B . Frequency of ISV ectopic branching occurring at the HM ( n = 4 experiments ; * P < 0 . 05 ; one - way ANOVA with Bonferroni ' s correction ) . + / + , + / ∆ and ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos , respectively ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Frequency of ISV ectopic branching occurring at the HM (n=4 experiments; *P<0.05; one-way ANOVA with Bonferroni's correction). +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively (B).


B . T6SS secretion assay of A . tumefaciens strains : wild type C58 , various mutants lacking one , two , or three toxin - immunity gene pairs , and a mutant lacking tssL .

O O O O O O O O O O O O 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. T6SS secretion assay of A. tumefaciens strains: wild type C58, various mutants lacking one, two, or three toxin-immunity gene pairs, and a mutant lacking tssL.


C . T6SS secretion assay of various A . tumefaciens strains : wild type C58 , ΔtssL , the tde double deletion mutant ( Δtdei ) containing pRL662 and pTrc200 empty vector ( V ) only or expression of pTdei1 ( tde1 - tdi1 expressed from pTrc200 ) , pTde2 * ( catalytic site mutated tde2 expressed on pRL662 ) , or pTdei1 + pTde2 * .

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

C. T6SS secretion assay of various A. tumefaciens strains: wild type C58, ΔtssL, the tde double deletion mutant (Δtdei) containing pRL662 and pTrc200 empty vector (V) only or expression of pTdei1 (tde1-tdi1 expressed from pTrc200), pTde2* (catalytic site mutated tde2 expressed on pRL662), or pTdei1+ pTde2*.


D . A . tumefaciens antibacterial activity assay against E . coli . The strains of A . tumefaciens were co - cultured at a ratio of 30 : 1 with E . coli DH10B ( + pRL662 ) on LB agar . The survival of target E . coli cells was quantified by counting CFUs on gentamicin - containing LB agar plates . Data represent mean ± SEM of 6 biological replicates from three independent experiments . One - way ANOVA followed by Turkey HSD test was used for statistical analysis . Two groups with significant differences ( P = 0 . 0010053 ) are indicated with different letters ( a and b ) .

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

D. A. tumefaciens antibacterial activity assay against E. coli. The strains of A. tumefaciens were co-cultured at a ratio of 30:1 with E. coli DH10B (+ pRL662) on LB agar. The survival of target E. coli cells was quantified by counting CFUs on gentamicin-containing LB agar plates. Data represent mean ± SEM of 6 biological replicates from three independent experiments. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Two groups with significant differences (P=0.0010053) are indicated with different letters (a and b).


A . T6SS secretion assay of A . tumefaciens strains : wild type C58 , ΔtssL , Δtap - 1 , and Δatu3641 harboring a pTrc200 vector ( V ) or derivatives pTap - 1 ( tap - 1 expressed from pTrc200 ) , or p3641 ( atu3641 expressed from pTrc200 ) .

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

A. T6SS secretion assay of A. tumefaciens strains: wild type C58, ΔtssL, Δtap-1, and Δatu3641 harboring a pTrc200 vector (V) or derivatives pTap-1 (tap-1 expressed from pTrc200), or p3641 (atu3641 expressed from pTrc200).


B . T6SS secretion assay of A . tumefaciens C58 Δtap - 1Δatu3641 containing an empty pTrc200 vector ( V ) or its derivatives expressing tap - 1 , atu3641 , tde1 - tdi1 , or tde2 - tdi2 .

O O O O O O O O O O O O O O O O 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. T6SS secretion assay of A. tumefaciens C58 Δtap-1Δatu3641 containing an empty pTrc200 vector (V) or its derivatives expressing tap-1, atu3641, tde1-tdi1, or tde2-tdi2.


C . T6SS secretion assay of A . tumefaciens C58ΔG2op mutant encoding full - length or truncated VgrG1 proteins .

C. T6SS secretion assay of A. tumefaciens C58ΔG2op mutant encoding full-length or truncated VgrG1 proteins.


A . Representative images of cells of A . tumefaciens C58 strains ΔtssB , ΔtssLΔtssB , and ΔtdeiΔtssB cells each expressing TssB - GFP from pTrc200 . Upper panel : phase contrast images ; Lower panel : green fluorescence images . Examples of GFP streaks were indicated by white arrowheads . Scale bar : 2 μm .

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

A. Representative images of cells of A. tumefaciens C58 strains ΔtssB, ΔtssLΔtssB, and ΔtdeiΔtssB cells each expressing TssB-GFP from pTrc200. Upper panel: phase contrast images; Lower panel: green fluorescence images. Examples of GFP streaks were indicated by white arrowheads. Scale bar: 2 μm.


B . Quantification of TssB - GFP foci in different genetic background . The number of the fluorescent foci were counted using the " analysis particle " function in Fiji with a manually set threshold . The number of cells were counted manually . For each strain , a total of three images were obtained from one independent experiment and each image contained more than 300 cells for quantification and statistical analysis . One - way ANOVA followed by Turkey HSD test was used for statistical analysis . Data are mean ± SEM and two groups with significant differences ( P = 0 . 000272 ) are indicated with different letters ( a and b ) . Similar results are reproduced in two independent experiments .

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

B. Quantification of TssB-GFP foci in different genetic background. The number of the fluorescent foci were counted using the "analysis particle" function in Fiji with a manually set threshold. The number of cells were counted manually. For each strain, a total of three images were obtained from one independent experiment and each image contained more than 300 cells for quantification and statistical analysis. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Data are mean ± SEM and two groups with significant differences (P=0.000272) are indicated with different letters (a and b). Similar results are reproduced in two independent experiments.


C . Western blots of the isolated sheaths , which were prepared via ultracentrifugation of cell lysates from A . tumefaciens C58 wild type , ΔtssL , and Δtdei . T : total proteins from the cell lysate . P : pellet samples containing TssBC sheaths after ultracentrifugation . S : supernatant after ultracentrifugation . Proteins were analyzed in western blots probed with indicated antibodies . Fha serves as a cytoplasmic control . Molecular weight markers ( in kDa ) are indicated on the left . Similar results were obtained from at least two independent experiments . Data are from one independent experiment and reproduced in at least two independent experiments .

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

C. Western blots of the isolated sheaths, which were prepared via ultracentrifugation of cell lysates from A. tumefaciens C58 wild type, ΔtssL, and Δtdei. T: total proteins from the cell lysate. P: pellet samples containing TssBC sheaths after ultracentrifugation. S: supernatant after ultracentrifugation. Proteins were analyzed in western blots probed with indicated antibodies. Fha serves as a cytoplasmic control. Molecular weight markers (in kDa) are indicated on the left. Similar results were obtained from at least two independent experiments. Data are from one independent experiment and reproduced in at least two independent experiments.


D . Pellet samples of wild type and Δtdei were visualized with transmission electron microscopy ( TEM ) . Sheaths are indicated by red arrowheads and flagella , which are distinguishable on the basis of smoother , more solid , and thinner structures ( ~ 13 nm in width ) , are indicated by green arrowheads [ 33 ] . Data are from one independent experiment and reproduced in at least two independent experiments . Scale bar : 0 . 5 μm ( upper panel ) , 100 nm ( upper panel ) .

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

D. Pellet samples of wild type and Δtdei were visualized with transmission electron microscopy (TEM). Sheaths are indicated by red arrowheads and flagella, which are distinguishable on the basis of smoother, more solid, and thinner structures (~13 nm in width), are indicated by green arrowheads [33]. Data are from one independent experiment and reproduced in at least two independent experiments. Scale bar: 0.5 μm (upper panel), 100 nm (upper panel).


B . Secretion assay of wild type and various mutants of 12D1 , 1D1108 and 15955 .

B. 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 .

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

C. 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 .

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

D. 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 .

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

E. Secretion assay of wild type 1D1108, ΔtssL, Δv4, Δv6 containing pRL662 empty vector (V) or its derivatives with v4 or v6.


A . T6SS secretion assay of A . tumefaciens wild type C58 , ΔtssL , and various single , double , and triple toxin - immunity deletion strains .

A. 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 .

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. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, Δtdei, and Δ3TIs harboring the indicated plasmids.


A . T6SS secretion assay of A . tumefaciens 12D1 wild type , ΔtssL , and various toxin - immunity deletion strains .

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

A. 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 .

O O O 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. 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 .

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 .

D. T6SS secretion assay of A. tumefaciens C58 wild type, ΔtssL, and various mutant strains.


( 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 .

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

(a) 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.


( 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 ) .

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

(a) 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).


( 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 .

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

(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.


( 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 .

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

(a) 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.


( 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 ) .

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

(a) 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).


D - 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

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

D-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 ganglion


I ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I) 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).


( 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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(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.


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 : ; ( 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 ) .

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

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: ; (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 ) .

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

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).


Human 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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Human 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.


Human 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 ) .

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

Human 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).


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 . ( 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 ) .

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

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. (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


Line 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Line 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).


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Volcano plot showing gene abbreviations for colonic transcripts that were significantly enriched in 14SM FF or 14SM FR after correction for multiple comparisons using DESeq2


mRNA 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA 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).


For 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 ) .

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

For 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


A 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 .

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A 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 < 0.0001


C 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 .

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C 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 < 0.0001


E 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 .

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E 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 < 0.0001


F 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 .

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F 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 .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 < 0.0001


H 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 .

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

H 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 < 0.0001


I 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 .

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

I 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 .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 < 0.0001


K 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 O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K 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


A 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 .

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

A 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 < 0.001


B 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 .

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

B 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 < 0.001


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

C 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


A 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 .

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

A 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 < 0.0001


D - 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 .

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D-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


A - 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 .

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

A-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 < 0.0001


E 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 .

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

E 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 < 0.0001


G 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 .

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

G 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 < 0.0001


H 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 .

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

H 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 < 0.0001


I 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I 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;


A - 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 .

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

A-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 < 0.0001


D 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 .

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D 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 < 0.0001


E 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 ) .

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

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


B 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 .

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

B 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 < 0.001


C 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 .

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

C 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 < 0.001


D 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 .

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

D 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 < 0.001


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

E 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


A 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 .

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

A 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 .

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

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.


TOPflash 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 .

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TOPflash 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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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.


E , 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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E,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.


A , 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 .

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A, 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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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.


D , 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 .

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D, 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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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.


E - 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 .

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E-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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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.


A , 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 .

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A, 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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C, qPCR analysis of Lgr5 and Axin2 expression levels in first passage mouse intestinal organoids cultured in EN + 5% RSPO1.


B . 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 .

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

B. 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 ) .

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

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).