Figure 5 ( A ) Image of touch neurons viewed by zdIs5 in different genetic backgrounds . Scale bar : 20 μm . ( B ) Quantification of the percentage of animals with 0 , 1 , 2 , 3 , 4 , 5 and 6 surviving touch neurons in different genetic backgrounds . Data show mean ± SEM . At least 50 animals per strain were analyzed in each experiment . Number of experiments n = 5 . ( C ) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds in ( B ) . The lid - 1 ( xd288 ) and atgl - 1 ( xd314 ) mutations significantly increase the number of surviving neurons . Neuron - specific expression of atgl - 1 or lid - 1 reverses the protective effect of atgl - 1 or lid - 1 mutation . The data were analyzed using one - way ANOVA with Turkey ' s multiple comparison test . Asterisks signify significant differences between the groups under the crossbars . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with at least 50 animals per strain analyzed in each experiment . ( D ) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds . Mutations of fat - 1 , fat - 3 and fat - 4 significantly enhance the suppression effect of atgl - 1 ( xd314 ) in preventing the neurodegeneration of mec - 4 ( d ) . The data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison test . # # # and # # # # denote significant differences as compared to atgl - 1 ( xd314 ) ; mec - 4 ( d ) . Asterisks signify significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * * * * P < 0 . 0001 . # # # P < 0 . 001 , # # # P < 0 . 0001 . Data show mean ± SEM . Number of experiments n ≥ 3 , with at least 50 animals per strain analyzed in each experiment . ( E ) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds supplemented with different fatty acids . AA and EPA but not LA significantly enhance the neurodegeneration triggered by mec - 4 ( d ) . The data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison test . Asterisks signify significant differences between the groups under the crossbars . ns : not statistically significant . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n ≥ 3 , with at least 50 animals per strain analyzed in each experiment .

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

Figure 5(A) Image of touch neurons viewed by zdIs5 in different genetic backgrounds. Scale bar: 20 μm.(B) Quantification of the percentage of animals with 0, 1, 2, 3, 4, 5 and 6 surviving touch neurons in different genetic backgrounds. Data show mean ± SEM. At least 50 animals per strain were analyzed in each experiment. Number of experiments n = 5.(C) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds in (B). The lid-1(xd288) and atgl-1(xd314) mutations significantly increase the number of surviving neurons. Neuron-specific expression of atgl-1 or lid-1 reverses the protective effect of atgl-1 or lid-1 mutation. The data were analyzed using one-way ANOVA with Turkey's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n = 5, with at least 50 animals per strain analyzed in each experiment.(D) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds. Mutations of fat-1, fat-3 and fat-4 significantly enhance the suppression effect of atgl-1(xd314) in preventing the neurodegeneration of mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. ### and #### denote significant differences as compared to atgl-1(xd314); mec-4(d). Asterisks signify significant differences as compared to mec-4(d). ns: not statistically significant. ****P < 0.0001. ###P < 0.001, ###P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment.(E) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds supplemented with different fatty acids. AA and EPA but not LA significantly enhance the neurodegeneration triggered by mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ns: not statistically significant. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment.


Figure 6 ( A ) Lipidomic data show that the relative content of total C20 : 3 is increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) and atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . The increase is greatest in mutants with the fat - 4 mutation . ( B ) Lipidomic data show that the relative content of total C20 : 4 is increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) and atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . The increase is greatest in mutants with the fat - 4 mutation . ( C ) Lipidomic data show that the relative content of total C20 : 5 is slightly increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) but is dramatically decreased in fat - 4 ( wa14 ) ; mec - 4 ( d ) and atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) , which matches the role of FAT - 4 in synthesizing C20 : 5 ( EPA ) . Data information The data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with about 10 , 000 animals per strain analyzed in each experiment . ( D ) The percentage of C20 : 3 - , C20 : 4 - or C20 : 5 - containing TAGs to total lipids containing the same PUFA in different genetic backgrounds . The percentage of C20 : 4 - containing TAGs to total lipids containing C20 : 4 is significantly increased in atgl - 1 and fat - 4 single mutants . The percentage is further increased in atgl - 1 and fat - 4 double mutant . Data information : FA : free fatty acids ; TAG : triacylglycerol ; PL : phospholipids . The data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with about 10 , 000 animals per strain analyzed in each experiment . ( E ) The percentage of C20 : 3 - , C20 : 4 - or C20 : 5 - containing phospholipids to total lipids containing the same PUFA in different genetic backgrounds . The percentage of C20 : 4 - containing phospholipids to total lipids containing C20 : 4 is significantly decreased in atgl - 1 and fat - 4 single mutants . The percentage is further decreased in atgl - 1 and fat - 4 double mutant . Data information : FA : free fatty acids ; TAG : triacylglycerol ; PL : phospholipids . The data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison test . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with about 10 , 000 animals per strain analyzed in each experiment . ( F ) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds . mboa - 7 mutation significantly increases the percentage of three or more surviving touch neurons in mec - 4 ( d ) , atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) and atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . The data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison test . Asterisks signify significant differences between the groups under the crossbars . * * P < 0 . 01 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n ≥ 4 , with at least 50 animals per strain analyzed in each experiment . ( G ) The percentage of C20 : 4 - containing phospholipids to total lipids that have C20 : 4 is dramatically decreased in mboa - 7 ( gk399 ) ; atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . The data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison test . Asterisks signify significant differences as compared to mec - 4 ( d ) . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± SEM . Number of experiments n = 5 , with about 10 , 000 animals per strain analyzed in each experiment .

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

Figure 6(A) Lipidomic data show that the relative content of total C20:3 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation. (B) Lipidomic data show that the relative content of total C20:4 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation. (C) Lipidomic data show that the relative content of total C20:5 is slightly increased in atgl-1(xd314); mec-4(d) but is dramatically decreased in fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d), which matches the role of FAT-4 in synthesizing C20:5 (EPA). Data information The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.(D) The percentage of C20:3-, C20:4- or C20:5-containing TAGs to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing TAGs to total lipids containing C20:4 is significantly increased in atgl-1 and fat-4 single mutants. The percentage is further increased in atgl-1 and fat-4 double mutant. Data information: FA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.(E) The percentage of C20:3-, C20:4- or C20:5-containing phospholipids to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing phospholipids to total lipids containing C20:4 is significantly decreased in atgl-1 and fat-4 single mutants. The percentage is further decreased in atgl-1 and fat-4 double mutant. Data information: FA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.(F) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds. mboa-7 mutation significantly increases the percentage of three or more surviving touch neurons in mec-4(d), atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. **P < 0.01, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 4, with at least 50 animals per strain analyzed in each experiment.(G) The percentage of C20:4-containing phospholipids to total lipids that have C20:4 is dramatically decreased in mboa-7(gk399); atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks signify significant differences as compared to mec-4(d). ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


Figure 1Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies . Fluorescence analyses of GFP - ATG5 or ATG16L1 - GFP stably expressed in ATG5 - / - or ATG16L1 - / - . Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence . Scale bar : 10 μm . Assessment of ATG16L1 levels in the cytosolic ( C ) and membrane ( M ) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1 . ATG16L1 - / - and ATG5 - / - stably expressed ATG16L1 - GFP while ATG3 - / - stably expressed Flag - S - ATG16L1 . Antibodies against α - tubulin and integrin β1 were used as controls for fractionation . Exogenous ( exo ) ATG16L1 was detected using antibodies against ATG16L1 . Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs ( WIPI2 + / + ) and WIPI2 - / - cells . Immunofluorescence analyses of ATG16L1 - / - and WIPI2 - / - stably expressing Flag - S - ATG16L1 . Cells were amino acid starved ( AA starve ) for 2 hr in the presence or absence of 3 ' MA ( and additional pretreatment for 30 min ) followed by fixation and immuno - staining using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

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

Figure 1Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies.Fluorescence analyses of GFP-ATG5 or ATG16L1-GFP stably expressed in ATG5-/- or ATG16L1-/-. Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm.Assessment of ATG16L1 levels in the cytosolic (C) and membrane (M) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1. ATG16L1-/- and ATG5-/- stably expressed ATG16L1-GFP while ATG3-/- stably expressed Flag-S-ATG16L1. Antibodies against α-tubulin and integrin β1 were used as controls for fractionation. Exogenous (exo) ATG16L1 was detected using antibodies against ATG16L1.Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs (WIPI2+/+) and WIPI2-/- cells.Immunofluorescence analyses of ATG16L1-/- and WIPI2-/- stably expressing Flag-S-ATG16L1. Cells were amino acid starved (AA starve) for 2 hr in the presence or absence of 3'MA (and additional pretreatment for 30 min) followed by fixation and immuno-staining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Figure 2Fragments depicted in ( A ) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S - ATG16L1 . Scale bar : 10 μm . U2OS cells expressing Flag - ATG16L1 ∆ 2 or Flag - ATG16L1 ∆ 2 ∆ 182 - 205 were treated as in ( B ) and stained using antibodies against Flag tag ( to detect ATG16L1 , green ) and FIP200 ( red ) . Scale bar : 10 μm . Protein - protein interaction assay in 293T cells transiently transfected with the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies . Homodimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies . Homodimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies . Flag - ATG16L1WT or Flag - ATG16L1 ∆ 182 - 205 were stably expressed in ATG5 - / - cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

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

Figure 2Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm.U2OS cells expressing Flag-ATG16L1∆2 or Flag-ATG16L1∆2∆182-205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm.Protein-protein interaction assay in 293T cells transiently transfected with the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.Flag-ATG16L1WT or Flag-ATG16L1∆182-205 were stably expressed in ATG5-/- cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Figure 3Microscopy - based protein - liposome binding assay . ATG16L1 - GFP immobilised on beads incubated in the presence of rhodamine - labelled liposome preparations containing the indicated phosphoinositides . Scale bar : 50 μm . Quantification of relative liposome bindingMicroscopy - based protein - liposome binding assay as in ( B ) . ATG16L1WT - and ATG16L1LD - GFP immobilised on beads were incubated with rhodamine - labelled , PI3P - positive liposome preparations . Scale bar : 50 μm . Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values . Protein - protein interaction assay in 293T cells transiently transfected with the indicated S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies . by immunoblotting using the indicated antibodies . Dimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated S - tagged ATG16L1 constructs and

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Figure 3Microscopy-based protein-liposome binding assay. ATG16L1-GFP immobilised on beads incubated in the presence of rhodamine-labelled liposome preparations containing the indicated phosphoinositides. Scale bar: 50 μm.Quantification of relative liposome bindingMicroscopy-based protein-liposome binding assay as in (B). ATG16L1WT- and ATG16L1LD-GFP immobilised on beads were incubated with rhodamine-labelled, PI3P-positive liposome preparations. Scale bar: 50 μm. Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values.Protein-protein interaction assay in 293T cells transiently transfected with the indicated S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.by immunoblotting using the indicated antibodies. Dimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated S-tagged ATG16L1 constructs and analysed


Figure 4ATG16L1 - / - stably expressing the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) and ATG5 ( red ) . Scale bar : 9 μm . Right panels show quantifications of average number of Flag - and ATG5 - positive dots per cell . ATG16L1 - / - stably expressing the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag ( to detect ATG16L1 , red ) and WIPI2 ( green ) . Scale bar : 9 μm . Lower panel shows quantification of average number of WIPI2 positive dots per cell . Average intensities of individual Flag - ATG16L1 dots in ( A ) . Underlying grey circles represent individual data points . Dots were quantified ( n = 1225 for ATG16L1WT ; n = 334 for ATG16L1LD ) . WIPI2 - / - cells reconstituted with the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) and FIP200 ( red ) . Scale bar : 9 μm . Lower panel shows quantification of average number of Flag positive dots per puncta - positive cell .

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Figure 4ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and ATG5 (red). Scale bar: 9 μm. Right panels show quantifications of average number of Flag- and ATG5-positive dots per cell.ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag (to detect ATG16L1, red) and WIPI2 (green). Scale bar: 9 μm. Lower panel shows quantification of average number of WIPI2 positive dots per cell.Average intensities of individual Flag-ATG16L1 dots in (A). Underlying grey circles represent individual data points. Dots were quantified (n=1225 for ATG16L1WT; n=334 for ATG16L1LD).WIPI2-/- cells reconstituted with the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and FIP200 (red). Scale bar: 9 μm. Lower panel shows quantification of average number of Flag positive dots per puncta-positive cell.


Figure 5ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Cells were amino acid starved ( AA starved ) to induce mTORC1 - dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis . ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Mitophagy was induced by CCCP ( 10 μM ) treatment for 6 hr prior to lysis . ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Glucose starvation ( Glu starved ) for 20 hr was used to induce mTORC1 - independent autophagy . BafA1 was added to all conditions 2 hr prior to lysis . ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . LC3 - associated phagocytosis ( LAP ) - like LC3 lipidation was induced by treating cells with CCCP ( 100 μM ) for 2 hr prior to lysis . Right panel shows quantification of LC3 - II / LC3 - I ratio . ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . LAP - like LC3 lipidation induced by monensin or ammonium chloride ( NH4Cl ) for 2 hr prior to lysis .

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Figure 5ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Cells were amino acid starved (AA starved) to induce mTORC1-dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis.ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Mitophagy was induced by CCCP (10 μM) treatment for 6 hr prior to lysis.ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Glucose starvation (Glu starved) for 20 hr was used to induce mTORC1-independent autophagy. BafA1 was added to all conditions 2 hr prior to lysis.ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LC3-associated phagocytosis (LAP)-like LC3 lipidation was induced by treating cells with CCCP (100 μM) for 2 hr prior to lysis. Right panel shows quantification of LC3-II/LC3-I ratio.ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LAP-like LC3 lipidation induced by monensin or ammonium chloride (NH4Cl) for 2 hr prior to lysis.


Figure 6Coomassie gel of recombinant ATG16L172 - 307 wild type and mutants . * indicates bacterial proteinLiposome co - sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes . Supernatant ( S ) and pellet ( P ) were analysed by immunoblotting against T7 tag to detect ATG16L1 . Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments ( right panel ) . Liposome co - sedimentation assay , as in ( C ) , using ATG16L172 - 307LE and sonicated liposomes that contain or lack PI3P . Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments ( right panel ) with error bars depicting SEM values .

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Figure 6Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial proteinLiposome co-sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes. Supernatant (S) and pellet (P) were analysed by immunoblotting against T7 tag to detect ATG16L1. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel).Liposome co-sedimentation assay, as in (C), using ATG16L172-307LE and sonicated liposomes that contain or lack PI3P. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel) with error bars depicting SEM values.


Figure 7ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LE - expression constructs and autophagy assessed during amino acid starvation ( AA starve ) followed by immunoblotting or immunofluorescence analyses . Immunofluorescence analyses of amino acid starved cells ( 2 hr ) using antibodies against WIPI2 ( red ) or ATG16L1 ( green ) . Scale bar : 9 μm . Cells as in ( A ) with the addition of 3 ' MA ( and pretreatment with the drug for 30 min ) . Scale bar : 9 μm . Quantification of percentage of cells positive for ATG16L1 punctaCells were left untreated , amino acid starved for 2 hr , or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr . Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1 . Scale bar : 10 μm . Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr . Cell lysates were analysed using the indicated antibodies . WIPI2 - / - cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr . BafA1 treatment was included in all conditions for 2 hr prior to lysis . Cell lysates were analysed by immunoblotting using the indicated antibodies . Quantification of LC3 - II / LC3 - I ratio of three independent experiments is shown on the right panel . Ferroptosis assay in ATG16L1 - / - cells stably expressing ATG16L1WT , ATG16L1LD or ATG16L1LE . Cells were cultured in amino acid free media ( AA starve ) in the presence of 10 % FBS or 10 % dialysed FBS ( diFBS ) for 24 hr . Quantification of percentage of PI - positive cells from at least three independent experiments is shown .

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Figure 7ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm.Quantification of percentage of cells positive for ATG16L1 punctaCells were left untreated, amino acid starved for 2 hr, or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1. Scale bar: 10 μm.Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr. Cell lysates were analysed using the indicated antibodies.WIPI2-/- cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr. BafA1 treatment was included in all conditions for 2 hr prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3-II/LC3-I ratio of three independent experiments is shown on the right panel.Ferroptosis assay in ATG16L1-/- cells stably expressing ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 hr. Quantification of percentage of PI-positive cells from at least three independent experiments is shown.


Figure 1B . Western blot analysis of purified autophagic vesicles . Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms , respectively , and are represented with total lysate ( TL ) . Depicted are representative blots of 14 independent approaches .

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Figure 1B. Western blot analysis of purified autophagic vesicles. Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms, respectively, and are represented with total lysate (TL). Depicted are representative blots of 14 independent approaches.


Figure 2A . Differential interference contrast microscopy images of purified autophagic vesicles at high ( I ) or low ( II ) dilution . Images are representative of 3 independent approaches . B . Negative stain electron microscopy images of isolated vesicles . C . Size evaluation of isolated vesicles . The diameters of approx . 60 individual vesicles were determined using EM images . D . Western blot analysis of isolated autophagic vesicles upon proteinase K digestion . Mechanically opened vesicles served as positive control . For negative control , isolates were incubated with BSA instead of proteinase K . Depicted are two different blots that are representative for 5 independent

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Figure 2A. Differential interference contrast microscopy images of purified autophagic vesicles at high (I) or low (II) dilution. Images are representative of 3 independent approaches. B. Negative stain electron microscopy images of isolated vesicles. C. Size evaluation of isolated vesicles. The diameters of approx. 60 individual vesicles were determined using EM images.D. Western blot analysis of isolated autophagic vesicles upon proteinase K digestion. Mechanically opened vesicles served as positive control. For negative control, isolates were incubated with BSA instead of proteinase K. Depicted are two different blots that are representative for 5 independent experiments


Figure 3E . Phospholipids identified in autophagic vesicles isolated upon different conditions . Relative amounts were calculated based on total levels of detected phospholipids . Abbreviations are depicted in A .

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Figure 3E. Phospholipids identified in autophagic vesicles isolated upon different conditions. Relative amounts were calculated based on total levels of detected phospholipids. Abbreviations are depicted in A.


Figure 4A . Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy . Log2 - transformed fold changes . For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles , no fold changes could be calculated and they are indicated as not determinable ( nd ) . B . Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy . Log2 - transformed fold changes . For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles , no fold changes could be calculated and they are indicated as not determinable ( nd ) .

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Figure 4A. Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd). B. Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd).


Figure 1Growth suppression in cancer cells after 72h of treatment with APR - 246 + / - MRP1 inhibitor MK - 571 as shown by the WST - 1 assay ( n ≥ 3 , n shown in Table S1 ) . Cross in heatmap indicates no data available . Growth suppression in human dermal fibroblasts ( HDF ) by WST - 1 assay after 72h of with APR - 246 + / - MK - 571 ( n = 3 ) . Mean IC50 values ( µM ) for APR - 246 + / - 20 μM MK - 571 of 21 cell lines . * p < 0 . 0001 , Wilcoxon test . Each dot represents one cell line . Mean IC50 values and n shown in Table S1 . ZIP synergy scores of most synergistic area sub - grouped according to TP53 gene status for 21 cell lines . Each dot represents one cell line . Scores and n are shown in Table S1 . Sub - G1 DNA content of OVCAR - 3 cells ( R248Q ) after APR - 246 + / - MK - 571 treatment as assessed by propidium iodide staining at 48h ( n = 3 ) . OVCAR - 3 cell confluency by IncuCyte ® during 72h of treatment with APR - 246 + / - MK - 571 ( n = 3 ) . Growth suppression in HCT116 WT ( n = 4 ) and R248W cells ( n = 3 ) after 72h treatment with APR - 246 + / - MRP1 inhibitor Reversan , as shown by WST - 1 . Western blot analysis of MRP1 ( Cell Signaling . ) , p53 ( DO - 1 ) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53 . Growth suppression ( WST - 1 assay ) of HCT116 WT and R248W cells transfected with MRP1 siRNA ( n ≥ 5 and n ≥ 2 respectively ) after 48h APR - 246 treatment . Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs . Data and n of individual siRNAs are shown in Fig S1J , data is also part of Fig 7B . Western blot analysis of MRP1 ( Cell Signaling . ) , p53 ( DO - 1 ) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector ( EV ) and MRP1 expression vector . Growth suppression ( WST - 1 assay ) of HCT116 WT and R248W cells transfected with empty vector ( EV ) and MRP1 vector after 72h APR - 246 treatment ( n = 4 ) . Data is also part of Fig S7E .

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Figure 1Growth suppression in cancer cells after 72h of treatment with APR-246 +/- MRP1 inhibitor MK-571 as shown by the WST-1 assay (n ≥ 3, n shown in Table S1). Cross in heatmap indicates no data available.Growth suppression in human dermal fibroblasts (HDF) by WST-1 assay after 72h of with APR-246 +/- MK-571 (n = 3). Mean IC50 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p < 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1. ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1. Sub-G1 DNA content of OVCAR-3 cells (R248Q) after APR-246 +/- MK-571 treatment as assessed by propidium iodide staining at 48h (n = 3).OVCAR-3 cell confluency by IncuCyte® during 72h of treatment with APR-246 +/- MK-571 (n = 3).Growth suppression in HCT116 WT (n = 4) and R248W cells (n = 3) after 72h treatment with APR-246 +/- MRP1 inhibitor Reversan, as shown by WST-1.Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53.Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B.Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector (EV) and MRP1 expression vector.Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E.


Figure 2Growth of Eso26 xenografts ( R248W mutant TP53 ) in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 and MK - 571 for 16 days . p values determined by one - way ANOVA with Tukey ' s correction at day 18 , n is indicated in the figure . Kaplan - Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 and MK - 571 for 16 days . Mice that were culled at ethical endpoints prior to reaching a tumor volume of > 1400mm3 are marked as censored observations . p values were determined by Log - rank ( Mantell - Cox ) test between different treatment groups . n is indicated in the figure . Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint ( 5 days ) or post treatment / late timepoint ( > 22 days ) after treatment initiation with APR - 246 ( 50mg / kg ) + / - MK - 571 ( 50mg / kg ) . Dots indicate individual tumors . Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig . 2C . Dots indicate individual tumors . Representative images of hematoxylin / eosin ( H & E ) staining and immunostaining of Ki67 , cleaved caspase 3 and p53 , post treatment / late timepoint ( > 22 days after treatment initiation ) with APR - 246 ( 50mg / kg ) + / - MK - 571 ( 50mg / kg ) . Growth suppression determined by the ATP - based CTG assay in colorectal cancer patient - derived organoids ( colo - PDO ) after treatment with APR - 246 + / - MK - 571 ( n = 3 ) . IC50 values ( µM ) APR - 246 + / - 20 μM MK - 571 in indicated PDOs . * p = 0 . 04 , paired t test ( n = 3 except colo - PDO2 and eso - PDO3 where n = 2 ) . Organoid area ( in black , left y - axis ) and PI intensity ( in red , right y - axis ) of colo - PDO1 and colo - PDO2 as determined by image analysis 72h after treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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Figure 2Growth of Eso26 xenografts (R248W mutant TP53) in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. p values determined by one-way ANOVA with Tukey's correction at day 18, n is indicated in the figure.   Kaplan-Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. Mice that were culled at ethical endpoints prior to reaching a tumor volume of >1400mm3 are marked as censored observations. p values were determined by Log-rank (Mantell-Cox) test between different treatment groups. n is indicated in the figure.   Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint (5 days) or post treatment / late timepoint (>22 days) after treatment initiation with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Dots indicate individual tumors.Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig. 2C. Dots indicate individual tumors. Representative images of hematoxylin/eosin (H&E) staining and immunostaining of Ki67, cleaved caspase 3 and p53, post treatment / late timepoint (>22 days after treatment initiation) with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Growth suppression determined by the ATP-based CTG assay in colorectal cancer patient-derived organoids (colo-PDO) after treatment with APR-246 +/- MK-571 (n = 3).IC50 values (µM) APR-246 +/- 20 μM MK-571 in indicated PDOs. *p = 0.04, paired t test (n = 3 except colo-PDO2 and eso-PDO3 where n = 2).Organoid area (in black, left y-axis) and PI intensity (in red, right y-axis) of colo-PDO1 and colo-PDO2 as determined by image analysis 72h after treatment with APR-246 +/- MK-571 (n = 3).


Figure 314C accumulation ( cpm / mg x ml - 1 ) in 11 cell lines after 24h 14C - APR - 246 + / - MK - 571 treatment ( n ≥ 3 ) . * p = 0 . 0006 , paired t - test . 14C - accumulation and n of individual cell lines as shown in Table S3 . 14C accumulation ( cpm / mg x ml - 1 ) in HCT116 ( WT and R248W ) cells after 24h + treatment with 14C - APR - 246 + / - MK - 571 or Reversan ( n ≥ 3 , each dots in figure ) . 14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W cells ( n ≥ 3 and ≥ 2 ) transfected with MRP1 siRNAs after 24h of 14C - APR - 246 treatment . Indicated values represent four individual MRP1 siRNAs and two individual negative control ( Ctrl ) siRNAs , where each dot is an individual siRNA . Mean of 14C - accumulation after treatment and n with individual siRNA sequences is shown in Table S4 . * p = 0 . 04 and for R248W p = 0 . 06 , Wilcoxon matched pairs signed rank test .

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

Figure 314C accumulation (cpm/mg x ml-1) in 11 cell lines after 24h 14C -APR-246 +/- MK-571 treatment (n ≥ 3). *p = 0.0006, paired t-test. 14C-accumulation and n of individual cell lines as shown in Table S3.14C accumulation (cpm/mg x ml-1) in HCT116 (WT and R248W) cells after 24h +treatment with 14C -APR-246 +/- MK-571 or Reversan (n ≥ 3, each dots in figure).14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test.


Figure 4APR - 246 content in OVCAR - 3 cells ( TP53 R248Q ) 24h after treatment with APR - 246 + / - MK - 571 , ( n = 3 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 . Glutathione - conjugated MQ ( GS - MQ ) in OVCAR - 3 cells after 24h treatment with APR - 246 + / - MK - 571 ( n = 2 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 . Amount of GS - MQ at indicated timepoints after incubation of GS - MQ + / - NAC ( n = 2 ) . See Fig . EV4 for chemical reaction . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 . Amount of GS - MQ by LC - MS ( green line , left axis ) and NAC - MQ ( purple line , right axis ) over time after incubation of GS - MQ with NAC ( n = 2 ) . Values on y - axes are not comparable because GS - MQ and NAC - MQ have different response signals on MS . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

Figure 4APR-246 content in OVCAR-3 cells (TP53 R248Q) 24h after treatment with APR-246 +/- MK-571, (n = 3). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.Glutathione-conjugated MQ (GS-MQ) in OVCAR-3 cells after 24h treatment with APR-246 +/- MK-571 (n = 2). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.Amount of GS-MQ at indicated timepoints after incubation of GS-MQ +/- NAC (n = 2). See Fig. EV4 for chemical reaction. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.Amount of GS-MQ by LC-MS (green line, left axis) and NAC-MQ (purple line, right axis) over time after incubation of GS-MQ with NAC (n = 2). Values on y-axes are not comparable because GS-MQ and NAC-MQ have different response signals on MS. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Figure 5Mean IC50 ( µM ) values of APR - 246 in 17 different cell lines ( n ≥ 3 ) as shown by WST - 1 . * p = 0 . 03 , Mann Whitney test . Mean IC50 ( µM ) values for APR - 246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 . Box - and - whisker plot of Pearson correlations between PRIMA - 1 area - under - the - curve ( AUC ) and the levels of 225 different metabolites from the DepMap portal . Central band indicates median , boxes indicate 25th and 75th percentile , and whiskers show 2 . 5th and 97 . 5th percentile outlier metabolites . High GSH and GSSG ( in red ) correlate with low PRIMA - 1 sensitivity . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 . Total intracellular glutathione ( GSH + GSSG ) as shown by glutathione reductase ( GR ) re - cycling assay in H1299 R175H ( tet - off ) ( + / - doxycycline ) and H1299 - / - cells 24h after seeding ( n = 3 ) . Indicated values are fold change compared to mutant TP53 cells . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 . Total intracellular glutathione ( GSH + GSSG ) as shown by GR re - cycling assay in untreated HCT116 ( R248W , WT and - / - ) cells 48h after seeding ( n = 3 , 5 or 2 respectively ) . Indicated values are fold change to mutant TP53 cells . Part of this data is shown in Fig . EV4 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 . Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells ( n = 2 ) Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 . Correlation between 14C accumulation ( cpm / mg x ml - 1 ) after treatment with 25 µM 14C - APR - 246 and IC50 ( µM ) values for APR - 246 in 11 cell lines ( n ≥ 3 ) ( r = - 0 . 66 , p = 0 . 03 , Pearson correlation ) . Each dot represents one cell line , 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 . ABCC1 mRNA Expression , RSEM ( Batch normalized from Illumina HiSeq _ RNASeqV2 ) in lung cancer ( luad & lusc studies , * p < 0 . 0001 ) and colon adenocarcinoma ( coad study , * p = 0 . 005 ) grouped into having no alterations or putative driver missense mutations in TP53 . Statistical analysis by Mann Whitney test , n is indicated in the figure . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 . 14C accumulation ( cpm / mg x ml - 1 ) in H1299 R175H cells ( + / - doxycycline ) and H1299 - / - cells after 24h treatment with 14C - APR - 246 ( n = 2 ) . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 . Growth suppression in H1299 R175H cells ( + / - doxycycline ) and H1299 - / - cells treated for 72h with APR - 246 as shown by WST - 1 assay ( n = 3 ) . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Figure 5Mean IC50 (µM) values of APR-246 in 17 different cell lines (n ≥ 3) as shown by WST-1. *p = 0.03, Mann Whitney test. Mean IC50 (µM) values for APR-246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.Box-and-whisker plot of Pearson correlations between PRIMA-1 area-under-the-curve (AUC) and the levels of 225 different metabolites from the DepMap portal. Central band indicates median, boxes indicate 25th and 75th percentile, and whiskers show 2.5th and 97.5th percentile outlier metabolites. High GSH and GSSG (in red) correlate with low PRIMA-1 sensitivity. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. Total intracellular glutathione (GSH+GSSG) as shown by glutathione reductase (GR) re-cycling assay in H1299 R175H (tet-off) (+/- doxycycline) and H1299 -/- cells 24h after seeding (n = 3). Indicated values are fold change compared to mutant TP53 cells. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.  Total intracellular glutathione (GSH+GSSG) as shown by GR re-cycling assay in untreated HCT116 (R248W, WT and -/-) cells 48h after seeding (n = 3, 5 or 2 respectively). Indicated values are fold change to mutant TP53 cells. Part of this data is shown in Fig. EV4. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2) Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. Correlation between 14C accumulation (cpm/mg x ml-1) after treatment with 25 µM 14C-APR-246 and IC50 (µM) values for APR-246 in 11 cell lines (n ≥ 3) (r = -0.66, p = 0.03, Pearson correlation). Each dot represents one cell line, 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.  ABCC1 mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) in lung cancer (luad & lusc studies, *p<0.0001) and colon adenocarcinoma (coad study, *p=0.005) grouped into having no alterations or putative driver missense mutations in TP53. Statistical analysis by Mann Whitney test, n is indicated in the figure. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.  14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.  Growth suppression in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells treated for 72h with APR-246 as shown by WST-1 assay (n = 3). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Figure 6 Total intracellular glutathione ( GSH + GSSG per 106 cells ) , as determined by GR re - cycling assay after 24h MK - 571 treatment in six different cell lines ( n ≥ 3 , except HCT116 WT n = 2 ) . * p = 0 . 014 , Paired t test . Each dot represents one cell line , see Fig . EV6A for individual cell lines and n . Intracellular oxidized glutathione ( GSSG per 106 cells ) as shown by LC - MS in OVCAR - 3 ( R248Q TP53 ) cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) . Intracellular reduced glutathione ( GSH per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) . Western blot analysis of MRP1 ( Cell Signaling ) , xCT and GAPDH of OVCAR - 3 cells treated with APR - 246 + / - MK - 571 for 24h . Western blot analysis of MRP1 ( Cell Signaling ) , xCT and GAPDH of HCT116 WT and R248W cells treated with APR - 246 + / - MK - 571 for 24h . Intracellular cystine ( CySS per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) . Intracellular cysteine ( Cys per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Figure 6 Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n. Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3). Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).  Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h. Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h. Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).  Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Figure 7 14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W mutant TP53 cells + - transfected with MRP1 or xCT siRNA following 24h of 14C - APR - 246 treatment + / - MK - 571 . Indicated values are mean values from four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) and two control siRNAs ( n ≥ 3 ) . Mean 14C - accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C - APR - 246 treatment is also shown in Fig 3D . Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR - 246 treatment as shown by WST - 1 assay . Indicated values are mean values of four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) and two control siRNAs ( n ≥ 3 ) . Data for control and MRP1 siRNA after APR - 246 single treatment are also included in Fig 1I . Values and n for individual siRNA shown in Fig . S7C . Total intracellular glutathione ( GSH + GSSG per 106 cells ) in HCT116 WT cells as shown by GR re - cycling assay 48h post transfection of siRNA . Indicated values are means from four MRP1 siRNAs ( n = 3 ) , two xCT siRNA ( n = 2 - 3 ) and two control siRNA ( n = 2 - 3 ) . Total intracellular glutathione ( GSH + GSSG per 106 cells ) in HCT116 WT and R248W cells measured by GR re - cycling assay after 24h of MK - 571 or sulfasalazine ( SSZ ) treatment ( n ≥ 3 , except SSZ where n = 2 , n indicated by dots ) . 14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W cells at 24h treatment with 14C - APR - 246 , combined with MK - 571 or sulfasalazine ( SSZ ) , ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots ) . Data for 14C - APR - 246 + / - MK - 571 is also shown in Fig 3C and S3D . Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) , or untreated ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots ) OVCAR - 3 ( R248Q TP53 ) confluence by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalzine ( SSZ ) ( n = 3 ) . Part of the data are shown in Fig 1G . Growth suppression in OVCAR - 3 cells as determined by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) depicted as heatmaps ( n = 3 ) .

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

Figure 7 14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D.  Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C.  Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3).  Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT and R248W cells measured by GR re-cycling assay after 24h of MK-571 or sulfasalazine (SSZ) treatment (n ≥ 3, except SSZ where n = 2, n indicated by dots).  14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells at 24h treatment with 14C -APR-246, combined with MK-571 or sulfasalazine (SSZ), (n ≥ 4, except SSZ where n = 2, n indicated by dots). Data for 14C -APR-246 +/- MK-571 is also shown in Fig 3C and S3D.  Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ), or untreated (n ≥ 4, except SSZ where n = 2, n indicated by dots)  OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G. Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3).


Figure 1Male , 12wk wildtype ( WT ; C57Bl / 6J ) or obese / diabetic monogenic ( db / db ; BKS . Cg - m + / + Lepr DB / J ; N = 4 / group , A - E ) , New Zealand Black ( NZB ) and polygenic obese / prediabetic New Zealand Obese ( NZO ; n = 4 / group ; F - J ) , as well as young ( i . e . 3 mo ) and aged ( i . e . 22 mo ; n = 5 / group ; K - O ) , mice were fed ad libitum ( fed ) or fasted for 24h ( fasted ) . Serum non - esterified fatty acids ( A , F , K ) , triglycerides ( B , G , L ) and ketone bodies ( C , H , M ) were measured . In addition , serumacylcarinitine profiling was conducted and medium - chain ( D , I , N ) and long - chain ( E , J , O ) acylcarnitine concentrations are shown . In another cohort of mice , ex vivo long - chain fatty acid ( LCFA ) metabolism , including uptake ( P ) , oxidation ( Q ) and non - oxidative LCFA disposal ( NOFAD ; R ) , in precision cut liver slices from fed and fasted WT and db / db mice ( n = 3 / group ; 4 slices per mouse ) , were determined . In addition , in slices from fasted mice , glucose output was determined in the presence of incubation with NEFA ( BSA - NEFA ) or vehicle ( BSA ) ( S ) .

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

Figure 1Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic monogenic (db/db; BKS.Cg-m+/+ Lepr DB/J; N = 4/group, A-E), New Zealand Black (NZB) and polygenic obese/prediabetic New Zealand Obese (NZO; n=4/group; F-J), as well as young (i.e. 3 mo) and aged (i.e.22 mo; n = 5/group; K-O), mice were fed ad libitum (fed) or fasted for 24h (fasted). Serum non-esterified fatty acids (A, F, K), triglycerides (B, G, L) and ketone bodies (C, H, M) were measured. In addition, serumacylcarinitine profiling was conducted and medium-chain (D, I, N) and long-chain (E, J, O) acylcarnitine concentrations are shown.In another cohort of mice, ex vivo long-chain fatty acid (LCFA) metabolism, including uptake (P), oxidation (Q) and non-oxidative LCFA disposal (NOFAD; R), in precision cut liver slices from fed and fasted WT and db/db mice (n = 3/ group; 4 slices per mouse), were determined. In addition, in slices from fasted mice, glucose output was determined in the presence of incubation with NEFA (BSA-NEFA) or vehicle (BSA) (S).


Figure 2Liver mRNA expression of Growth Arrest and DNA Damage inducible 45 alpha ( Gadd45a ; A , D , G ) , beta ( Gadd45b ; B , E , H ) and gamma ( Gadd45g ; C , F , I ) was measured in fed and fasted obese / diabetic monogenic ( db / db ( n = 4 / group ) ; A , - C ) , obese / pre - diabetic polygenic New Zealand Obese ( NZO ( n = 4 / group ; D - F ) , and aged C57Bl / 6J ( 22mo ; n = 5 / group ; G - I ) as well as corresponding lean , young wildtype ( WT ) mice ( n = 4 / group ) . Matched controls for the NZO and aged mice were New Zealand Black ( NZB ) and 12wk old C57Bl / 6J ( 12wk ) mice , respectively . Liver GADD45β as well as the housekeeping protein Valosin containing protein ( VCP ) protein expression was measured from liver samples ( J - L ) . Data are mean ± SEM . Effect of genotype , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Effect of nutritional state : # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 . The statistical test used and respective p - value outputs can be found in Appendix Table S1 .

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

Figure 2Liver mRNA expression of Growth Arrest and DNA Damage inducible 45 alpha (Gadd45a; A, D, G), beta (Gadd45b; B, E, H) and gamma (Gadd45g; C, F, I) was measured in fed and fasted obese/diabetic monogenic (db/db (n=4/group); A,-C), obese/pre-diabetic polygenic New Zealand Obese (NZO (n=4/group; D-F), and aged C57Bl/6J (22mo; n = 5/group; G-I) as well as corresponding lean, young wildtype (WT) mice (n=4/group). Matched controls for the NZO and aged mice were New Zealand Black (NZB) and 12wk old C57Bl/6J (12wk) mice, respectively.Liver GADD45β as well as the housekeeping protein Valosin containing protein (VCP) protein expression was measured from liver samples (J-L). Data are mean ± SEM. Effect of genotype, * p < 0.05, ** p < 0.01, *** p < 0.001. Effect of nutritional state: # p < 0.05, ## p < 0.01, ### p < 0.001. The statistical test used and respective p-value outputs can be found in Appendix Table S1.


Figure 3Male , GADD45β + / + ( WT ; n = 6 ) or GADD45β - / - mice ( KO ; n = 4 ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) , and subsequently refed for 24h . O2 consumption rate ( A ) , CO2 production rate ( B ) and respiratory exchange ratio ( C ) were measured by indirect calorimetry . In a distinct cohort , male GADD45β + / + ( WT ; n = 5 ) or GADD45β - / - mice ( KO ; n = 8 ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) . Serumnon - esterified fatty acids ( D ) and ketone bodies ( E ) as well as serum ( F ) and liver ( G ) triglycerides ( TG ) were measured . Serum TG ( H ) , NEFA ( I ) , and blood glucose ( BG , J ) concentrations during an oral lipid tolerance test in overnight fasted GADD45β + / + ( WT ; n = 5 ) or GADD45β - / - ( KO ; n = 5 ) mice . Bloodglucose excursion during and intraperitoneal insulin tolerance test ( K ) as well as fasting bloodglucose ( L ) , seruminsulin ( M ) , and HOMA - IR ( N ) in GADD45β + / + ( WT ; n = 6 ) or GADD45β - / - mice ( KO , n = 9 ) chronically fed a normal - ( NFD ) or high ( HFD ) fat diet . Data are mean ± SEM .

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

Figure 3Male, GADD45β+/+ (WT; n=6) or GADD45β-/- mice (KO; n=4) were fed ad libitum (fed) or fasted for 24h (fasted), and subsequently refed for 24h. O2 consumption rate (A), CO2 production rate (B) and respiratory exchange ratio (C) were measured by indirect calorimetry.In a distinct cohort, male GADD45β+/+ (WT; n=5) or GADD45β-/- mice (KO; n= 8) were fed ad libitum (fed) or fasted for 24h (fasted). Serumnon-esterified fatty acids (D) and ketone bodies (E) as well as serum (F) and liver (G) triglycerides (TG) were measured.Serum TG (H), NEFA (I), and blood glucose (BG, J) concentrations during an oral lipid tolerance test in overnight fasted GADD45β+/+ (WT; n = 5) or GADD45β-/- (KO; n=5) mice.Bloodglucose excursion during and intraperitoneal insulin tolerance test (K) as well as fasting bloodglucose (L), seruminsulin (M), and HOMA-IR (N) in GADD45β+/+ (WT; n = 6) or GADD45β-/- mice (KO, n = 9) chronically fed a normal- (NFD) or high (HFD) fat diet. Data are mean ± SEM.


Figure 4Male , GADD45β + / + ( WT ) or GADD45β - / - mice ( KO ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) and ex vivo liver slice long - chain fatty acid ( LCFA ) metabolism was measured including LCFA uptake ( A ) , oxidation ( B ) and non - oxidative LCFA disposal ( NOFAD ) was calculated ( C ) . In addition , glucose production was measured ( D ) in the presence ( BSA - NEFA ) or absence ( BSA ) of extracellular fatty acids ( n = 4 / group with 4 liver slices per mouse liver ) . Gadd45b mRNA expression was measured from fractionated parenchymal hepatocytes ( Hepas ) as well as non - parenchymal cells ( NP ) from C57Bl / 6J mice fed ad libitum ( fed ) or fasted for 24h ( fasted ) ; n = 3 / group ( E ) . Male , C57Bl / 6J mice with ( AAV - G45b miR ) or without ( AAV - NC miR ) liver - hepatocyte - restricted GADD45β silencing were fed or fasted for 24h ( n = 6 / group ) and serum levels of non - esterified fatty acids ( NEFA ; F ) as well as liver triglyceride ( TG ) concentration ( G ) were measured . Male , GADD45β + / + ( WT ; n = 16 ) or GADD45β - / - mice ( KO ; n = 15 ) fasted for 24h ( fasted ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 7 - 8 / group ) . LiverTG concentration ( H ) was measured . Male , C57Bl / 6J mice with ( AAV - G45b miR ; n = 15 ) or without ( AAV - NC miR , n = 13 ) liver - hepatocyte - restricted GADD45β silencing were chronically fed a normal ( NFD ) or high ( HFD ) fat diet ( n = 6 - 8 / group ) . Fasting blood glucose ( I ) and serum insulin ( J ) were measured from which HOMA - IR was calculated ( K ) .

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

Figure 4Male, GADD45β+/+ (WT) or GADD45β-/- mice (KO) were fed ad libitum (fed) or fasted for 24h (fasted) and ex vivo liver slice long-chain fatty acid (LCFA) metabolism was measured including LCFA uptake (A), oxidation (B) and non-oxidative LCFA disposal (NOFAD) was calculated (C).In addition, glucose production was measured (D) in the presence (BSA-NEFA) or absence (BSA) of extracellular fatty acids (n=4/group with 4 liver slices per mouse liver).Gadd45b mRNA expression was measured from fractionated parenchymal hepatocytes (Hepas) as well as non-parenchymal cells (NP) from C57Bl/6J mice fed ad libitum (fed) or fasted for 24h (fasted); n=3/group (E).Male, C57Bl/6J mice with (AAV-G45b miR) or without (AAV-NC miR) liver-hepatocyte-restricted GADD45β silencing were fed or fasted for 24h (n = 6/group) and serum levels of non-esterified fatty acids (NEFA; F) as well as liver triglyceride (TG) concentration (G) were measured.Male, GADD45β+/+ (WT; n=16) or GADD45β-/- mice (KO; n=15) fasted for 24h (fasted) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 7-8/group). LiverTG concentration (H) was measured.Male, C57Bl/6J mice with (AAV-G45b miR; n= 15) or without (AAV-NC miR, n = 13) liver-hepatocyte-restricted GADD45β silencing were chronically fed a normal (NFD) or high (HFD) fat diet (n = 6-8/group). Fasting blood glucose (I) and serum insulin (J) were measured from which HOMA-IR was calculated (K).


Figure 5Male , 12wk wildtype ( WT ; C57Bl / 6J ) or obese / diabetic ( db / db ; BKS . Cg - m + / + Lepr DB / J ) with ( AD - G45b OE ) or without ( AD - NC ) prior liver - restricted GADD45β over - expression were fasted and blood glucose ( A ) and serum insulin ( B ) were measured from which HOMA - IR was calculated ( C ) ( n = 4 - 6 / group ) . Data are mean ± SEM . Effect of genotype / viral manipulation , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Effect of nutritional state : # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 . Liver GADD45B mRNA expression from men with ( T2D ) or without ( NGT ) type 2 diabetes ( n = 14 - 23 / group ) . Scatter plots fasting plasma triglycerides ( E ) and glucose ( F ) in correlation with liver GADD45B mRNA expression ( n = 37 ) . Inserts show r2 values and p values from Spearman ' s correlation test . The statistical test used and respective p - value outputs can be found in Appendix Table S1 .

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

Figure 5Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) prior liver-restricted GADD45β over-expression were fasted and blood glucose ( A) and serum insulin (B) were measured from which HOMA-IR was calculated (C) (n = 4-6/group). Data are mean ± SEM. Effect of genotype/viral manipulation, * p < 0.05, ** p < 0.01, *** p < 0.001. Effect of nutritional state: # p < 0.05, ## p < 0.01, ### p < 0.001.Liver GADD45B mRNA expression from men with (T2D) or without (NGT) type 2 diabetes (n = 14-23/group).Scatter plots fasting plasma triglycerides (E) and glucose (F) in correlation with liver GADD45B mRNA expression (n = 37). Inserts show r2 values and p values from Spearman's correlation test. The statistical test used and respective p-value outputs can be found in Appendix Table S1.


Figure 6Male , GADD45β + / + ( WT ; n = 16 ) or GADD45β - / - mice ( KO ; n = 15 ) fasted for 24h ( fasted ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 7 - 8 / group ) . Liver mRNA expression of Gadd45b ( A ) as well as fatty acid metabolic genes ( B ) encompassing transport ( Slc28a2 , Slc27a5 , Cd36 ) , intracellular binding ( Fabp1 , Dbi ) and metabolism ( Acly , Dgat1 , Atgl , Hsl ) . C : Representative immunoblots of FATP2 , CD36 , FABP1 and GADD45β from liver whole tissue lysate ( W ) as well as fractionated organelles / intraceuular structures including nuclei ( N ) , mitochondria ( MT ) , microsomes ( MS ) and cytoplasm ( C ) , from GADD45β + / + ( WT ) and GADD45β - / - ( KO ) mice . D : Quantified band densities of FABP1 enrichment from fractions in C ( n = 4 / group ) . E : Liver fraction enrichment of FABP1 from male , GADD45β + / + ( WT ) or GADD45β - / - ( KO ) fasted for 24h with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 4 / group ) . Insert shows a representative FABP1 immunoblot . F : Liver fraction enrichment of FABP1 from obese / diabetic male db / db mice fasted for 24h with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 4 / group ) . Insert shows a representative FABP1 immunoblot . G : FABP1 and GADD45B immunoblots from Flag - immunoprecipitations ( IP - FLAG ) or mock IP ( IP - HA ) from liver input samples from mice with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression . Shown is a representative immunoblot from 3 separate experiments using 3 different input samples per condition . H : Liver long chain acyl - CoA ( LC - acyl - CoA ) concentrations were determined in GADD45β + / + ( WT ) or GADD45β - / - ( KO ) mice ( H ; n = 6 / group ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( I ; n = 5 / group ) . Liver LC - acyl - CoA concentrations were determined in wildtype ( WT ; C57Bl / 6J ) or obese / diabetic ( db / db ; BKS . Cg - m + / + Lepr DB / J ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( J ; n = 4 / group ) .

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

Figure 6Male, GADD45β+/+ (WT; n=16) or GADD45β-/- mice (KO; n=15) fasted for 24h (fasted) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 7-8/group). Liver mRNA expression of Gadd45b (A) as well as fatty acid metabolic genes (B) encompassing transport (Slc28a2, Slc27a5, Cd36), intracellular binding (Fabp1, Dbi) and metabolism (Acly, Dgat1, Atgl, Hsl).C: Representative immunoblots of FATP2, CD36, FABP1 and GADD45β from liver whole tissue lysate (W) as well as fractionated organelles/intraceuular structures including nuclei (N), mitochondria (MT), microsomes (MS) and cytoplasm (C), from GADD45β+/+ (WT) and GADD45β-/- (KO) mice. D: Quantified band densities of FABP1 enrichment from fractions in C (n = 4/group).E: Liver fraction enrichment of FABP1 from male, GADD45β+/+ (WT) or GADD45β-/- (KO) fasted for 24h with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 4/group). Insert shows a representative FABP1 immunoblot.F: Liver fraction enrichment of FABP1 from obese/diabetic male db/db mice fasted for 24h with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 4/group). Insert shows a representative FABP1 immunoblot.G: FABP1 and GADD45B immunoblots from Flag-immunoprecipitations (IP-FLAG) or mock IP (IP-HA) from liver input samples from mice with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression. Shown is a representative immunoblot from 3 separate experiments using 3 different input samples per condition.H: Liver long chain acyl-CoA (LC-acyl-CoA) concentrations were determined in GADD45β+/+ (WT) or GADD45β-/- (KO) mice (H; n=6/ group) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (I; n =5/group).Liver LC-acyl-CoA concentrations were determined in wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (J; n=4/group).


Figure 1Concentration of total cell - free DNA ( cfDNA ) in 13 CSF samples . Samples in which SCNAs were detected are shown in dark blue , and tended to have higher levels of total cfDNARelative copy number estimation in 20 genes of interest , and 18 large genomic regions , determined by sWGS of CSF from 13 glioma patients . Genes are ordered by genomic position , and their chromosomal locations are indicated in parentheses . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in grey . The top green bar indicates the tumor largest length dimension ( < 42 mm vs > 42 mm ) , and the top violet bar indicates the glioma subtype . SCNAs were more frequently detected in CSF from patients with large glioblastoma tumorsSCNAs determined by sWGS in 4 tumor subparts ( T1 - T4 ) and the CSF sample , collected from patient G1 . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in dark grey . sWGS from plasma and urine samples collected at the same time as the CSF sample showed no

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

Figure 1Concentration of total cell-free DNA (cfDNA) in 13 CSF samples. Samples in which SCNAs were detected are shown in dark blue, and tended to have higher levels of total cfDNARelative copy number estimation in 20 genes of interest, and 18 large genomic regions, determined by sWGS of CSF from 13 glioma patients. Genes are ordered by genomic position, and their chromosomal locations are indicated in parentheses. Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in grey. The top green bar indicates the tumor largest length dimension (<42 mm vs >42 mm), and the top violet bar indicates the glioma subtype. SCNAs were more frequently detected in CSF from patients with large glioblastoma tumorsSCNAs determined by sWGS in 4 tumor subparts (T1 - T4) and the CSF sample, collected from patient G1. Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in dark grey. sWGS from plasma and urine samples collected at the same time as the CSF sample showed no SCNAs


Figure 2Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 ( 4 tumor subparts and 1 CSF sample ) . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in light greyHeat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples . Shared detection in tissue and CSF is indicated in green , detection of the alteration only in tissue in orange , and non - detection in blue . The top bars indicate the cfDNA concentration ( copies / mL ; in a range of purples ) , the size of the tumors ( in a range of browns ) , the type of glioma ( in a range of blues ) , and whether the tumor was in direct contact with the CSF or not ( based on MRI , green or red , respectively ) . Samples are ranked from the left to right by decreasing concentration of cfDNA ( copies / mL )

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

Figure 2Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 (4 tumor subparts and 1 CSF sample). Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in light greyHeat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples. Shared detection in tissue and CSF is indicated in green, detection of the alteration only in tissue in orange, and non-detection in blue. The top bars indicate the cfDNA concentration (copies/mL; in a range of purples), the size of the tumors (in a range of browns), the type of glioma (in a range of blues), and whether the tumor was in direct contact with the CSF or not (based on MRI, green or red, respectively). Samples are ranked from the left to right by decreasing concentration of cfDNA (copies/mL)