Figure 4B . Secretion assay of wild type and various mutants of 12D1 , 1D1108 and 15955 . C . Antibacterial activity assay of wild type and mutants of 12D1 , 1D1108 and 15955 was carried out in a ratio of 30 : 1 against E . coli harboring the plasmid pRL662 . The target E . coli cells were serially - diluted and grown overnight on gentamicin - containing LB agar prior to photographing . Each competition was done at least four times and reproduced in three independent experiments . D . Secretion assay of wild type 12D1 , ΔtssL , Δv1 , Δv3 - 4 containing pRL662 empty vector ( V ) or its derivatives with v1 or v3 - 4 were analyzed for secretion . E . Secretion assay of wild type 1D1108 , ΔtssL , Δv4 , Δv6 containing pRL662 empty vector ( V ) or its derivatives with v4 or v6 .

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Figure 4B. Secretion assay of wild type and various mutants of 12D1, 1D1108 and 15955.C. Antibacterial activity assay of wild type and mutants of 12D1, 1D1108 and 15955 was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in three independent experiments.D. Secretion assay of wild type 12D1, ΔtssL, Δv1, Δv3-4 containing pRL662 empty vector (V) or its derivatives with v1 or v3-4 were analyzed for secretion.E. Secretion assay of wild type 1D1108, ΔtssL, Δv4, Δv6 containing pRL662 empty vector (V) or its derivatives with v4 or v6.


Figure 5A . T6SS secretion assay of A . tumefaciens wild type C58 , ΔtssL , and various single , double , and triple toxin - immunity deletion strains . B . T6SS secretion assay of A . tumefaciens wild type C58 , ΔtssL , Δtdei , and Δ3TIs harboring the indicated plasmids .

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Figure 5A. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, and various single, double, and triple toxin-immunity deletion strains.B. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, Δtdei, and Δ3TIs harboring the indicated plasmids.


Figure 6A . T6SS secretion assay of A . tumefaciens 12D1 wild type , ΔtssL , and various toxin - immunity deletion strains . B . T6SS secretion assay of A . tumefaciens 12D1 wild type , ΔtssL , and various toxin - immunity deletion strains harboring the indicated plasmids . C . Antibacterial activity assay of A . tumefaciens 12D1 wild type , ΔtssL , and various toxin - immunity deletion strains harboring the indicated plasmids was carried out in a ratio of 30 : 1 against E . coli harboring the plasmid pRL662 . The target E . coli cells were serially - diluted and grown overnight on gentamicin - containing LB agar prior to photographing . Each competition was done at least four times and reproduced in two independent experiments . D . T6SS secretion assay of A . tumefaciens C58 wild type , ΔtssL , and various mutant strains .

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Figure 6A. T6SS secretion assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains.B. T6SS secretion assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids.C. Antibacterial activity assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in two independent experiments.D. T6SS secretion assay of A. tumefaciens C58 wild type, ΔtssL, and various mutant strains.


Figure 1 ( a ) Transverse slice of live E10 . 5 Venus : : HES5 homozygous knock - in mouse showing the ventral HES5 domain in spinal cord ex vivo ( left panel ) ; Draq5 live nuclear stain with nuclear segmentation overlay ( right panel ) ; scale bar 30 μm .

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Figure 1(a) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo (left panel); Draq5 live nuclear stain with nuclear segmentation overlay (right panel); scale bar 30 μm.


Figure 2 ( a ) 20x snapshot of an ex - vivo slice culture of E10 . 5 spinal cord from Venus : : HES5 heterozygous knock - in mouse , transverse section ; delineated region ( blue ) correspond to data shown in ( b - c ) . D - dorsal , V - ventral . ( b ) Spatial profile of Venus : : HES5 intensity averaged over 2 . 5hrs with 0 distance representing the ventral end of kymograph ; black line represents the trend in Venus : : HES5 data across the domain produced using an polynomial order 6 ( see Materials and Methods ) . ( c ) Detrended spatial profile of Venus : : HES5 corresponding to data shown in ( b ) .

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Figure 2(a) 20x snapshot of an ex-vivo slice culture of E10.5 spinal cord from Venus::HES5 heterozygous knock-in mouse, transverse section; delineated region (blue) correspond to data shown in (b-c). D- dorsal, V- ventral. (b) Spatial profile of Venus::HES5 intensity averaged over 2.5hrs with 0 distance representing the ventral end of kymograph; black line represents the trend in Venus::HES5 data across the domain produced using an polynomial order 6 (see Materials and Methods). (c) Detrended spatial profile of Venus::HES5 corresponding to data shown in (b).


Figure 4 ( a ) Start : Finish Venus : : HES5 intensity ratio in E10 . 5 Venus : : HES5 heterozygous spinal cord slices treated with control ( DMSO ) and Notch inhibitor DBZ ( 2 μM ) observed over 16h ; bars indicate mean and standard deviation of DMS0 ( n = 3 experiments ) and DBZ ( n = 4 experiments ) ; 2 - tailed t - test * * * * p = 0 . 0001 .

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Figure 4(a) Start:Finish Venus::HES5 intensity ratio in E10.5 Venus::HES5 heterozygous spinal cord slices treated with control (DMSO) and Notch inhibitor DBZ (2 μM) observed over 16h; bars indicate mean and standard deviation of DMS0 (n=3 experiments) and DBZ (n=4 experiments); 2-tailed t-test ****p=0.0001.


Figure 7 ( a ) Transverse cryosection of E10 . 5 Venus : : HES5 spinal cord . Venus : : HES5 endogenous signal , OLIG2 - motorneuron progenitor marker , NGN2 - early marker of neuronal commitment ; scale bar 20 μm . ( b ) Relative nuclear intensities of Venus : : HES5 and NGN2 in motorneuron and interneuron progenitors ; bars show mean and SD of at least 494 cells per domain from 5 slices in 2 experiments ; Kruskal - Wallis with Dunn ' s multiple comparison test adjusted p - values * * p = 0 . 0032 , * * * p < 0 . 001 .

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Figure 7(a) Transverse cryosection of E10.5 Venus::HES5 spinal cord. Venus::HES5 endogenous signal, OLIG2 - motorneuron progenitor marker, NGN2 - early marker of neuronal commitment; scale bar 20 μm. (b) Relative nuclear intensities of Venus::HES5 and NGN2 in motorneuron and interneuron progenitors; bars show mean and SD of at least 494 cells per domain from 5 slices in 2 experiments; Kruskal-Wallis with Dunn's multiple comparison test adjusted p-values **p=0.0032, ***p<0.001.


Figure 8 ( a ) Detrended spatial profile of NGN2 : : mScarlet - I intensity from transverse slice of E10 . 5 spinal cord from heterozygous knock - in mouse in ventral - dorsal direction ; red indicates motorneuron ( MN ) domain , blue interneuron domain ( IN ) .

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Figure 8(a) Detrended spatial profile of NGN2::mScarlet-I intensity from transverse slice of E10.5 spinal cord from heterozygous knock-in mouse in ventral-dorsal direction;red indicates motorneuron (MN) domain, blue interneuron domain (IN).


Figure 4D - E ) Immunofluorescence against CITRINE ( electroporated cells ) , SOX10 ( Schwann cell precursors and Schwann cells ) and ISL1 ( sensory and sympathetic neurons ) on embryos electroporated with either control CRISPR plasmid ( D ) or a CRISPR plasmid containing a guide - RNA against Sox8 ( E ) . CITRINE + cells populate the developing dorsal root ganglia ( DRG ) and peripheral nerves . Examples of CITRINE + / SOX10 + cells shown by arrowheads . Asterisks show ventral boundary cap glia . Scale bar is 50 µm in overviews and 10 µm in insets . F ) Quantification of the fate distribution of CITRINE + cells as a % between glial ( SOX10 + ) cells , sensory neurons ( ISL1 + ) or neither ( SOX10 - / ISL1 - ) in the DRG of control and Sox8KD chick embryos . Biological replicates - N = 3 embryos per condition ( wild type versus Sox8 KD ) . Data represented as mean ± SEM . Statistical significance determined using the Holm - Sidak method ( alpha = 0 . 05 ) ( multiple t - tests , unpaired ) . SOX10 + cells : p = 0 . 7901 , ISL1 + cells : p = 0 . 9206 , SOX10 - / ISL1 - cells : p = 0 . 9206 . For statistical significance : non - significant - p - value ≥ 0 . 05 . G ) Quantification of ( left ) the % of SOX10 + cells in the peripheral nerves that are CITRINE + in control and Sox8KD chick embryos and ( right ) the % of PH3 + CITRINE + cells corresponding to proliferative cells . Biological replicates - N = 4 embryos per condition for SOX10 + distribution and N = 2 for PH3 quantification ( wild type versus Sox8 KD ) . Data represented as mean ± SEM . Statistical significance determined unpaired t - test with two - tailed p value . SOX10 + cells : p = 0 . 0044 , PH3 + cells : p = 0 . 4929 . For statistical significance : non - significant - p - value ≥ 0 . 05 , * * - p - value < 0 . 01 . Data information : In total , 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean ± SEM per embryo ( corresponding to biological replicates ) : 3 - 4 electroporated embryos were analyzed per condition ( control and SOX8 knock down ) with 4 - 5 sections stained and analyzed per embryo per region of interest ( DRG , DRG : dorsal root ganglia , sg : sympathetic ganglionI ) Immunofluorescence against CITRINE ( electroporated cells ) , SOX10 ( Schwann cell precursors and Schwann cells ) and TH ( sympathetic neurons and chromaffin cells ) of the sympathoadrenal domain on control and Sox8KD embryos . CITRINE + / SOX10 + cells shown by empty arrowheads while CITRINE + / TH + cells are shown by filled arrowheads . Scale bar = 50 µm . J ) Quantification of the fate distribution of CITRINE + cells as a % between glial ( SOX10 + ) cells , chromaffin cells ( TH + ) or neither ( SOX10 - / TH - ) in the proximity of the dorsal aorta and visceral nerve of wild type and Sox8 KD chick embryos . Biological replicates - N = 4 embryos per condition . Data represented as mean ± SEM . Statistical significance determined using the Holm - Sidak method ( alpha = 0 . 05 ) ( multiple t - tests , unpaired ) . SOX10 + cells : p = 0 . 0067 , TH + cells : p = 0 . 0067 , SOX10 - / TH - cells : p = 0 . 8819 . For statistical significance : non - significant - p - value ≥ 0 . 05 , * - p - value < 0 . 05 . Data information : In total , 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean ± SEM per embryo ( corresponding to biological replicates ) : 3 - 4 electroporated embryos were analyzed per condition ( control and SOX8 knock down ) with 4 - 5 sections stained and analyzed per embryo per region of interest Sympathoadrenal domain ) .

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

Figure 4D-E) Immunofluorescence against CITRINE (electroporated cells), SOX10 (Schwann cell precursors and Schwann cells) and ISL1 (sensory and sympathetic neurons) on embryos electroporated with either control CRISPR plasmid (D) or a CRISPR plasmid containing a guide-RNA against Sox8 (E). CITRINE+ cells populate the developing dorsal root ganglia (DRG) and peripheral nerves. Examples of CITRINE+/SOX10+ cells shown by arrowheads. Asterisks show ventral boundary cap glia. Scale bar is 50 µm in overviews and 10 µm in insets. F) Quantification of the fate distribution of CITRINE+ cells as a % between glial (SOX10+) cells, sensory neurons (ISL1+) or neither (SOX10-/ISL1-) in the DRG of control and Sox8KD chick embryos. Biological replicates - N=3 embryos per condition (wild type versus Sox8 KD). Data represented as mean±SEM. Statistical significance determined using the Holm-Sidak method (alpha = 0.05) (multiple t-tests, unpaired). SOX10+ cells: p=0.7901, ISL1+ cells: p=0.9206, SOX10-/ISL1- cells: p=0.9206. For statistical significance: non-significant - p-value≥0.05. G) Quantification of (left) the % of SOX10+ cells in the peripheral nerves that are CITRINE+ in control and Sox8KD chick embryos and (right) the % of PH3+ CITRINE+ cells corresponding to proliferative cells. Biological replicates - N=4 embryos per condition for SOX10+ distribution and N=2 for PH3 quantification (wild type versus Sox8 KD). Data represented as mean±SEM. Statistical significance determined unpaired t-test with two-tailed p value. SOX10+ cells: p=0.0044, PH3+ cells: p=0.4929. For statistical significance: non-significant - p-value≥0.05, ** - p-value<0.01. Data information: In total, 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean±SEM per embryo (corresponding to biological replicates): 3-4 electroporated embryos were analyzed per condition (control and SOX8 knock down) with 4-5 sections stained and analyzed per embryo per region of interest (DRG, DRG: dorsal root ganglia, sg: sympathetic ganglionI) Immunofluorescence against CITRINE (electroporated cells), SOX10 (Schwann cell precursors and Schwann cells) and TH (sympathetic neurons and chromaffin cells) of the sympathoadrenal domain on control and Sox8KD embryos. CITRINE+/SOX10+ cells shown by empty arrowheads while CITRINE+/TH+ cells are shown by filled arrowheads. Scale bar = 50 µm. J) Quantification of the fate distribution of CITRINE+ cells as a % between glial (SOX10+) cells, chromaffin cells (TH+) or neither (SOX10-/TH-) in the proximity of the dorsal aorta and visceral nerve of wild type and Sox8 KD chick embryos. Biological replicates - N=4 embryos per condition. Data represented as mean±SEM. Statistical significance determined using the Holm-Sidak method (alpha = 0.05) (multiple t-tests, unpaired). SOX10+ cells: p=0.0067, TH+ cells: p=0.0067, SOX10-/TH- cells: p=0.8819. For statistical significance: non-significant - p-value≥0.05, * - p-value<0.05. Data information: In total, 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean±SEM per embryo (corresponding to biological replicates): 3-4 electroporated embryos were analyzed per condition (control and SOX8 knock down) with 4-5 sections stained and analyzed per embryo per region of interest Sympathoadrenal domain).


Figure 1 ( b ) shows succinate quantification by NMR from tracheal aspirates collected in mechanically - ventilated patients with ( pink bar , n = 9 ) or without ( orange bar , n = 7 ) diagnostic of IAV pneumonia . * P < 0 . 05 . ( c ) IL - 6 and IL - 8 measurements by ELISA from these same tracheal aspirates . Data information : Data are mean ± SEM and statistical analysis was performed using the Mann - Whitney U - test .

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Figure 1(b) shows succinate quantification by NMR from tracheal aspirates collected in mechanically-ventilated patients with (pink bar, n=9) or without (orange bar, n=7) diagnostic of IAV pneumonia. *P < 0.05. (c) IL-6 and IL-8 measurements by ELISA from these same tracheal aspirates. Data information: Data are mean ± SEM and statistical analysis was performed using the Mann-Whitney U-test.


Figure 48 - week - old female C57Bl / 6 mice were infected intranasally with 150 pfu of influenza A / Scotland / 20 / 74 ( H3N2 ) virus ( IAV ) and treated or not simultaneously with 4 mg of succinate ( Suc ; by the intranasal route ) . Some mice were euthanized at 4 days post - infection and lungs were collected to determine : ; ( b ) the viral protein expression by Western blotting followed by a relative quantification ( c ) , Data information : Statistical analysis was performed using the Mann - Whitney test c , ( * P < 0 . 05 ) . 8 - week - old female C57Bl / 6 mice were infected intranasally with 150 pfu of influenza A / Scotland / 20 / 74 ( H3N2 ) virus ( IAV ) and treated or not simultaneously with 4 mg of succinate ( Suc ; by the intranasal route ) . Some mice were euthanized at 4 days post - infection and lungs were collected to determine : ( h ) tissue lesions in lung sections stained with hematoxylin - eosin and further assessed by microscopy . Scale bar : x6 / 20 µm . All data are represented as the mean or the mean ± SEM and are cumulative of 2 independent experiments with 5 animals each . Data information : Statistical analysis was performed using the Mann - Whitney test , h , , ( * P < 0 . 05 ) .

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Figure 48-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice were euthanized at 4 days post-infection and lungs were collected to determine: ; (b) the viral protein expression by Western blotting followed by a relative quantification (c), Data information: Statistical analysis was performed using the Mann-Whitney test c, (*P < 0.05).8-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice were euthanized at 4 days post-infection and lungs were collected to determine: (h) tissue lesions in lung sections stained with hematoxylin-eosin and further assessed by microscopy. Scale bar: x6/20 µm. All data are represented as the mean or the mean ± SEM and are cumulative of 2 independent experiments with 5 animals each. Data information: Statistical analysis was performed using the Mann-Whitney test , h, , (*P < 0.05).


Figure 5Human bronchial epithelial BEAS - 2B cells were infected with A / Scotland / 20 / 74 ( H3N2 ) virus ( IAV ) at MOI = 1 . After 4 h , cells were treated or not with succinate ( Suc ; 4 mg / ml ) up to 20 h . protein expression ( c ) were assessed by Western blotting to detect viral proteins ( β - actin was used as a loading control ) , respectively .

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Figure 5Human bronchial epithelial BEAS-2B cells were infected with A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1. After 4 h, cells were treated or not with succinate (Suc; 4 mg/ml) up to 20 h. protein expression (c) were assessed by Western blotting to detect viral proteins (β-actin was used as a loading control), respectively.


Figure 6Human bronchial epithelial BEAS - 2B cells were infected with influenza A / Scotland / 20 / 74 ( H3N2 ) virus ( IAV ) at MOI = 1 for 4 h , then washed and treated or not with succinate ( Suc ; 4 mg / mL ) for 20 h . ( a Expression of IAV proteins ( i . e . NP , NS1 ) was analyzed by confocal microscopy using specific antibodies . Viral proteins are stained in green , DNA in blue and actin in red . For a given antibody , pictures were taken on the exact same day with the same laser power settings , Pictures are representative of five independent experiments . Scale bar 10 µM . ( b ) Relative nuclear intensity of NS1 or NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool . Seven ( NS1 ) or nine ( NP ) random images were collected per treatment with at least 5 cells per field . Data are represented as the mean ± SEM . Statistical analysis was performed using the Mann - Whitney test , ( * P < 0 . 05 ) .

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Figure 6Human bronchial epithelial BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, then washed and treated or not with succinate (Suc; 4 mg/mL) for 20 h. (a Expression of IAV proteins (i.e. NP, NS1) was analyzed by confocal microscopy using specific antibodies. Viral proteins are stained in green, DNA in blue and actin in red. For a given antibody, pictures were taken on the exact same day with the same laser power settings, Pictures are representative of five independent experiments. Scale bar 10 µM. (b) Relative nuclear intensity of NS1 or NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Seven (NS1) or nine (NP) random images were collected per treatment with at least 5 cells per field. Data are represented as the mean ± SEM. Statistical analysis was performed using the Mann-Whitney test, (*P < 0.05).


Figure 8Human bronchial epithelial BEAS - 2B cells were infected with the influenza A / Scotland / 20 / 74 ( H3N2 ) virus ( IAV ) at MOI = 1 for 4 h , and subsequently treated or not with 4 mg / mL of succinate ( Suc ) for 20 h . ( a , b ) Cells were stained using a monoclonal antibody that specifically recognizes " NP - vRNA " complexes , but not RNA - free NP , and confocal microscopy ( a ) was performed ( green staining ) . The value raw integrated density ( RawIntDen , which is the sum of all pixel values in the ROI ( region of interest ) ) was further measured ( b ) . Data information : Data are represented as the mean ± SEM of 3 biological replicates or without ( a 2 technical replicates each . 8 ( b ) random images were analysed per treatment with a minimum of 5 cells per field . Statistical analysis was performed using the Mann - Whitney test ( b , ( * P < 0 . 05 ) . Human bronchial epithelial BEAS - 2B cells were infected with the influenza A / Scotland / 20 / 74 ( H3N2 ) virus ( IAV ) at MOI = 1 for 4 h , and subsequently treated or not with 4 mg / mL of succinate ( Suc ) for 20 h . ( e ) Human bronchial epithelial BEAS - 2B cells were infected with a A / Scotland / 20 / 74 ( H3N2 ) virus ( IAV ) carrying a wild - type NP ( NP WT ) or with the corresponding mutated virus bearing a NP with a K87R substitution ( NP K87R ) . Cells were infected by either virus at MOI = 1 for 4 h , then washed and treated or not with 4 mg / mL of succinate for 20 h . Localization of NP proteins was analyzed by confocal immunofluorescence microscopy . Scale bar : 10 µM . Pictures are representative of three independent experiments . ( f ) Relative nuclear intensity of NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool . Data information : Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each . at least 10 ( f ) random images were analysed per treatment with a minimum of 5 cells per field . Statistical analysis was performed using the Mann - Whitney test ( * P < 0 . 05 ) .

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 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

Figure 8Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (a, b) Cells were stained using a monoclonal antibody that specifically recognizes "NP-vRNA" complexes, but not RNA-free NP, and confocal microscopy (a) was performed (green staining). The value raw integrated density (RawIntDen, which is the sum of all pixel values in the ROI (region of interest)) was further measured (b). Data information: Data are represented as the mean ± SEM of 3 biological replicates or without (a 2 technical replicates each. 8 (b) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (b, (*P < 0.05).Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (e) Human bronchial epithelial BEAS-2B cells were infected with a A/Scotland/20/74 (H3N2) virus (IAV) carrying a wild-type NP (NP WT) or with the corresponding mutated virus bearing a NP with a K87R substitution (NP K87R). Cells were infected by either virus at MOI=1 for 4 h, then washed and treated or not with 4 mg/mL of succinate for 20 h. Localization of NP proteins was analyzed by confocal immunofluorescence microscopy. Scale bar: 10 µM. Pictures are representative of three independent experiments. (f) Relative nuclear intensity of NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Data information: Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each. at least 10 (f) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (*P < 0.05).


Figure 1Line plots of the mean relative abundances and SD of each bacterial strain from ages 10 to 30 days . For non - AM strains , relative abundances of the 14SM groups were recalculated excluding AM in order to compare abundances with the 13SM group . Data information : Strain abundances were normally distributed within each group according to the Kolmogorov - Smirnov test . Statistical significance compared to the 14SM FR control at each time point was calculated by two - way ANOVA with false - discovery adjustment using the Benjamini and Hochberg method . Adjusted p values are indicated for 14SM FF vs 14SM FR ( red ) and 13SM FR vs 14SM FR ( orange ) . Data are from two independent experiments and represent biological replicates ( n = 2 - 11 / 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 O O O O O O 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 1Line plots of the mean relative abundances and SD of each bacterial strain from ages 10 to 30 days. For non-AM strains, relative abundances of the 14SM groups were recalculated excluding AM in order to compare abundances with the 13SM group. Data information: Strain abundances were normally distributed within each group according to the Kolmogorov-Smirnov test. Statistical significance compared to the 14SM FR control at each time point was calculated by two-way ANOVA with false-discovery adjustment using the Benjamini and Hochberg method. Adjusted p values are indicated for 14SM FF vs 14SM FR (red) and 13SM FR vs 14SM FR (orange). Data are from two independent experiments and represent biological replicates (n = 2-11/group).


Figure 2Volcano plot showing gene abbreviations for colonic transcripts that were significantly enriched in 14SM FF or 14SM FR after correction for multiple comparisons using DESeq2mRNA expression of Ifng , Tnfa , Il17f , and Il22 . Colonic transcription levels were normalized by Hprt , which was the most stable among the three housekeeping genes tested ( Hprt , Hsp90 , Gapdh ) . Longitudinal data of mean ± SEM is shown for 14SM FF , 14SM FR , and 13SM FR pups at age 10 - 20 days ( circles ) with statistical significance calculated using two - way ANOVA factored on group and time point compared to the 14SM FR control . To the right of each longitudinal plot , we also report colonic transcript levels for pups born to germ - free ( GF ) FF or FR dams at age 15 days ( squares ) with statistical significance based on a one - way ANOVA with false - discovery adjustment using the Benjamini and Hochberg method . Outliers removed using ROUT method with Q = 10 % . Data are from two independent experiments and represent biological replicates ( n = 3 - 20 / 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 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

Figure 2Volcano plot showing gene abbreviations for colonic transcripts that were significantly enriched in 14SM FF or 14SM FR after correction for multiple comparisons using DESeq2mRNA expression of Ifng, Tnfa, Il17f, and Il22. Colonic transcription levels were normalized by Hprt, which was the most stable among the three housekeeping genes tested (Hprt, Hsp90, Gapdh). Longitudinal data of mean ± SEM is shown for 14SM FF, 14SM FR, and 13SM FR pups at age 10-20 days (circles) with statistical significance calculated using two-way ANOVA factored on group and time point compared to the 14SM FR control. To the right of each longitudinal plot, we also report colonic transcript levels for pups born to germ-free (GF) FF or FR dams at age 15 days (squares) with statistical significance based on a one-way ANOVA with false-discovery adjustment using the Benjamini and Hochberg method. Outliers removed using ROUT method with Q = 10%. Data are from two independent experiments and represent biological replicates (n = 3-20/group).


Figure 3For mice at age 15 days , the proportion of cytotoxic natural killer ( cNK ) , innate lymphoid cell ( ILC ) type 1 , ILC type 2 , ILC type 3 , natural cytotoxicity receptor ( NCR ) positive ILC3 , and NCR negative ILC3 populations among CD45 + cells . Data information : Statistical significance is based on a one - way ANOVA with false - discovery adjustment using the Benjamini and Hochberg method . Boxplots show IQR with error bars showing min . and max . values . Data are from two independent experiments and represent biological replicates ( n = 8 - 20 / group ) . For mice at age 15 days , the proportion of CD8 + T cells , CD4 + T cells , Tregs ( total FoxP3 + ) , Th1 cells ( T - bet + FoxP3 - ) , Th2 ( GATA3 + FoxP3 - ) , and Th17 ( RORgt + FoxP3 - ) populations among CD45 + cells . Data information : Statistical significance is based on a one - way ANOVA with false - discovery adjustment using the Benjamini and Hochberg method . Boxplots show IQR with error bars showing min . and max . values . Data are from two independent experiments and represent biological replicates ( n = 8 - 20 / 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 O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3For mice at age 15 days, the proportion of cytotoxic natural killer (cNK), innate lymphoid cell (ILC) type 1, ILC type 2, ILC type 3, natural cytotoxicity receptor (NCR) positive ILC3, and NCR negative ILC3 populations among CD45+ cells. Data information: Statistical significance is based on a one-way ANOVA with false-discovery adjustment using the Benjamini and Hochberg method. Boxplots show IQR with error bars showing min. and max. values. Data are from two independent experiments and represent biological replicates (n = 8-20/group).For mice at age 15 days, the proportion of CD8+ T cells, CD4+ T cells, Tregs (total FoxP3+), Th1 cells (T-bet+FoxP3-), Th2 (GATA3+FoxP3-), and Th17 (RORgt+FoxP3-) populations among CD45+ cells. Data information: Statistical significance is based on a one-way ANOVA with false-discovery adjustment using the Benjamini and Hochberg method. Boxplots show IQR with error bars showing min. and max. values. Data are from two independent experiments and represent biological replicates (n = 8-20/group).


Figure 1A Representative 3 - color STED images of synapses of cultured hippocampal neurons fixed and stained for Synapsin1 / 2 ( presynaptic marker ) , PI ( 3 ) P and Homer ( postsynaptic marker ) . Scale bar , 0 . 5 µm . PI ( 3 ) P - positive structures appear in both pre - and postsynaptic compartments . B Line scan fluorescence intensity profiles of single synapses from the images in A , normalized to maximum value for each channel . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001C Representative 3 - color confocal images of cultured hippocampal neurons fixed and stained for VGlut1 , VGAT and PI ( 3 ) P . Arrows indicate PI ( 3 ) P puncta in VGlut1 + or VGAT + presynaptic terminals . Scale bar , 10 µm . D PI ( 3 ) P intensity in VGlut1 + and VGAT + presynaptic terminals . PI ( 3 ) P is equally abundant in excitatory and inhibitory presynaptic terminals . N = 3 independent experiments ( ≥ 15 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001E Relative PI ( 3 ) P levels in the MAP2 + ( Soma ) and Syp + ( Synapse ) areas in DMSO and VPS34IN1 - treated neurons . N = 3 independent experiments ( ≥ 35 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM ; n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001F Cultured hippocampal neurons were field - stimulated with four trains of 200 APs ( 40 Hz , 5 s ; 90 s gap between each train ) ( Stim ) or left unstimulated ( - ) , fixed and stained for MAP2 , Synaptophysin ( Syp ) , and PI ( 3 ) P . Arrows indicate PI ( 3 ) P puncta in synapses . Scale bar , 10 µm . PI ( 3 ) P puncta that do not overlap with neuronal soma and synapses belong to astrocytes in culture and are disregarded from the quantification . G Relative intensity of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) from the images in F . N = 5 independent experiments ; ( ≥ 40 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001H Relative intensity of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) of cultured hippocampal neurons treated with DMSO or Gabazine ( 10 µM ) for 24 h . N = 3 independent experiments ( ≥ 65 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001I Cultured hippocampal neurons treated with AP5 ( 20 µM ) , CNQX ( 10 µM ) or TTX ( 0 . 2 µM ) for 24 h , fixed and stained for MAP2 , Syp and PI ( 3 ) P . Arrows indicate PI ( 3 ) P puncta in synapses . Scale bar , 10 µm . J Relative intensity of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) from the images in I . N ≥ 4 independent experiments ( ≥ 60 images per condition ) ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001K Relative intensity of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) of cultured hippocampal neurons were treated with Roscovitine ( 10 µM ) for 1 h and field - stimulated with four trains of 200 APs ( 40 Hz , 5 s ; 90 s gap between each train ) ( Stim ) or left unstimulated ( - ) . N = 3 independent experiments ( ≥ 40 images per condition ) ; Two - way ANOVA ; Fisher ' s Least Significant Difference Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

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 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 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 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 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 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 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 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

Figure 1A Representative 3-color STED images of synapses of cultured hippocampal neurons fixed and stained for Synapsin1/2 (presynaptic marker), PI(3)P and Homer (postsynaptic marker). Scale bar, 0.5 µm. PI(3)P-positive structures appear in both pre- and postsynaptic compartments. B Line scan fluorescence intensity profiles of single synapses from the images in A, normalized to maximum value for each channel. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001C Representative 3-color confocal images of cultured hippocampal neurons fixed and stained for VGlut1, VGAT and PI(3)P. Arrows indicate PI(3)P puncta in VGlut1+ or VGAT+ presynaptic terminals. Scale bar, 10 µm. D PI(3)P intensity in VGlut1+ and VGAT+ presynaptic terminals. PI(3)P is equally abundant in excitatory and inhibitory presynaptic terminals. N = 3 independent experiments (≥ 15 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001E Relative PI(3)P levels in the MAP2+ (Soma) and Syp+ (Synapse) areas in DMSO and VPS34IN1-treated neurons. N = 3 independent experiments (≥ 35 images per condition); Student's t-test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001F Cultured hippocampal neurons were field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for MAP2, Synaptophysin (Syp), and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm. PI(3)P puncta that do not overlap with neuronal soma and synapses belong to astrocytes in culture and are disregarded from the quantification.G Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in F. N = 5 independent experiments; (≥ 40 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001H Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons treated with DMSO or Gabazine (10 µM) for 24 h. N = 3 independent experiments (≥ 65 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001I Cultured hippocampal neurons treated with AP5 (20 µM), CNQX (10 µM) or TTX (0.2 µM) for 24 h, fixed and stained for MAP2, Syp and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm.J Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in I. N ≥ 4 independent experiments (≥ 60 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001K Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons were treated with Roscovitine (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-). N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Fisher's Least Significant Difference Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


Figure 2A Basal excitatory synaptic transmission of CA3 - CA1 connections in acute hippocampal slices treated with DMSO ( 0 . 03 % ) or the VPS34 inhibitors , VPS34IN1 ( 3 µM ) or SAR405 ( 20 µM ) . Representative fEPSP traces ( above ) and the relative slope of fEPSPs ( below ) are shown . VPS34 inhibition causes reduced excitatory synaptic transmission down to 46 . 0 ± 4 . 1 % and 55 . 0 ± 4 . 4 % for VPS34IN1 and SAR405 at the end of 60 - min treatment . N = 6 slices per condition from ≥ 4 mice ; Student ' s t - test . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001B Increased paired paired - pulse facilitation ( PPF ) at hippocampal CA3 - CA1 synapses after VPS34 inhibition . Representative traces of fEPSPs with a 20 ms interpulse interval is shown above . The ratio of the slope of the second to the first response over a range of interstimulus intervals ( 10 - 500 ms ) is plotted below , indicating an increased facilitation of the second response in VPS34IN1 - treated acute hippocampal slices . PI ( 3 ) P depletion causes a significant facilitation of the second fEPSP , indicative of reduced initial release probability . N = 6 slices per condition from 6 mice ; Two - way ANOVA . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001C Synaptophysin - pHluorin responses normalized to baseline from neurons stimulated with 12 consecutive trains of 20 APs ( 20 Hz , 1 s ) with 30 s gap between each stimulus . Neurons are acutely treated with DMSO ( 0 . 1 % ) or VPS34 inhibitor1 ( 50 µM ) starting from the onset of 2nd stimulation . The timing of the treatment and the stimulation trains are indicated . D Quantification of data from C . Net difference of Synaptophysin - pHluorin fluorescence 2 s before and 28 s after each stimulation ( 2 s before the next stimulation ) to estimate relative surface retention of SVs . SV endocytosis is blocked within 5 minutes of treatment with VPS34 Inhibitor1 , leading to progressive accumulation of Synaptophysin - pHluorin signal . N = 4 or 5 independent measurements per condition ; Student ' s t - test . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

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 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 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

Figure 2A Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%) or the VPS34 inhibitors, VPS34IN1 (3 µM) or SAR405 (20 µM). Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. VPS34 inhibition causes reduced excitatory synaptic transmission down to 46.0 ± 4.1 % and 55.0 ± 4.4 % for VPS34IN1 and SAR405 at the end of 60-min treatment. N = 6 slices per condition from ≥ 4 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001B Increased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses after VPS34 inhibition. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below, indicating an increased facilitation of the second response in VPS34IN1-treated acute hippocampal slices. PI(3)P depletion causes a significant facilitation of the second fEPSP, indicative of reduced initial release probability. N = 6 slices per condition from 6 mice; Two-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001C Synaptophysin-pHluorin responses normalized to baseline from neurons stimulated with 12 consecutive trains of 20 APs (20 Hz, 1 s) with 30 s gap between each stimulus. Neurons are acutely treated with DMSO (0.1 %) or VPS34 inhibitor1 (50 µM) starting from the onset of 2nd stimulation. The timing of the treatment and the stimulation trains are indicated. D Quantification of data from C. Net difference of Synaptophysin-pHluorin fluorescence 2 s before and 28 s after each stimulation (2 s before the next stimulation) to estimate relative surface retention of SVs. SV endocytosis is blocked within 5 minutes of treatment with VPS34 Inhibitor1, leading to progressive accumulation of Synaptophysin-pHluorin signal. N = 4 or 5 independent measurements per condition; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001


Figure 3A Electron micrographs of VPS34IN1 - and DMSO - treated synapses following four consecutive trains of 200 AP ( 40 Hz , 5 s ; 90 s gap between each train ) stimulation . The postsynaptic compartment and the postsynaptic density are highlighted in green and purple , respectively . Note the reduced SV numbers and plasma membrane - derived endocytic intermediates in VPS34IN1 - treated synapses . Scale bars , 500 nm ( left ) and 200 nm ( right ) . B High magnification views of endocytic intermediates accumulated in stimulated hippocampal neurons treated with VPS34IN1 . Scale bar , 200 nm . C 3D - reconstruction of a synaptic terminal treated with VPS34IN1 and stimulated as in A . The plasma membrane is colored in turquoise . Synaptic vesicles ( yellow ) , plasma membrane - derived invaginations ( blue ) , endosome - like vacuoles ( green ) and clathrin - coated vesicles ( red ) are highlighted . Scale bar , 500 nm ( top ) and 200 nm ( bottom ) . Data information : Data presented as mean ± SEM n . s . not significant ; * * * p < 0 . 001 ; * * * * p < 0 . 0001D - H Morphometric quantification of data shown in A . Synapses of VPS34IN1 - treated stimulated neurons display reduced numbers of SVs / bouton area in ( D ) and an accumulation of endocytic membrane invaginations ( E ) , endosome like vacuoles ( ELV ) ( F ) , clathrin - coated pits ( CCP ) ( G ) , and clathrin - coated vesicles ( CCV ) ( H ) . n = 100 ( SV ) and n = 200 ( invagination , ELV , CCP , CCV ) profiles per condition ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * * * p < 0 . 001 ; * * * * p < 0 .

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 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

Figure 3A Electron micrographs of VPS34IN1- and DMSO-treated synapses following four consecutive trains of 200 AP (40 Hz, 5 s; 90 s gap between each train) stimulation. The postsynaptic compartment and the postsynaptic density are highlighted in green and purple, respectively. Note the reduced SV numbers and plasma membrane-derived endocytic intermediates in VPS34IN1-treated synapses. Scale bars, 500 nm (left) and 200 nm (right). B High magnification views of endocytic intermediates accumulated in stimulated hippocampal neurons treated with VPS34IN1. Scale bar, 200 nm. C 3D-reconstruction of a synaptic terminal treated with VPS34IN1 and stimulated as in A. The plasma membrane is colored in turquoise. Synaptic vesicles (yellow), plasma membrane-derived invaginations (blue), endosome-like vacuoles (green) and clathrin-coated vesicles (red) are highlighted. Scale bar, 500 nm (top) and 200 nm (bottom). Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001D-H Morphometric quantification of data shown in A. Synapses of VPS34IN1-treated stimulated neurons display reduced numbers of SVs/ bouton area in (D) and an accumulation of endocytic membrane invaginations (E), endosome like vacuoles (ELV) (F), clathrin-coated pits (CCP) (G), and clathrin-coated vesicles (CCV) (H). n = 100 (SV) and n = 200 (invagination, ELV, CCP, CCV) profiles per condition; Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001


Figure 4A - D Normalized Synaptophysin - pHluorin responses from neurons treated with VPS34IN1 ( 10 µM for 1h prior to imaging ) and stimulated with four consecutive trains of 50 APs ( 10 Hz , 5 s ) in A or 200 APs ( 40 Hz , 5 s ) in C . PI ( 3 ) P depletion in VPS34IN1 - treated neurons causes the progressive accumulation of Synaptophysin - pHluorin on the neuronal surface in neurons stimulated with consecutive trains of 50 APs in B or 200 APs in D . Surface retention of Synaptophysin - pHluorin 90 s post - stimulus is plotted for each of the four successive stimulation trains . N ≥ 4 independent experiments ; Two - way ANOVA ; Sidak ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001E Cultured hippocampal neurons pre - treated with DMSO ( 0 . 1 % ) or VPS34IN1 ( 10 µM , 1 h ) were either field - stimulated with four trains of 200 APs ( 40 Hz , 5 s ; 90 s gap between each train ) ( Stim ) or left unstimulated ( - ) , fixed and stained for surface - stranded Synaptotagmin - 1 , total Synaptotagmin - 1 , and vGlut1 . Scale bar , 10 µm . F Fraction of endogenous Synaptotagmin - 1 stranded on the surface of presynaptic nerve terminals from the images shown in E . N = 3 independent experiments ( ≥ 40 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001G Surface retention of Synaptophysin - pHluorin 90 s post - stimulus is plotted for each of the four successive 200 AP stimulation trains in neurons treated with SAR405 ( 20 µM ) . Data are similar to those shown in C and D . N = 3 independent experiments ( ≥ 11 images per condition ) ; Two - way ANOVA ; Sidak ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001H Exogenous supply of membrane - permeant PI ( 3 ) P acutely rescues defective Synaptophysin - pHluorin endocytosis induced by pharmacological inhibition of VPS34 - mediated PI ( 3 ) P synthesis in hippocampal neurons . Data are similar to those shown in C and D ; where indicated exogenous PI ( 3 ) P - AM ( 20 µM ) was supplied in parallel to VPS34IN1 ( 10 µM ) for 1 h prior to imaging . N = 3 independent experiments ( 15 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001I Kinetic block of Synaptophysin - pHluorin endocytosis at hippocampal synapses depleted of VPS34 . Hippocampal neurons depleted of endogenous VPS34 by specific shRNA ( shVPS34 ) or treated with non - targeting control shRNA ( shCtrl ) and stimulated as in C and analyzed as in D . N = 3 independent experiments ( ≥ 10 images per condition ) ; Two - way ANOVA ; Sidak ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ;

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 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 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 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 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

Figure 4A-D Normalized Synaptophysin-pHluorin responses from neurons treated with VPS34IN1 (10 µM for 1h prior to imaging) and stimulated with four consecutive trains of 50 APs (10 Hz, 5 s) in A or 200 APs (40 Hz, 5 s) in C. PI(3)P depletion in VPS34IN1-treated neurons causes the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface in neurons stimulated with consecutive trains of 50 APs in B or 200 APs in D. Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive stimulation trains. N ≥ 4 independent experiments; Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001E Cultured hippocampal neurons pre-treated with DMSO (0.1%) or VPS34IN1 (10 µM, 1 h) were either field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for surface-stranded Synaptotagmin-1, total Synaptotagmin-1, and vGlut1. Scale bar, 10 µm. F Fraction of endogenous Synaptotagmin-1 stranded on the surface of presynaptic nerve terminals from the images shown in E. N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001G Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive 200 AP stimulation trains in neurons treated with SAR405 (20 µM). Data are similar to those shown in C and D. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001H Exogenous supply of membrane-permeant PI(3)P acutely rescues defective Synaptophysin-pHluorin endocytosis induced by pharmacological inhibition of VPS34-mediated PI(3)P synthesis in hippocampal neurons. Data are similar to those shown in C and D; where indicated exogenous PI(3)P-AM (20 µM) was supplied in parallel to VPS34IN1 (10 µM) for 1 h prior to imaging. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001I Kinetic block of Synaptophysin-pHluorin endocytosis at hippocampal synapses depleted of VPS34. Hippocampal neurons depleted of endogenous VPS34 by specific shRNA (shVPS34) or treated with non-targeting control shRNA (shCtrl) and stimulated as in C and analyzed as in D. N =3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001;


Figure 5A - C The kinetic block of SV endocytosis at excitatory synapses depleted of PI ( 3 ) P depends on prior neuronal activity and Ca2 + . VPS34IN1 - induced defects in SV endocytosis are reverted by parallel treatment with TTX ( 0 . 2 µM ) in A and EGTA ( 2 mM ) in B and AP5 ( 20 µM ) in C , in Synaptophysin - pHluorin - expressing neurons N = 3 independent experiments ( ≥ 10 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM ; n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001D Overexpression of dominant - negative Rab5 ( Rab5DN ) , but not constitutively active Rab5 ( Rab5CA ) , rescues the progressive accumulation of Synaptophysin - pHluorin on the surface of VPS34IN1 - treated hippocampal neurons stimulated and analyzed as in A - C . N = 3 independent experiments ( ≥ 10 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001E Levels of various synaptic proteins in DMSO ( 0 . 1 % ) and VPS34IN1 ( 10 µM ) treated cultured cerebellar granule neurons analyzed by western blot . VPS34 inhibition results in p35 processing to hyperactive p25 , a reaction blocked by Calpain inhibitor ALLN ( 100 µM ) . F - H Calpain inhibition by ALLN ( 100 µM ) in F or Calpain 2 knockdown via lentiviral shRNA ( shCAPN2 ) in H rescue the progressive accumulation of Synaptophysin - pHluorin on the neuronal surface induced by VPS34IN1 . Calpain 1 knockdown via lentiviral shRNA ( shCAPN1 ) in G had no impact on VPS34IN1 - induced kinetic delay of SV endocytosis . N = 3 independent experiments ( ≥ 11 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM ; n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

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 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 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 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

Figure 5A-C The kinetic block of SV endocytosis at excitatory synapses depleted of PI(3)P depends on prior neuronal activity and Ca2+. VPS34IN1-induced defects in SV endocytosis are reverted by parallel treatment with TTX (0.2 µM) in A and EGTA (2 mM) in B and AP5 (20 µM) in C, in Synaptophysin-pHluorin-expressing neurons N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001D Overexpression of dominant-negative Rab5 (Rab5DN), but not constitutively active Rab5 (Rab5CA), rescues the progressive accumulation of Synaptophysin-pHluorin on the surface of VPS34IN1-treated hippocampal neurons stimulated and analyzed as in A-C. N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001E Levels of various synaptic proteins in DMSO (0.1%) and VPS34IN1 (10 µM) treated cultured cerebellar granule neurons analyzed by western blot. VPS34 inhibition results in p35 processing to hyperactive p25, a reaction blocked by Calpain inhibitor ALLN (100 µM).F-H Calpain inhibition by ALLN (100 µM) in F or Calpain 2 knockdown via lentiviral shRNA (shCAPN2) in H rescue the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1. Calpain 1 knockdown via lentiviral shRNA (shCAPN1) in G had no impact on VPS34IN1-induced kinetic delay of SV endocytosis. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001


Figure 6B Cdk5 inhibition by Roscovitine ( Ros , 10 µM ) rescues the progressive accumulation of Synaptophysin - pHluorin on the neuronal surface induced by VPS34IN1 ( 10 µM ) . N = 4 independent experiments ( 20 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparisons Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001C Calcineurin inhibition by Cyclosporin A ( 10 µM ) phenocopies VPS34IN1 - induced loss of PI ( 3 ) P with respect to the accumulation of Synaptophysin - pHluorin on the neuronal surface after four successive stimulation trains ( 40 Hz , 5 s ) . Data were analyzed as in B . N = 3 independent experiments ( 15 images per condition ) ; Two - way ANOVA ; Tukey ' s Multiple Comparison Test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001D Basal excitatory synaptic transmission of CA3 - CA1 connections in acute hippocampal slices treated with DMSO ( 0 . 03 % ) , Roscovitine ( Ros , 10 µM ) or VPS34IN1 ( 3 µM ) plus Roscovitine . Representative fEPSP traces ( above ) and the relative slope of fEPSPs ( below ) are shown . Concomitant application of VPS34IN1 and Roscovitine facilitates excitatory synaptic transmission , akin to Roscovitine alone , and in contrast to VPS34IN1 alone N = 6 slices per condition from 6 mice ; Student ' s t - test . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001E Decreased paired paired - pulse facilitation ( PPF ) at hippocampal CA3 - CA1 synapses treated with Roscovitine alone ( Ros , 10 µM ) or VPS34IN1 ( 3 µM ) plus Roscovitine . Representative traces of fEPSPs with a 20 ms interpulse interval is shown above . The ratio of the slope of the second to the first response over a range of interstimulus intervals ( 10 - 500 ms ) is plotted below . The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine - treated acute hippocampal slices , unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice ; Two - way RM ANOVA . Data information : Data presented as mean ± SEM n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

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

Figure 6B Cdk5 inhibition by Roscovitine (Ros, 10 µM) rescues the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1 (10 µM). N = 4 independent experiments (20 images per condition); Two-way ANOVA; Tukey's Multiple Comparisons Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001C Calcineurin inhibition by Cyclosporin A (10 µM) phenocopies VPS34IN1-induced loss of PI(3)P with respect to the accumulation of Synaptophysin-pHluorin on the neuronal surface after four successive stimulation trains (40 Hz, 5 s). Data were analyzed as in B. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001D Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%), Roscovitine (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. Concomitant application of VPS34IN1 and Roscovitine facilitates excitatory synaptic transmission, akin to Roscovitine alone, and in contrast to VPS34IN1 alone N = 6 slices per condition from 6 mice; Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001E Decreased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses treated with Roscovitine alone (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below. The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine-treated acute hippocampal slices, unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice; Two-way RM ANOVA. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001


Figure 7A Activity - dependent polyspiking of population spikes ( PS ) during 1 Hz stimulation is reduced by VPS34IN1 and facilitated by Roscovitine compared to DMSO - treated control slices . Top , representative traces of the 1st and 30th stimulus responses with polyspikes indicated by arrows . N = 12 slices per condition from 12 mice . B Quantification of polyspiking amplitudes in response to the 30th stimulus in the presence of DMSO , VPS34IN1 , or Roscovitine . Polyspiking amplitudes are increased by Roscovitine and reduced by VPS34IN1 . N = 12 slices per condition from 12 mice ; One - way ANOVA . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * * p < 0 . 001 . C The relative changes in the PS and polyspike areas in the presence of picrotoxin ( 50 µM ) and a subsequent treatment with DMSO or VPS34IN1 ( 10 µM ) . VPS34IN1 significantly reduced the PS and polyspike area compared to DMSO . N = 6 slices per condition from 6 mice . D Mean area of PS and polyspikes in picrotoxin - treated slices 40 min post - application of DMSO or VPS34IN1 . N ≥ 6 slices per condition from ≥ 6 mice ; Student ' s t - test . Data information : Data presented as mean ± SEM ; * p < 0 . 05 ; * * * p < 0 . 001 .

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Figure 7A Activity-dependent polyspiking of population spikes (PS) during 1 Hz stimulation is reduced by VPS34IN1 and facilitated by Roscovitine compared to DMSO-treated control slices. Top, representative traces of the 1st and 30th stimulus responses with polyspikes indicated by arrows. N = 12 slices per condition from 12 mice. B Quantification of polyspiking amplitudes in response to the 30th stimulus in the presence of DMSO, VPS34IN1, or Roscovitine. Polyspiking amplitudes are increased by Roscovitine and reduced by VPS34IN1. N = 12 slices per condition from 12 mice; One-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001.C The relative changes in the PS and polyspike areas in the presence of picrotoxin (50 µM) and a subsequent treatment with DMSO or VPS34IN1 (10 µM). VPS34IN1 significantly reduced the PS and polyspike area compared to DMSO. N = 6 slices per condition from 6 mice. D Mean area of PS and polyspikes in picrotoxin-treated slices 40 min post-application of DMSO or VPS34IN1. N ≥ 6 slices per condition from ≥ 6 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001.


Figure 1TOPflash reporter assays in HEK293T cells stimulated with either control , Wnt3a , Wnt3a and RSPO1 - 4 conditioned media , or by transfection with the indicated constructs . Where indicated , cells were co - transfected with the indicated siRNAs . K , Representative co - immunoprecipitation experiments in HEK293T cells that were transfected with GFP - USP42 or the indicated GFP - tagged control , together with the stated Wnt signalling component ( N = 3 independent experiments ) . IgG heavy chain is indicated with an asterisk . Dvl1 functions as a positive control that binds both ZNRF3 and Axin1 .

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Figure 1TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1-4 conditioned media, or by transfection with the indicated constructs. Where indicated, cells were co-transfected with the indicated siRNAs.K, Representative co-immunoprecipitation experiments in HEK293T cells that were transfected with GFP-USP42 or the indicated GFP-tagged control, together with the stated Wnt signalling component (N = 3 independent experiments). IgG heavy chain is indicated with an asterisk. Dvl1 functions as a positive control that binds both ZNRF3 and Axin1.


Figure 2E , F , Co - immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in ( A ) . In ( F ) cells were co - transfected with DVL2 as indicated . Unspecific bands resulting from antibody cross - reaction are marked with asterisks . Representative blots of n ≥ 3 independent experiments are shown .

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Figure 2E,F, Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). In (F) cells were co-transfected with DVL2 as indicated. Unspecific bands resulting from antibody cross-reaction are marked with asterisks. Representative blots of n ≥ 3 independent experiments are shown.


Figure 3A , Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation . Consecutive immunoprecipitations were carried out as indicated in the scheme . The Avidin pulldown was carried under denaturing conditions ( 1st IP ) . The unrelated receptor CD147 was used as loading control for the avidin pull down , and CUL1 for the total input of cell lysate . F , G , Western blots of lysates from HEK293T cells transfected with the indicated constructs . The asterisk marks an unspecific band . Representative blots from at least three independent experiments are shown .

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Figure 3A, Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation. Consecutive immunoprecipitations were carried out as indicated in the scheme. The Avidin pulldown was carried under denaturing conditions (1st IP). The unrelated receptor CD147 was used as loading control for the avidin pull down, and CUL1 for the total input of cell lysate.F,G, Western blots of lysates from HEK293T cells transfected with the indicated constructs. The asterisk marks an unspecific band. Representative blots from at least three independent experiments are shown.


Figure 4D , TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs . Cells were stimulated with control or Wnt3a conditioned medium . Data is displayed as mean ± SD , and show one representative of n = 3 independent experiments with three biological replicates . E , F , qPCR analysis of USP42 , LGR5 and AXIN2 expression levels in HCT116 cells . Data are displayed as mean ± SD and show n = 4 ( E ) or n = 3 ( F ) independent experiments .

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Figure 4D, TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs. Cells were stimulated with control or Wnt3a conditioned medium. Data is displayed as mean ± SD, and show one representative of n = 3 independent experiments with three biological replicates.E,F, qPCR analysis of USP42, LGR5 and AXIN2 expression levels in HCT116 cells. Data are displayed as mean ± SD and show n = 4 (E) or n = 3 (F) independent experiments.


Figure 5E - H , Immunofluorescence microscopy showing endogenous E - cadherin levels in wt and WLS ( Evi ) KO HCT116 cells upon transfection with siUSP42 ( E ) , or electroporation with eSpCas9 empty plasmid ( non - edited ) or eSpCas9 - USP42 gRNAs ( G ) . Scale bar = 20 μm . In ( F , G ) , quantification of the fluorescent signal ( Alexa - 488 ) in n ≥ 4 biological replicates with n ≥ 15 cells per replicate are shown . I - L , Spheroid formation experiments with HCT116 cells upon transfection with siUSP42 ( I - K ) or electroporation with eSpCas9 empty plasmid ( non - edited ) or eSpCas9 - USP42 gRNAs ( L ) . Scale bar = 500 μm . Where indicated , cells were treated with 5 μM IWP - 2 or 20 μM LGK - 974 ( PORCN inhibitors ) . The median size was quantified for n ≥ 13 spheroids from a representative experiment repeated independently n ≥ 3 is shown .

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Figure 5E-H, Immunofluorescence microscopy showing endogenous E-cadherin levels in wt and WLS (Evi) KO HCT116 cells upon transfection with siUSP42 (E), or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs(G). Scale bar = 20 μm. In (F,G), quantification of the fluorescent signal (Alexa-488) in n ≥ 4 biological replicates with n ≥ 15 cells per replicate are shown.I-L, Spheroid formation experiments with HCT116 cells upon transfection with siUSP42 (I-K) or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs (L). Scale bar = 500 μm. Where indicated, cells were treated with 5 μM IWP-2 or 20 μM LGK-974 (PORCN inhibitors). The median size was quantified for n ≥ 13 spheroids from a representative experiment repeated independently n ≥ 3 is shown.


Figure 6A , Representative images of mouse small intestinal organoids generated with empty ( Non - edited ) or USP42gRNA containing CRISPR - concatemers , and cultured for 3 days in matrigel with the indicated growth factors , or the Wnt secretion inhibitor IWP - 2 . EN , EGF and noggin . Note that knockout of Usp42 ( Usp42 gRNAs ) rendered the organoids insensitive to RSPO1 withdrawal . Scale bar = 1 mm . C , qPCR analysis of Lgr5 and Axin2 expression levels in first passage mouse intestinal organoids cultured in EN + 5 % RSPO1 .

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Figure 6A, Representative images of mouse small intestinal organoids generated with empty (Non-edited) or USP42gRNA containing CRISPR-concatemers, and cultured for 3 days in matrigel with the indicated growth factors, or the Wnt secretion inhibitor IWP-2. EN, EGF and noggin. Note that knockout of Usp42 (Usp42 gRNAs) rendered the organoids insensitive to RSPO1 withdrawal. Scale bar = 1 mm.C, qPCR analysis of Lgr5 and Axin2 expression levels in first passage mouse intestinal organoids cultured in EN + 5% RSPO1.


Figure 1B . MASTL was ablated in MDA - MB - 231 cells using the isgMASTL system . 72 h after Dox , cells were starved of glucose in the media in presence of 10 % dFBS for 1 h ( - ) and then re - stimulated with 25 mM of glucose for 10 min before recovery ( + ) . Whole - cell lysates were blotted with the indicated antibodies . * in indicates unspecific band . β - Actin was used as a loading control . C . MASTL was depleted in MDA - MB - 231 cells using shRNAs against MASTL ( + ) or scrambled shRNAs ( − ) as control . Cells were starved from glucose for 2 h and re - stimulated with 5 mM glucose for 15 min and 1 h . Total extracts were recovered and tested for the indicated antibodies . β - Actin was used as a loading control . D . Quantification of phospho - AKT T308 levels in glucose deprivation or glucose stimulation ( 15 min ) conditions in MDA - MB - 231 control cells and MASTL - depleted cells either using shRNA or the inducible sgRNA . Bars displays mean data + SEM from three independent experiments . Significance determined by Student ' s t - test comparing feedback activation in MASTL - depleted cells versus control cells ( ns , not significant ; * P < 0 . 05 ) . E . Immunoblot analysis with the indicated antibodies in BT - 549 cells upon glucose deprivation and 15 min glucose stimulation . MASTL knockdown was performed by infection with a shRNA for MASTL ( + ) or a Scramble shRNA ( − ) as a control . β - Actin was used as a loading control . Bar chart displays mean data + SEM from 3 experiments . Significance determined by Student ' s t - test comparing feedback activation in MASTL - depleted cells versus control cells ( ns , not significant ) . F . Immunoblot analysis with the indicated antibodies in MCF - 7 cells upon glucose deprivation and glucose stimulation for 15 min . MASTL knockdown was performed by infection with a shRNA for MASTL ( + ) or a Scramble shRNA ( − ) as a control . Bar chart displays mean data + SEM from 3 experiments . Significance determined by Student ' s t - test comparing feedback activation in MASTL - depleted cells versus control cells ( ns , not significant ; * P < 0 . 05 ) .

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Figure 1B. MASTL was ablated in MDA-MB-231 cells using the isgMASTL system. 72 h after Dox, cells were starved of glucose in the media in presence of 10% dFBS for 1 h (-) and then re-stimulated with 25 mM of glucose for 10 min before recovery (+). Whole-cell lysates were blotted with the indicated antibodies. * in indicates unspecific band. β-Actin was used as a loading control.C. MASTL was depleted in MDA-MB-231 cells using shRNAs against MASTL (+) or scrambled shRNAs (−) as control. Cells were starved from glucose for 2 h and re-stimulated with 5 mM glucose for 15 min and 1 h. Total extracts were recovered and tested for the indicated antibodies. β-Actin was used as a loading control. D. Quantification of phospho-AKT T308 levels in glucose deprivation or glucose stimulation (15 min) conditions in MDA-MB-231 control cells and MASTL-depleted cells either using shRNA or the inducible sgRNA. Bars displays mean data + SEM from three independent experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).E. Immunoblot analysis with the indicated antibodies in BT-549 cells upon glucose deprivation and 15 min glucose stimulation. MASTL knockdown was performed by infection with a shRNA for MASTL (+) or a Scramble shRNA (−) as a control. β-Actin was used as a loading control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant). F. Immunoblot analysis with the indicated antibodies in MCF-7 cells upon glucose deprivation and glucose stimulation for 15 min. MASTL knockdown was performed by infection with a shRNA for MASTL (+) or a Scramble shRNA (−) as a control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).


Figure 2A . Time course experiment at the indicated time points after 100 nM insulin stimulation in 6 h serum - starved control and MASTL knockout MDA - MB - 231 cells using the isgMASTL system . The plot shows the quantification of phospho - AKT T308 after 30 min of insulin stimulation . Plots displays mean data + SEM from 5 independent experiments . * P < 0 . 05 , Student ' s t - test . B . TSC2 was knocked down in control or MASTL - null MDA - MB - 231 cells using specific ( + ) or Scrambled ( − ) shRNAs . Cells were starved for glucose for 1 h . The plot shows the quantification of phospho - AKT T308 . Data are mean + SEM from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA . C . MDA - MB - 231 control ( − ) or MASTL null ( + ) cells were treated with 100 nM rapamycin or vehicle as control . Cells were starved for glucose for 1 h and re - stimulated with glucose for 15 min ; rapamycin was added 15 min before glucose stimulation . The plot shows the quantification of phospho - AKT T308 . Data are mean + SEM from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA .

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Figure 2A. Time course experiment at the indicated time points after 100 nM insulin stimulation in 6 h serum-starved control and MASTL knockout MDA-MB-231 cells using the isgMASTL system. The plot shows the quantification of phospho-AKT T308 after 30 min of insulin stimulation. Plots displays mean data + SEM from 5 independent experiments. *P<0.05, Student's t-test.B. TSC2 was knocked down in control or MASTL-null MDA-MB-231 cells using specific (+) or Scrambled (−) shRNAs. Cells were starved for glucose for 1 h. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.C. MDA-MB-231 control (−) or MASTL null (+) cells were treated with 100 nM rapamycin or vehicle as control. Cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min; rapamycin was added 15 min before glucose stimulation. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.


Figure 3A . Immunofluorescence for GLUT4 ( magenta ) in MDA - MB - 231 cells in conditions of serum starvation and insulin stimulation . DAPI ( DNA ) is in cyan and a cell mask is in yellow . Arrowheads indicate positive cells for GLUT4 enrichment at the plasma membrane ( PM ) . Representative magnifications are also shown . Scale bars , 10 μm The plot shows the quantification of the percentage of cells positive for GLUT4 translocation at the PM . Data are mean fold increase vs serum deprivation in scramble condition + SEM from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA . Control inmunoblotting in conditions of insulin stimulation is also shown for the indicated antibodies .

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Figure 3A. Immunofluorescence for GLUT4 (magenta) in MDA-MB-231 cells in conditions of serum starvation and insulin stimulation. DAPI (DNA) is in cyan and a cell mask is in yellow. Arrowheads indicate positive cells for GLUT4 enrichment at the plasma membrane (PM). Representative magnifications are also shown. Scale bars, 10 μm The plot shows the quantification of the percentage of cells positive for GLUT4 translocation at the PM. Data are mean fold increase vs serum deprivation in scramble condition + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA. Control inmunoblotting in conditions of insulin stimulation is also shown for the indicated antibodies.


Figure 4E . GLUT4 staining ( magenta ) in skeletal muscle sections . Vinculin ( membrane ) and DAPI ( DNA ) staining is shown in yellow and cyan , respectively . Scale bars , 50 µm . Mice were fasted overnight for 16 h , and re - fed for 2 h before sample collection . Quantification of the mean intensity [ relative units x103 ; n = 3 Mastl ( + / + ) and 3 Mastl ( ∆ / ∆ ) ] is shown . Data points reflect the mean + SEM ; * P < 0 . 05 ; * * * P < 0 . 001 ; unpaired Student ' s t - test . G . Immunoblot with the indicated antibodies in liver tissues from Mastl ( + / + ) ( n = 6 ) and Mastl ( Δ / Δ ) ( n = 7 ) mice . Mice were fasted overnight for 16 h , injected intraperitoneally with glucose ( 2 g / kg body ) , and sacrificed 30 min later for sample collection . Quantification of the relative fold change signal of phospho - AKT T308 ( normalized to total AKT level ) . n = 6 Mastl ( + / + ) and 7 Mastl ( Δ / Δ ) mice . Charts reflect the mean + SEM ; * P < 0 . 05 ; unpaired Student ' s t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 4E. GLUT4 staining (magenta) in skeletal muscle sections. Vinculin (membrane) and DAPI (DNA) staining is shown in yellow and cyan, respectively. Scale bars, 50 µm. Mice were fasted overnight for 16 h, and re-fed for 2 h before sample collection. Quantification of the mean intensity [relative units x103; n=3 Mastl(+/+) and 3 Mastl(∆/∆)] is shown. Data points reflect the mean + SEM; *P<0.05; ***P <0.001; unpaired Student's t-test.G. Immunoblot with the indicated antibodies in liver tissues from Mastl(+/+) (n=6) and Mastl(Δ/Δ) (n=7) mice. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of phospho-AKT T308 (normalized to total AKT level). n=6 Mastl(+/+) and 7 Mastl(Δ/Δ) mice. Charts reflect the mean + SEM; *P<0.05; unpaired Student's t-test.