification of ATG12-VAMP2 co-localization is shown on the graph as the Pearson's coefficient (data are mean ±s.d.;*P0.05; two-tailed t-test). Scale bars, 5 μm.


( b ) Western blot analysis of VAMP2 , actin and LC3 - II in HeLa cells where VAMP2 was knocked down , as indicated . The cells were starved in Hanks balanced salt solution ( HBSS ) and treated with Baf A1 as indicated . ( SE , short exposure ; LE , longer exposure . ) Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; NS , not significant , two - tailed t - test ) .

O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells where VAMP2 was knocked down, as indicated. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure.) Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; NS, not significant, two-tailed t-test).


( c ) LC3 dot counting in CALM and VAMP2 knockdown cells . CALM or VAMP2 were knocked down in cells expressing GFP - LC3 . The cells were kept in full medium or starved in HBSS for 4 h , fixed and subjected to automated fluorescence microscopy to score the number of LC3 dots per cell . The number of LC3 dots per cell ( shown as mean ± s . d . ) is shown on the graph for each condition ( n ≥ 300 cells per condition ; BC , basal conditions ) . ( * P0 . 01 ; NS , not significant , two - tailed t - test ) .

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(c) LC3 dot counting in CALM and VAMP2 knockdown cells. CALM or VAMP2 were knocked down in cells expressing GFP-LC3. The cells were kept in full medium or starved in HBSS for 4 h, fixed and subjected to automated fluorescence microscopy to score the number of LC3 dots per cell. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). (*P0.01; NS, not significant, two-tailed t-test).


( d ) Number of p62 dots per cell in VAMP2 knockdown . HeLa cells where VAMP2 was knocked down were fixed and subjected to microscopy after labelling endogenous p62 using specific antibody . The data represent the number of p62 dots per cell shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ; n ≥ 300 cells per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Number of p62 dots per cell in VAMP2 knockdown. HeLa cells where VAMP2 was knocked down were fixed and subjected to microscopy after labelling endogenous p62 using specific antibody. The data represent the number of p62 dots per cell shown as mean ±s.d. (*P0.05; two-tailed t-test; n≥300 cells per condition).


( e ) Percentage of huntingtinQ74 - expressing cells with aggregates in VAMP2 knockdown HeLa cells . Data are from one representative experiments performed in triplicate , out of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Percentage of huntingtinQ74-expressing cells with aggregates in VAMP2 knockdown HeLa cells. Data are from one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


( f ) Western blot analysis of VAMP2 , actin and LC3 - II in HeLa cells stably expressing VAMP2 - HA wild type or VAMP2 - HA mutated in the CALM - binding site at different levels ( wild - type clone 6 , wt Cl6 : low level ; wild - type clone 11 , wt Cl11 : high level ; mutant clone 11 , AA Cl11 : low level ; mutant clone 12 , AA Cl12 : high level ) . The cells were treated with Baf A1 as indicated . ( SE , short exposure ; LE , longer exposure ) . Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells stably expressing VAMP2-HA wild type or VAMP2-HA mutated in the CALM-binding site at different levels (wild-type clone 6, wt Cl6: low level; wild-type clone 11, wt Cl11: high level; mutant clone 11, AA Cl11: low level; mutant clone 12, AA Cl12: high level). The cells were treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


( a ) Size of endogenous ATG12 vesicles in HeLa cells stably expressing either wild - type or mutant VAMP2 . Confocal pictures are presented with magnified areas showing ATG12 vesicles . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two tail t - test ) . Scale bars , 5 μm .

(a) Size of endogenous ATG12 vesicles in HeLa cells stably expressing either wild-type or mutant VAMP2. Confocal pictures are presented with magnified areas showing ATG12 vesicles. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two tail t-test). Scale bars, 5 μm.


( b ) Co - localization between ATG12 and LC3 in HeLa cells stably expressing either wild - type clone 11 or mutant clone 12 VAMP2 . Confocal pictures are presented with magnified areas showing ATG12 - LC3 co - localization . The Pearson ' s coefficient between ATG12 and LC3 is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm .

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(b) Co-localization between ATG12 and LC3 in HeLa cells stably expressing either wild-type clone 11 or mutant clone 12 VAMP2. Confocal pictures are presented with magnified areas showing ATG12-LC3 co-localization. The Pearson's coefficient between ATG12 and LC3 is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). Scale bars, 5 μm.


( c ) Size and number of endogenous ATG12 vesicles in VAMP2 - knockdown HeLa cells in starvation condition . Confocal pictures are shown with arrows indicating ATG12 vesicles . Data are representative of three independent experiments and shown as mean ± s . d . for the size of ATG12 vesicle . ( n ≥ 100 vesicles ; * P0 . 05 ; based two - tailed t - test ) . Scale bars , 5 μm .

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(c) Size and number of endogenous ATG12 vesicles in VAMP2-knockdown HeLa cells in starvation condition. Confocal pictures are shown with arrows indicating ATG12 vesicles. Data are representative of three independent experiments and shown as mean ±s.d. for the size of ATG12 vesicle. (n≥100 vesicles; *P0.05; based two-tailed t-test). Scale bars, 5 μm.


( d ) Live - cell imaging of ATG16L1 - GFP in VAMP2 - knockdown HeLa cells . Confocal pictures from various time points of a 5 - min movie are shown in inverted greyscale . Arrows indicate ATG16L1null . The number of fusion events per vesicles is shown . Data are representative of five movies and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm .

(d) Live-cell imaging of ATG16L1-GFP in VAMP2-knockdown HeLa cells. Confocal pictures from various time points of a 5-min movie are shown in inverted greyscale. Arrows indicate ATG16L1null. The number of fusion events per vesicles is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.


( e ) In vitro fusion assay of post - nuclear supernatants from control and VAMP2 knockdown HeLa cells expressing either GFP - ATG16L1 or mStrawberry - ATG16L1 . Confocal pictures are shown where ATG16L1 - mStrawberry signal is shown in purple to enable better visualization . Fused vesicles appear in white . The ATP - negative condition , which prevents SNARE - dependent fusion , is also shown as a control of reaction . Magnified areas are shown to allow visualization of the vesicles . The percentage of fused vesicles ( white ) is represented . Data are representative of two independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ) . Scale bars , 5 μm . ( * P0 . 05 ; two - tailed t - test ) . n = numbers of vesicles scored per field .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) In vitro fusion assay of post-nuclear supernatants from control and VAMP2 knockdown HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control of reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles (white) is represented. Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test). n=numbers of vesicles scored per field.


( a ) Western blotting showing downregulation of lap ( i ) or increase in Atg8a - II ( ii ) expression level in adult fly heads on lap downregulation using the UAS - RNAi lines lapGD12732 or lapKK105767 , or the heterozygous allele lap1 . Quantification of lap / actin and Atg8a - II / actin is shown . Genotypes : Control w ; elav - GAL4 / + ; for the RNAi lines : w ; elav - GAL4 / lapKK105767 and w ; elav - GAL4 / + ; lapGD12732 / + ; for the lap1 allele : w ; elav - GAL4 / + ; lap1 / + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Western blotting showing downregulation of lap (i) or increase in Atg8a-II (ii) expression level in adult fly heads on lap downregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or the heterozygous allele lap1. Quantification of lap/actin and Atg8a-II/actin is shown. Genotypes: Control w; elav-GAL4/+; for the RNAi lines: w; elav-GAL4/ lapKK105767 and w; elav-GAL4/+; lapGD12732/+; for the lap1 allele: w; elav-GAL4/+; lap1/+.


( b ) Western blotting showing the accumulation of tau in Drosophila adult flyheads on lap downregulation using the UAS - RNAi lines lapGD12732 or lapKK105767 , or the heterozygous allele lap1 . Quantification of tau / lapGD12732 is shown . Genotypes : Control w ; elav - GAL4 / + ; for tau - WT : w ; elav - GAL4 / + ; UAS - tau - WT / + ; for RNAi lines : w ; elav - GAL4 / lapKK105767 ; UAS - tau - WT / + and w ; elav - GAL4 / + ; lapGD12732 / tau - WT ; for the lap1 allele : w ; elav - GAL4 / + ; lap1 / + or w ; elav - GAL4 / + ; lap1 / UAS - tau - WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting showing the accumulation of tau in Drosophila adult flyheads on lap downregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or the heterozygous allele lap1. Quantification of tau/lapGD12732 is shown. Genotypes: Control w; elav-GAL4/+; for tau-WT: w; elav-GAL4/+; UAS-tau-WT/+; for RNAi lines: w; elav-GAL4/ lapKK105767; UAS-tau-WT/+ and w; elav-GAL4/+; lapGD12732/tau-WT; for the lap1 allele: w; elav-GAL4/+; lap1/+ or w; elav-GAL4/+; lap1/UAS-tau-WT.


( ai ) Western blot analysis of tubulin , LC3 - I and LC3 - II in zebrafish larvae where ATG7 was downregulated , as indicated . ( ii ) Western blot analysis of CALM - HA , actin and LC3 - II in zebrafishlarvae where CALM - HA was expressed , as indicated . The larvae were treated with ammonium chloride ( NH4Cl ) , as indicated . ( SE , short exposure ; LE , longer exposure ) . Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafish larvae where ATG7 was downregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II in zebrafishlarvae where CALM-HA was expressed, as indicated. The larvae were treated with ammonium chloride (NH4Cl), as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


( b ) Modulation of autophagy alters Dendra - tau clearance dynamics . The fluorescence intensity of each individual cell was quantified at each timepoint ( n ≥ 31 cells , ≥ 9 larvae per treatment group ) and mean cell intensity values for each drug treatment at each timepoint were calculated . Images were taken immediately after photoconversion and at 24 , 30 and 48 h intervals thereafter . Rapamycin treatment significantly increased the rate of Dendra - tau clearance . Ammonium chloride ( NH4Cl ) treatment significantly decreases the rate of Dendra - tau clearance ( * * P0 . 01 , * * * P0 . 001 , one - way analysis of variance ( ANOVA ) ) . Error bars are ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) Modulation of autophagy alters Dendra-tau clearance dynamics. The fluorescence intensity of each individual cell was quantified at each timepoint (n≥ 31 cells, ≥9 larvae per treatment group) and mean cell intensity values for each drug treatment at each timepoint were calculated. Images were taken immediately after photoconversion and at 24, 30 and 48 h intervals thereafter. Rapamycin treatment significantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatment significantly decreases the rate of Dendra-tau clearance (**P0.01, ***P0.001, one-way analysis of variance (ANOVA)). Error bars are ±s.e.m.


( c ) Dendra - tau clearance in the presence of CALM : representative images of CALM with mosaic expression of Dendra - tau and full - length Dendra - tau taken immediately after photoconversion and at 6 , 24 , 30 , 48 and 54 h after conversion . The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co - expressing Dendra - tau and either full - length CALM or Δ - ANTH CALM ( larvae - ANTH - HA ) constructs ( n ≥ 100 cells , ≥ 9 larvae per treatment group ) at each time point was calculated . ( i ) Expression of full - length HA significantly delayed the clearance of CALM at all time points compared with Δ - ANTH CALM ( * * * P0 . 001 , one - way ANOVA ) . One graph , representative of three independent experiments , is presented . ( CALM - ANTH - HA : 132 cells , 20 fishes ; CALM - CALM : 18 cells , 8 fishes ) . Another two experiments are shown in Supplementary Fig . S5C . Error bars are mean ± s . d . ( ii ) Treatment with CALM alters the dynamics of CALM clearance . Expression of full - length CALM significantly delayed the clearance of CALM at all time points compared with Δ - ANTH CALM , as observed in i ( P0 . 001 , one - way ANOVA ) . However , treatment of Δ - ANTH CALM injected CALM with CALM slows the CALM clearance by 70 % to a level comparable to that observed in larvae injected with full - length Dendra - tau . Ammonium chloride treatment of Dendra - tau injected with full - length null results in a modest ( 22 % ) decrease in Dendra - tau clearance , suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect ( n ≥ 25 cells , n ≥ 9 Dendra - tau per treatment group ) . Error bars are mean ± s . d . Note that i and ii are distinct experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Dendra-tau clearance in the presence of CALM: representative images of CALM with mosaic expression of Dendra-tau and full-length Dendra-tau taken immediately after photoconversion and at 6, 24, 30, 48 and 54 h after conversion. The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co-expressing Dendra-tau and either full-length CALM or Δ-ANTH CALM (larvae-ANTH-HA) constructs (n≥ 100 cells, ≥9 larvae per treatment group) at each time point was calculated. (i) Expression of full-length HA significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM (***P0.001, one-way ANOVA). One graph, representative of three independent experiments, is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-CALM: 18 cells, 8 fishes). Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii) Treatment with CALM alters the dynamics of CALM clearance. Expression of full-length CALM significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM, as observed in i (P0.001, one-way ANOVA). However, treatment of Δ-ANTH CALM injected CALM with CALM slows the CALM clearance by 70% to a level comparable to that observed in larvae injected with full-length Dendra-tau. Ammonium chloride treatment of Dendra-tau injected with full-length null results in a modest (22%) decrease in Dendra-tau clearance, suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect (n≥25 cells, n≥9 Dendra-tau per treatment group). Error bars are mean ±s.d. Note that i and ii are distinct experiments.


( a ) rho : : GFP - tau fishes were incubated from 3 to 9 d . p . f . in either dimethyl sulphoxide ( DMSO ) or rapamycin ( i ) or from 3 to 7 d . p . f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin ( ii ) . Images through the central retina at 9 d . p . f . ( top panel ; ( i ) ) reveal rod degeneration ( arrow ) throughout the retina in control ( DMSO - treated ) larvae , whereas rod photoreceptors are present throughout the retina , particularly in the central region following treatment with rapamycin ( arrow ) . Images taken through the central retina at 7 d . p . f . ( bottom panel ; ( ii ) ) show normal photoreceptors in the marginal zones ( arrows ) and only limited numbers in the central region . NH4Cl exacerbates degeneration - photoreceptors are absent from the central retina and reduced / absent from marginal zones ( arrows ) . Sections were stained with anti - rhodopsin ( 1D1 ) antibody . GFP labels whole rod photoreceptors , whereas rhodopsin is present in the rod outer segment . GFP co - localizes with the red rhodopsin label in all experimental conditions . Scale bars , 50 μm . Quantification of rodphotoreceptor degeneration ( n = 10 larvae per group ; * * * P0 . 001 ; # # # P0 . 001 , two - tailed unpaired t - test ) . Error bars are mean ± s . d .

O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) rho::GFP-tau fishes were incubated from 3 to 9 d.p.f. in either dimethyl sulphoxide (DMSO) or rapamycin (i) or from 3 to 7 d.p.f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin (ii). Images through the central retina at 9 d.p.f. (top panel; (i)) reveal rod degeneration (arrow) throughout the retina in control (DMSO-treated) larvae, whereas rod photoreceptors are present throughout the retina, particularly in the central region following treatment with rapamycin (arrow). Images taken through the central retina at 7 d.p.f. (bottom panel; (ii)) show normal photoreceptors in the marginal zones (arrows) and only limited numbers in the central region. NH4Cl exacerbates degeneration-photoreceptors are absent from the central retina and reduced/absent from marginal zones (arrows). Sections were stained with anti-rhodopsin (1D1) antibody. GFP labels whole rod photoreceptors, whereas rhodopsin is present in the rod outer segment. GFP co-localizes with the red rhodopsin label in all experimental conditions. Scale bars, 50 μm. Quantification of rodphotoreceptor degeneration (n=10 larvae per group; ***P0.001; ###P0.001, two-tailed unpaired t-test). Error bars are mean ±s.d.


( b ) Full - length CALM electroporation into rho : : GFP larvae did not cause degeneration . , while full - length CALM electroporation into rho : : GFP - tau larvae exacerbated photoreceptor degeneration . Central retina sections in the region of the optic nerve head are presented . Scale bars , 50 μm . Data represent the ratio ( in % ) of the intensity of the GFP signal between CALM - HA electroporated eye versus control eye for five individuals per transgenic line . * * * P0 . 05 ; two - tailed t - test . Error bars are mean ± s . d .

(b) Full-length CALM electroporation into rho::GFP larvae did not cause degeneration., while full-length CALM electroporation into rho::GFP-tau larvae exacerbated photoreceptor degeneration. Central retina sections in the region of the optic nerve head are presented. Scale bars, 50 μm. Data represent the ratio (in %) of the intensity of the GFP signal between CALM-HA electroporated eye versus control eye for five individuals per transgenic line. ***P0.05; two-tailed t-test. Error bars are mean ±s.d.


( c ) NH4Cl treatment of rho : : GFP - tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control ( non - electroporated ) side but did not alter the degeneration caused by CALM electroporation . Rapamycin treatment of Rapamycin : : GFP - tau immediately after unilateral CALM electroporation rescued photoreceptors on the control ( non - electroporated ) side but not on the CALM - electroporated side . To demonstrate that loss of GFP corresponds to loss of photoreceptors , sections were stained with anti - rhodopsin ( 1D1 ) antibody . Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish ; Mann - Whitney test was used to compare drug treatment in the right eye of different fishes . * P0 . 05 . Scale bars , 50 μm . Error bars are mean ± s . e . m .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) NH4Cl treatment of rho::GFP-tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control (non-electroporated) side but did not alter the degeneration caused by CALM electroporation. Rapamycin treatment of Rapamycin::GFP-tau immediately after unilateral CALM electroporation rescued photoreceptors on the control (non-electroporated) side but not on the CALM-electroporated side. To demonstrate that loss of GFP corresponds to loss of photoreceptors, sections were stained with anti-rhodopsin (1D1) antibody. Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish; Mann-Whitney test was used to compare drug treatment in the right eye of different fishes. *P0.05. Scale bars, 50 μm. Error bars are mean ±s.e.m.


( a ) Histological section to demonstrate the individual cell layers of the zebrafish retina . The photoreceptor layer ( PR , comprising rod and cone photoreceptors ) lies immediately adjacent to the retinal pigment epithelium ( RPE ) at the outermost surface of the eye . Thioflavin - S labelling of retinal sections was used to identify neurofibrillary tangles in the photoreceptor layer ( marked with yellow dotted lines ) . No labelling was observed in the retina of rho : : GFP at 8 d . p . f . , whereas distinct thioflavin - S - positive tangles ( arrows ) were observed in the photoreceptor layer of rho : : GFP - tau fish . Note , the RPE is highly autofluorescent due to the presence of silver pigment . High power regions are shown in the top right of each panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Histological section to demonstrate the individual cell layers of the zebrafish retina. The photoreceptor layer (PR, comprising rod and cone photoreceptors) lies immediately adjacent to the retinal pigment epithelium (RPE) at the outermost surface of the eye. Thioflavin-S labelling of retinal sections was used to identify neurofibrillary tangles in the photoreceptor layer (marked with yellow dotted lines). No labelling was observed in the retina of rho::GFP at 8 d.p.f., whereas distinct thioflavin-S-positive tangles (arrows) were observed in the photoreceptor layer of rho::GFP-tau fish. Note, the RPE is highly autofluorescent due to the presence of silver pigment. High power regions are shown in the top right of each panel.


( b ) Unilateral electroporation of CALM into the retina of rho : : GFP - tau zebrafish resulted in a marked increase in thioflavin - S positive tangles in the electroporated retina in the photoreceptor layer ( PR ) compared with the control side . Top panel are lower magnification images to show the retinal cell layers . Thioflavin - S labelling is restricted to the PR layer . Note the RPE is highly autofluorescent due to the presence of silver pigment . Bottom panel are higher magnification images to show individual thioflavin - S tangles the largest of which are indicated by arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Unilateral electroporation of CALM into the retina of rho::GFP-tau zebrafish resulted in a marked increase in thioflavin-S positive tangles in the electroporated retina in the photoreceptor layer (PR) compared with the control side. Top panel are lower magnification images to show the retinal cell layers. Thioflavin-S labelling is restricted to the PR layer. Note the RPE is highly autofluorescent due to the presence of silver pigment. Bottom panel are higher magnification images to show individual thioflavin-S tangles the largest of which are indicated by arrows.


A LOUCY cells were treated in vitro with the indicated SMs with or without vincristine ( VCR ) for 48h . Apoptosis was determined by flow cytometry upon staining with Annexin - V and Propidium Iodide ( PI ) . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A LOUCY cells were treated in vitro with the indicated SMs with or without vincristine (VCR) for 48h. Apoptosis was determined by flow cytometry upon staining with Annexin-V and Propidium Iodide (PI). Mean ± SD of 3 independent experiments is shown; ** p <0.01, *** p <0.001 and **** p <0.0001 by unpaired Student's t-test.


B Fresh primary leukemic cells from two pediatric patients , one with common B - ALL at relapse ( left ) and one with T - ALL at diagnosis ( right ) , both with poor response to second line chemotherapy , were treated with LBW242 and VCR at the indicated concentrations . Apoptosis was measured after 24 h as described in A .

O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

B Fresh primary leukemic cells from two pediatric patients, one with common B-ALL at relapse (left) and one with T-ALL at diagnosis (right), both with poor response to second line chemotherapy, were treated with LBW242 and VCR at the indicated concentrations. Apoptosis was measured after 24 h as described in A.


The SM - drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cells were stained with respective fluorescent substrates for caspase - 8 ( C ) and - 9 ( D ) , and were subsequently analyzed by flow cytometry . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The SM-drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with respective fluorescent substrates for caspase-8 (C) and -9 (D) , and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.


The SM - drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cells were stained with an antibody detecting the active caspase - 3 subunit ( E ) , and were subsequently analyzed by flow cytometry . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The SM-drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with an antibody detecting the active caspase-3 subunit (E), and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.


The SM - drug combination induces PARP cleavage . LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cleavage of PARP was measured by Western blot using Actin as loading control ( F ) . One representative immunoblot out 3 independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The SM-drug combination induces PARP cleavage. LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cleavage of PARP was measured by Western blot using Actin as loading control (F). One representative immunoblot out 3 independent experiments is shown.


A Necrostatin - 1 ( Nec ) does not affect LBW - mediated sensitization to VCR - induced apoptosis . Apoptosis induction in LOUCY cells was determined by flow cytometry 48h after treatment with SM and / or VCR ( left ) and / or TNFα ( right ) in the presence or absence of the RIPK1 - inhibitor Nec . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 and * * p < 0 . 01 by unpaired Student ' s t - test .

O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Necrostatin-1 (Nec) does not affect LBW-mediated sensitization to VCR-induced apoptosis. Apoptosis induction in LOUCY cells was determined by flow cytometry 48h after treatment with SM and/or VCR (left) and/or TNFα (right) in the presence or absence of the RIPK1-inhibitor Nec. Mean ± SD of 3 independent experiments is shown; * p <0.05 and ** p<0.01 by unpaired Student's t-test.


B RIPK1 is dispensable for apoptosis induction by LBW242 and VCR . LOUCY cells were transfected with a non - targeting siRNA ( neg - siRNA ) or a RIPK1 - specific siRNA . 6h post transfection , cells were treated with the indicated drugs and apoptosis was measured 40h thereafter . Mean ± SD of 3 independent experiments is shown ; * * * p < 0 . 001 by unpaired Student ' s t - test . Knockdown ( KD ) was verified by Western blot at the time of apoptosis measurement and KD efficiency calculated compared to the control . One representative immunoblot out of 3 biological replicates is shown .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RIPK1 is dispensable for apoptosis induction by LBW242 and VCR. LOUCY cells were transfected with a non-targeting siRNA (neg-siRNA) or a RIPK1-specific siRNA. 6h post transfection, cells were treated with the indicated drugs and apoptosis was measured 40h thereafter. Mean ± SD of 3 independent experiments is shown; *** p<0.001 by unpaired Student's t-test. Knockdown (KD) was verified by Western blot at the time of apoptosis measurement and KD efficiency calculated compared to the control. One representative immunoblot out of 3 biological replicates is shown.


C The SM - drug combination does not activate the canonical NF - κB pathway . Phosphorylation and degradation of IκBα upon treatment with VCR or TNFα and / or LBW242 was determined in LOUCY cells by Western blot following treatment with the indicated drugs for 5 , 15 or 60 minutes using Actin as loading control . One representative immunoblot out of 3 independent biological replicates is shown .

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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C The SM-drug combination does not activate the canonical NF-κB pathway. Phosphorylation and degradation of IκBα upon treatment with VCR or TNFα and/or LBW242 was determined in LOUCY cells by Western blot following treatment with the indicated drugs for 5, 15 or 60 minutes using Actin as loading control. One representative immunoblot out of 3 independent biological replicates is shown.


D KD of NEMO does not affect the SM - mediated sensitization to chemotherapy - induced apoptosis . Experiment was performed as described in B , except that an siRNA targeting NEMO was used . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 by unpaired Student ' s t - test .

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D KD of NEMO does not affect the SM-mediated sensitization to chemotherapy-induced apoptosis. Experiment was performed as described in B, except that an siRNA targeting NEMO was used. Mean ± SD of 3 independent experiments is shown; * p<0.05 by unpaired Student's t-test.


E The SM - drug combination activates the non - canonical NF - κB pathway . Cleavage of p100 NF - κB2 to the p52 fragment was determined by Western blot 24h after treatment of LOUCY cells with chemotherapy or TNFα in combination with SM . Asterisk indicates an unspecific signal . One representative immunoblot out of 3 biological replicates is shown .

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E The SM-drug combination activates the non-canonical NF-κB pathway. Cleavage of p100 NF-κB2 to the p52 fragment was determined by Western blot 24h after treatment of LOUCY cells with chemotherapy or TNFα in combination with SM. Asterisk indicates an unspecific signal. One representative immunoblot out of 3 biological replicates is shown.


F KD of RelB does not inhibit apoptosis . Experiment was performed as described in B , except that an siRNA targeting RelB was used . Mean ± SD of 3 independent experiments is shown ; differences are not significant , by unpaired Student ' s t - test .

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F KD of RelB does not inhibit apoptosis. Experiment was performed as described in B, except that an siRNA targeting RelB was used. Mean ± SD of 3 independent experiments is shown; differences are not significant, by unpaired Student's t-test.


A Expression of XIAP mRNA in samples obtained from BM of healthy individuals ( Ctrl , n = 10 ) or patients with AML ( n = 517 ) or ALL ( n = 306 ) . Data are expressed as median , 25th and 75th percentile , whiskers indicate min / max ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by Games - Howell post - hoc test .

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A Expression of XIAP mRNA in samples obtained from BM of healthy individuals (Ctrl, n=10) or patients with AML (n=517) or ALL (n=306). Data are expressed as median, 25th and 75th percentile, whiskers indicate min/max; ** p<0.01; **** p<0.0001 by Games-Howell post-hoc test.


B Protein levels of XIAP normalized to loading control in PDX ALL samples from pediatric and adult patients , analyzed by protein immunoassay ( Simple Western ) and depicted as mean ( n = 2 , biological replicates ) ; PBMC = peripheral blood mononuclear

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B Protein levels of XIAP normalized to loading control in PDX ALL samples from pediatric and adult patients, analyzed by protein immunoassay (Simple Western) and depicted as mean (n=2, biological replicates); PBMC = peripheral blood mononuclear cells


Parental and XIAP over - expressing NALM - 6 cells ( C ) were treated with VCR apoptosis ( Annexin - V / PI ) was measured at the indicated time points ( C ) by flow cytometry . Mean ± SEM of 3 independent experiments is shown ; * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test . XIAP protein level was analyzed in parental and XIAP - overexpressing NALM - 6 cells by protein immunoassay ( Simple Western ) ; Actin was used as loading control . One representative immunoassay out of 3 independent experiments is shown .

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Parental and XIAP over-expressing NALM-6 cells (C) were treated with VCR apoptosis (Annexin-V / PI) was measured at the indicated time points (C) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed in parental and XIAP-overexpressing NALM-6 cells by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.


D Parental and XIAP over - expressing REH cells ( D ) were treated with VCR or LBW242 ; apoptosis ( Annexin - V / PI ) was measured after 72h ( D ) by flow cytometry . Mean ± SEM of 3 independent experiments is shown ; * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test . XIAP protein level was analyzed by protein immunoassay ( Simple Western ) ; Actin was used as loading control . One representative immunoassay out of 3 independent experiments is shown .

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D Parental and XIAP over-expressing REH cells (D) were treated with VCR or LBW242; apoptosis (Annexin-V / PI) was measured after 72h (D) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.


B shRNA expression control . Equal expression levels of ds - RED in the sh - CTRL / mTagBFP and sh - XIAP / eGFP subpopulation indicates identical expression levels of control and XIAP shRNA . One representative histogram out of 3 independent experiments is shown .

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B shRNA expression control. Equal expression levels of ds-RED in the sh-CTRL/mTagBFP and sh-XIAP/eGFP subpopulation indicates identical expression levels of control and XIAP shRNA. One representative histogram out of 3 independent experiments is shown.


C Expression of IAP . Western blot analysis of XIAP in NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP under the SFFV promoter ; GAPDH was used as loading control . One representative immunoblot out of 3 independent experiments is shown .

C Expression of IAP. Western blot analysis of XIAP in NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP under the SFFV promoter; GAPDH was used as loading control. One representative immunoblot out of 3 independent experiments is shown.


D Western blot analysis of NALM - 6 cells with moderate ( 69 % , EF1α promoter ) or strong ( 92 % , SFFV promoter ) KD of XIAP ; GAPDH was used as loading control . The XIAP KD levels ( % KD ) were quantified relative to sh - CTRL . One representative immunoblot out of 3 independent experiments is shown .

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D Western blot analysis of NALM-6 cells with moderate (69%, EF1α promoter) or strong (92%, SFFV promoter) KD of XIAP; GAPDH was used as loading control. The XIAP KD levels (% KD) were quantified relative to sh-CTRL. One representative immunoblot out of 3 independent experiments is shown.


F Moderate knockdown of XIAP sensitizes towards chemotherapy . NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were mixed at a 1 : 1 ratio and injected into groups of mice . 4 days after injection , mice were either treated with PBS ( n = 4 ) , or with Vincristin ( VCR , n = 8 ) , 0 . 3mg / kg i . v . once a week for 3 consecutive weeks . 7 days after last treatment , mice were sacrificed , and relative proportion of sh - CTRL and sh - XIAP subpopulations re - isolated from the BM was quantified by flow cytometry . Data are depicted as mean ± SEM of all mice analyzed with * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test with Welch ' s correction .

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F Moderate knockdown of XIAP sensitizes towards chemotherapy. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice. 4 days after injection, mice were either treated with PBS (n=4), or with Vincristin (VCR, n=8), 0.3mg/kg i.v. once a week for 3 consecutive weeks. 7 days after last treatment, mice were sacrificed, and relative proportion of sh-CTRL and sh-XIAP subpopulations re-isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test with Welch's correction.


G Strong knockdown of XIAP impairs NALM - 6 growth in vivo . NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were mixed at a 1 : 1 ratio and injected into groups of mice ( n = 8 ) . 38 or 46 days after injection , mice were sacrificed , and relative proportion of sh - CTRL and sh - XIAP subpopulations re - isolated from the BM was quantified by flow cytometry . Data are depicted as mean ± SEM of all mice analyzed with * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test .

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G Strong knockdown of XIAP impairs NALM-6 growth in vivo. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice (n=8). 38 or 46 days after injection, mice were sacrificed, and relative proportion of sh-CTRL and sh-XIAP subpopulations re-isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test.


H PARP cleavage in NALM - 6 cells with strong XIAP inhibition . NALM - 6 cells were lentivirally transduces with sh - CTRL or sh - XIAP ( SFFV ) . 4 days later , cells were lysed and relative cleavage level of PARP ( % ) was analyzed by protein immunoassay ( Simple Western ) . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 by unpaired Student ' s t - test .

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H PARP cleavage in NALM-6 cells with strong XIAP inhibition. NALM-6 cells were lentivirally transduces with sh-CTRL or sh-XIAP (SFFV). 4 days later, cells were lysed and relative cleavage level of PARP (%) was analyzed by protein immunoassay (Simple Western). Mean ± SD of 3 independent experiments is shown; ** p <0.01 by unpaired Student's t-test.


I XIAP reconstitution in NALM - 6 cells . NALM - 6 cells were transduced with shCTRL , shXIAP . 1 alone or shXIAP . 1 together with the XIAP overexpression vector . At day 0 , transduced cells were mixed in a 1 : 1 ratio with parental ( untransduced ) NALM - 6 cells . Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 by unpaired Student ' s t - test .

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I XIAP reconstitution in NALM-6 cells. NALM-6 cells were transduced with shCTRL, shXIAP.1 alone or shXIAP.1 together with the XIAP overexpression vector. At day 0, transduced cells were mixed in a 1:1 ratio with parental (untransduced) NALM-6 cells. Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time. Mean ± SD of 3 independent experiments is shown; ** p <0.01 by unpaired Student's t-test.


XIAP plays an essential role for ALL - 265 growing in mice . Experimental scheme and stability of XIAP knockdown over passaging . ALL - 265 PDX cells produced as described in A were mixed at a 1 : 1 ratio and injected into mice ( P1 , n = 4 ) . At clinical signs of illness , cells were isolated and re - injected into next recipient mice ( P2 , n = 4 ) . XIAP expression levels of P1 and P2 PDX cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were analyzed by Western blot at the end of each passage ; GAPDH was used as loading control ( B ) .

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XIAP plays an essential role for ALL-265 growing in mice. Experimental scheme and stability of XIAP knockdown over passaging. ALL-265 PDX cells produced as described in A were mixed at a 1:1 ratio and injected into mice (P1, n=4). At clinical signs of illness, cells were isolated and re-injected into next recipient mice (P2, n=4). XIAP expression levels of P1 and P2 PDX cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were analyzed by Western blot at the end of each passage; GAPDH was used as loading control (B).


XIAP plays an essential role for ALL - 265 growing in mice . ALL - 265 PDX cells produced as described in A were mixed at a 1 : 1 ratio and injected into mice ( P1 , n = 4 ) . At clinical signs of illness , cells were isolated and re - injected into next recipient mice ( P2 , n = 4 ) . Relative proportion of ALL - 265 GEPDX cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP re - isolated from the BM at the end of each passage was quantified by flow cytometry ( C ) . Data are depicted as mean ± SEM of all mice analyzed ( n = 4 , each passage ) with * * * * p < 0 . 0001 by unpaired Student ' s t - test .

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XIAP plays an essential role for ALL-265 growing in mice. ALL-265 PDX cells produced as described in A were mixed at a 1:1 ratio and injected into mice (P1, n=4). At clinical signs of illness, cells were isolated and re-injected into next recipient mice (P2, n=4). Relative proportion of ALL-265 GEPDX cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP re-isolated from the BM at the end of each passage was quantified by flow cytometry (C). Data are depicted as mean ± SEM of all mice analyzed (n=4, each passage) with **** p<0.0001 by unpaired Student's t-test.


D Dose - response relationship between strength of XIAP knockdown and growth disadvantage ; ALL - 265 PDX cells of a representative mouse were analyzed separately for dsRED / shRNA high and low expressing cells .

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D Dose-response relationship between strength of XIAP knockdown and growth disadvantage; ALL-265 PDX cells of a representative mouse were analyzed separately for dsRED/shRNA high and low expressing cells.


Orthotopic MPNST xenografts at passages 1 ( OT P1 ) and 4 ( OT P4 ) were histopathologically similar to their corresponding primary MPNST ( PT ) in hematoxylin - eosin staining of paraffin - embedded tumor sections from patients MPNST - NF1 - 001 and MPNST - SP - 002 . Main panels show a general view of the tumors at low magnification ( 40 × ) ; inset pictures were taken at higher magnification ( 400 × ) .

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Orthotopic MPNST xenografts at passages 1 (OT P1) and 4 (OT P4) were histopathologically similar to their corresponding primary MPNST (PT) in hematoxylin-eosin staining of paraffin-embedded tumor sections from patients MPNST-NF1-001 and MPNST-SP-002. Main panels show a general view of the tumors at low magnification (40×); inset pictures were taken at higher magnification (400×).


Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features . A representative immunostained section of vimentin , CD34 , S100 , and Ki - 67 is shown for primary tumors ( PT ) and orthotopic tumors ( OT ) from patientsMPNST - NF1 - 001 and MPNST - SP - 002 . Positive antibody signals are shown in brown , and the hematoxylin counterstain in blue . Main panels show pictures at high magnification ( 400 × ) ; inset pictures show mitotic cells present in these tumors .

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Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features. A representative immunostained section of vimentin, CD34, S100, and Ki-67 is shown for primary tumors (PT) and orthotopic tumors (OT) from patientsMPNST-NF1-001 and MPNST-SP-002. Positive antibody signals are shown in brown, and the hematoxylin counterstain in blue. Main panels show pictures at high magnification (400×); inset pictures show mitotic cells present in these tumors.


Stromal elements of the primary tumors were labeled with anti - humanCD34 but not anti - mouseCD34 ; patient - derived xenografts are labeled with anti - mouseCD34 only and no anti - human marker . Representative sections ( at 40 × and 400 × magnification ) of the primary tumors ( PT ) and the orthoxenograft tumors at passages 1 ( OT P1 ) and 4 ( OT P4 ) were labeled with anti - humanCD34 ( H ) and anti - mouseCD34 ( M ) .

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Stromal elements of the primary tumors were labeled with anti-humanCD34 but not anti-mouseCD34; patient-derived xenografts are labeled with anti-mouseCD34 only and no anti-human marker. Representative sections (at 40× and 400× magnification) of the primary tumors (PT) and the orthoxenograft tumors at passages 1 (OT P1) and 4 (OT P4) were labeled with anti-humanCD34 (H) and anti-mouseCD34 (M).


Sanger sequencing of the germline NF1 mutation c . 350T > A present in the patient that developed two different tumors ( MPNST - NF1 - 001 and MPNST - NF1 - 002 ) . Sequencing results from normal tissue , primary tumor ( PT ) , and orthotopic xenograft tumors ( OT ) are shown . The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples , and not in the corresponding derived orthoxenografts , indicating probable loss of the human stromal cells .

Sanger sequencing of the germline NF1 mutation c.350T>A present in the patient that developed two different tumors (MPNST-NF1-001 and MPNST-NF1-002). Sequencing results from normal tissue, primary tumor (PT), and orthotopic xenograft tumors (OT) are shown. The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples, and not in the corresponding derived orthoxenografts, indicating probable loss of the human stromal cells.


SNP array of primary tumor ( PT ) and orthotopic xenograft passages 1 ( OT P1 ) and 4 ( OT P4 ) for MPNST - NF1 - 001 and MPNST - NF1 - 002 tumors . Results correspond to chromosome 17 . Images show the B - allele frequency ( BAF ) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines : 2n regions are shown in gray , gained regions in orange , lost regions in green , and LOH regions are represented in blue . The vertical red line indicates the location of the NF1 locus . In addition , the percentage of tumor versus stromal cells for each sample is represented in a pie chart ( blue , stromal cells ; red , tumor cells ) .

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SNP array of primary tumor (PT) and orthotopic xenograft passages 1 (OT P1) and 4 (OT P4) for MPNST-NF1-001 and MPNST-NF1-002 tumors. Results correspond to chromosome 17. Images show the B-allele frequency (BAF) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines: 2n regions are shown in gray, gained regions in orange, lost regions in green, and LOH regions are represented in blue. The vertical red line indicates the location of the NF1 locus. In addition, the percentage of tumor versus stromal cells for each sample is represented in a pie chart (blue, stromal cells; red, tumor cells).


Tumor growth effects of treatment with doxorubicin , sorafenib , rapamycin and combinations thereof in the five MPNST xenograft models . Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value . Statistically significant differences are shown as * P < 0 . 05 and * * P < 0 . 001 versus control group by the Bonferroni test .

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Tumor growth effects of treatment with doxorubicin, sorafenib, rapamycin and combinations thereof in the five MPNST xenograft models. Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value. Statistically significant differences are shown as *P < 0.05 and **P < 0.001 versus control group by the Bonferroni test.


For long - term studies , a subgroup of treated mice ( n = 3 - 5 mice / group ) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses ( usually 1 , 500 - 2 , 000 mm3 ) . The graph illustrates differences in the time delay ( in days ) of relapsed tumor masses for the different treatments .

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For long-term studies, a subgroup of treated mice (n = 3-5 mice/group) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses (usually 1,500-2,000 mm3). The graph illustrates differences in the time delay (in days) of relapsed tumor masses for the different treatments.


A Immunoblot of DDX3 expression in lung cancer cell lines .

A Immunoblot of DDX3 expression in lung cancer cell lines.


B , C Colony - forming assays in H1299 ( B ) and A549 ( C ) lung cancer cells after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control . Corresponding immunoblots displaying knockdown levels of DDX3 . Mean from 3 replicates with SD .

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B, C Colony-forming assays in H1299 (B) and A549 (C) lung cancer cells after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control. Corresponding immunoblots displaying knockdown levels of DDX3. Mean from 3 replicates with SD.


D Proliferation of A549 and H1299 cells after knockdown of DDX3 . Mean from 3 replicates with SD . ( A549 P = 0 . 011 , H1299 P = 0 . 014 ; exponential curve fit , extra sum of squares F - test ) .

D Proliferation of A549 and H1299 cells after knockdown of DDX3. Mean from 3 replicates with SD. (A549 P = 0.011, H1299 P = 0.014; exponential curve fit, extra sum of squares F-test).


E β - galactosidase staining in parental A549 cells and A549 DDX3 knockdown cells displaying senescent cells identified by the blue color .

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E β-galactosidase staining in parental A549 cells and A549 DDX3 knockdown cells displaying senescent cells identified by the blue color.


F Expression of DDX3 by immunohistochemistry in normal lung tissue . G DDX3 expression in squamous cell carcinoma . H DDX3 expression in adenocarcinoma . I DDX3 expression in small cell carcinoma . J Expression of DDX3 in different histological types of lung cancer . All data sets were compared against each other ( chi - square test , P = 0 . 481 ) .

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F Expression of DDX3 by immunohistochemistry in normal lung tissue.G DDX3 expression in squamous cell carcinoma.H DDX3 expression in adenocarcinoma.I DDX3 expression in small cell carcinoma.J Expression of DDX3 in different histological types of lung cancer. All data sets were compared against each other (chi-square test, P = 0.481).


K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors ( Kaplan - Meier curve and log - rank test , P = 0 . 016 ) .

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K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors (Kaplan-Meier curve and log-rank test, P = 0.016).


F Immunoblots of pull - down assay of DDX3 with biotin , AK - 298 , and SK - 153 . Lower panels display results using DDX5 and DDX17 antibodies .

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F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.


H Immunoblot displaying increasing concentrations of RK - 33 ( 50 , 100 , 200 nM ) resulting in increased inhibition of unwinding of oligomer products ( lanes 4 - 6 ) .

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H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4-6).


I MTS viability assay of various lung cancer cell lines treated with RK - 33 for 72 h . Mean from 3 replicates with SD .

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I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.


J , K Colony - forming assay of A549 and H3255 cells treated with RK - 33 and with various doses of radiation 4 h later . Curves were fitted with a quadratic polynomial equation . Mean from 2 replicates with SD . P - values were determined by the extra sum of squares F - test .

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J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.


Following injection of 20 mg / kg of RK - 33 , twice a week for 7 weeks , extensive histopathological examination was carried out following necropsy . Identical patterns were observed both in the control and in the treated mice ( n = 2 ) . Samples were stained with H & amp ; amp ; E . Scale bar is 50 μm .

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Following injection of 20 mg/kg of RK-33, twice a week for 7 weeks, extensive histopathological examination was carried out following necropsy. Identical patterns were observed both in the control and in the treated mice (n = 2). Samples were stained with H&amp;E. Scale bar is 50 μm.


Pharmacokinetics of RK - 33 in SCID mice at various time intervals . Results are mean ± SD from 5 mice . LC - MS / MS method was used to determine concentration of RK - 33 in mouse plasma and tissue .

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Pharmacokinetics of RK-33 in SCID mice at various time intervals. Results are mean ± SD from 5 mice. LC-MS/MS method was used to determine concentration of RK-33 in mouse plasma and tissue.


HAPI cells were treated with RK - 33 ( 10 μM ) , HA14 - 1 ( 25 μM ) , or DMSO vehicle , followed by oligomycin ( oligo , 0 . 5 μg / ml ) , FCCP ( 3 μM ) , and antimycin A ( AA , 1 μM ) while oxygen consumption rate ( OCR ) was measured . Pyruvate ( 10 mM ) was added in combination with FCCP to ensure that substrate supply was not rate - limiting for maximal OCR . Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages . OCR is baseline - normalized to the point prior to drug or vehicle addition .

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HAPI cells were treated with RK-33 (10 μM), HA14-1 (25 μM), or DMSO vehicle, followed by oligomycin (oligo, 0.5 μg/ml), FCCP (3 μM), and antimycin A (AA, 1 μM) while oxygen consumption rate (OCR) was measured. Pyruvate (10 mM) was added in combination with FCCP to ensure that substrate supply was not rate-limiting for maximal OCR. Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages. OCR is baseline-normalized to the point prior to drug or vehicle addition.


HAPI microglial cells were incubated for 1 h in glucose - free XF24 assay medium that was supplemented with 2 - deoxyglucose ( 50 mM ) and pyruvate ( 10 mM ) . RK - 33 ( 10 μM ) , oligomycin ( 0 . 5 μg / ml ) , or vehicle control was additionally present as indicated . Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages . Significance was assessed by two - sided , unpaired t - test .

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HAPI microglial cells were incubated for 1 h in glucose-free XF24 assay medium that was supplemented with 2-deoxyglucose (50 mM) and pyruvate (10 mM). RK-33 (10 μM), oligomycin (0.5 μg/ml), or vehicle control was additionally present as indicated. Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages. Significance was assessed by two-sided, unpaired t-test.


Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1 / KrasG12D mouse . Scale bar is 100 μm .

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Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1/KrasG12D mouse. Scale bar is 100 μm.


Micro - CT images of transgenic Twist1 / KrasG12D mice treated as in ( C ) , before treatment and 1 week after treatment . Tumors are indicated by arrows and confirmed by H & amp ; amp ; E staining of lung sections ( lower panel ) . Scale bar is 250 μm . Quantification of tumor volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( D ) . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD .

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Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (C), before treatment and 1 week after treatment. Tumors are indicated by arrows and confirmed by H&amp;E staining of lung sections (lower panel). Scale bar is 250 μm.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (D). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.


An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in ( C ) . Figure displays H & amp ; amp ; E staining of lung sections from radiation - treated ( upper panel ) and RK - 33 - and radiation - treated mice ( lower panel ) . Scale bar is 2 mm . Quantification of tumor burden ( as tumor surface divided by total lung surface ) in orthotopic A549 lung cancer mouse model , as shown in ( F ) . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD .

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An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in (C). Figure displays H&amp;E staining of lung sections from radiation-treated (upper panel) and RK-33- and radiation-treated mice (lower panel). Scale bar is 2 mm.Quantification of tumor burden (as tumor surface divided by total lung surface) in orthotopic A549 lung cancer mouse model, as shown in (F). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.


Micro - CT images of transgenic Twist1 / KrasG12D mice treated as in ( H ) , before treatment and 1 week after treatment . Quantification of tumor volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( I ) and expressed as relative tumor size . Significance was assessed by two - sided , unpaired t - test . Error bars represent SEM .

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Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (H), before treatment and 1 week after treatment.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (I) and expressed as relative tumor size. Significance was assessed by two-sided, unpaired t-test. Error bars represent SEM.


A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry . Knockdown of DDX3 led to a decrease of cells in S - phase and an increase of cells in G1 - phase , indicative of a G1 arrest . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD .

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A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry. Knockdown of DDX3 led to a decrease of cells in S-phase and an increase of cells in G1-phase, indicative of a G1 arrest. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.


B , C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK - 33 ( 0 , 2 , 4 , and 6 μM ) . RK - 33 induced a G1 cell cycle arrest in both cell lines . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD .

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B, C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK-33 (0, 2, 4, and 6 μM). RK-33 induced a G1 cell cycle arrest in both cell lines. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.


D , E Immunoblot of cell cycle - related proteins ( Cyclin D1 and Cyclin E1 ) and cell death - related proteins ( cleaved caspase 7 , cleaved caspase 9 ) in A549 and H1299 cells after treatment with RK - 33 ( 10 μM ) . Initially , a strong decrease of Cyclin D1 was observed . After 8 and 24 h , cleaved caspases 9 and 7 were apparent .

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D, E Immunoblot of cell cycle-related proteins (Cyclin D1 and Cyclin E1) and cell death-related proteins (cleaved caspase 7, cleaved caspase 9) in A549 and H1299 cells after treatment with RK-33 (10 μM). Initially, a strong decrease of Cyclin D1 was observed. After 8 and 24 h, cleaved caspases 9 and 7 were apparent.


F Immunoblot of MAPK pathway - related proteins ERK1 / 2 and phosphorylated ERK1 / 2 in A549 , H1299 , and H3255 ( RK - 33 resistant ) cells 24 h after treatment with RK - 33 ( 7 . 5 μM or 10 μM ) . ERK2 and especially ERK1 become dephosphorylated after treatment with RK - 33 in A549 and H1299 cells but not in H3255 cells . Outlined boxes indicate spliced lanes .

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F Immunoblot of MAPK pathway-related proteins ERK1/2 and phosphorylated ERK1/2 in A549, H1299, and H3255 (RK-33 resistant) cells 24 h after treatment with RK-33 (7.5 μM or 10 μM). ERK2 and especially ERK1 become dephosphorylated after treatment with RK-33 in A549 and H1299 cells but not in H3255 cells. Outlined boxes indicate spliced lanes.


A β - catenin ( red ) and DDX3 ( green ) expression in H1299 cells . After overexpressing β - catenin , both DDX3 and β - catenin accumulate in the nucleus . Scale bar is 10 μm .

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A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.


B Immunoprecipitation with DDX3 or H3K4Me3 ( control ) and immunoblotted with β - catenin in A549 and H1299 cells . Outlined boxes indicate spliced lanes .

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B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.


C Immunoprecipitation with β - catenin or H3K4Me3 ( control ) and immunoblotted with DDX3 in A549 and H1299 cells . Outlined boxes indicate spliced lanes .

C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.


D , E β - catenin / TCF4 activity was determined by the TOP / FOP reporter assay . Co - transfection with β - catenin is indicated below .

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D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.


F - I H1299 and A549 cells were treated with RK - 33 ( 0 , 1 , 2 , and 3 μM ) and co - transfected with β - catenin in ( F , H ) . Treatment with RK - 33 decreased TCF4 activity in both cell lines .

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F-I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.


J , K Normalized mRNA expression of TCF4 - regulated proteins ( Axin - 2 , c - Myc , Cyclin D1 ) and DDX3 were measured by qRT - PCR in H1299 cells after knockdown of DDX3 ( J ) and treatment with RK - 33 ( K ) . All experiments were repeated three times .

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J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT-PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.


A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2 - Gy radiation and A549 cells pre - treated with 6 μM RK - 33 , 12 h before radiation . Overlap of 53BP1 and γH2AX is seen in the merged picture of the co - immunofluorescence staining . Scale bar is 2 μm .

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A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.


B A549 cells were pre - treated with RK - 33 and radiated with 2 Gy , and 53BP1 and γH2AX foci were counted as a measure of DNA damage . Cells with more than 10 foci 53BP1 or γH2AX were counted . More than 400 cells per sample were evaluated .

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B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.


C H1299 cells stably transfected with a homologous recombination ( HR ) reporter construct were treated with RK - 33 . Reporter constructs expressed GFP , which was quantified by flow cytometry . Experiments were repeated three times .

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C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.


D H1299 cells , containing a stable non - homologous end - joining ( NHEJ ) reporter construct , were treated with RK - 33 and knockdown of DDX3 . Reporter construct expressed GFP , which was quantified by flow cytometry . All experiments were repeated three times .

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D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.


E Microarray results from MDA - MB - 231 cells treated with RK - 33 and shDDX3 were validated by qRT - PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates ( Bio - Rad ) and performed in biological triplicates .

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E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT-PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.


F , G DNA repair - related proteins ( ATR and XRCC4 ) , DDX3 , and actin were assessed by immunoblotting in A549 ( F ) and H1299 ( G ) cells . Cells were pretreated for 4 h with vehicle control or 6 μM RK - 33 and then radiated with 0 or 5 Gy . Outlined boxes indicate spliced lanes .

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F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.


ATPase staining of GAS and SOL muscles of WT and DKO mice ( left ) . Corresponding percentage of type I fibers were determined ( right ) ( n = 5 ) . Scale bars : 100 μm .

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ATPase staining of GAS and SOL muscles of WT and DKO mice (left). Corresponding percentage of type I fibers were determined (right) (n=5). Scale bars: 100 μm.