Suppressed basal excitatory synaptic transmission in Ptprd - / - hippocampal SLM layer , but not SO or SR layer ( 2 months ; ) , as shown by I / O ( n = 14 slices from 5 mice [ WT - SO ] , 14 , 5 [ KO - SO ] , 11 , 4 [ WT - SR ] , 12 , 5 [ KO - SR ] , 14 , 6 [ WT - SLM ] , 12 , 6 [ KO - SLM ] , mean ± , * p < 0 . 05 ; * * p < 0 . 01 , two - way repeated - measures / RM ANOVA , Holm - Sidak post - hoc multiple comparisons ) . ( F , H , J ) Normal presynaptic release in Ptprd - / - hippocampal SLM , SO , SR layers ( P22 - 27 ; ) , as shown by PPF ( n = 13 slices from 5 mice [ WT - SO ] , 13 , 5 [ KO - SO ] , 10 , 4 [ WT - SR ] , 12 , 5 [ KO - SR ] , 15 , 6 [ WT - SLM ] , 14 , 6 [ KO - SLM ] , mean ± , ns , not significant , two - way RM ANOVA ) .

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Suppressed basal excitatory synaptic transmission in the Ptprd-/- hippocampal SLM layer, but not SO or SR layer (2 months; male), as shown by the I/O ratio (n = 14 slices from 5 mice [WT-SO], 14, 5 [KO-SO], 11, 4 [WT-SR], 12, 5 [KO-SR], 14, 6 [WT-SLM], 12, 6 [KO-SLM], mean ± SEM, *p < 0.05; **p < 0.01, two-way repeated-measures/RM ANOVA, Holm-Sidak post-hoc multiple comparisons test). (F, H, and J) Normal presynaptic release in the Ptprd-/- hippocampal SLM, SO, and SR layers (P22-27; male), as shown by the PPF ratio (n = 13 slices from 5 mice [WT-SO], 13, 5 [KO-SO], 10,4 [WT-SR], 12, 5 [KO-SR], 15, 6 [WT-SLM], 14, 6 [KO-SLM], mean ± SEM, ns, not significant, two-way RM ANOVA).


Decreased PSD density , but not PSD , thickness , or perforation ( % ) , in Ptprd - / - SLM ( P21 ; ) , revealed by EM . Arrows in panel K indicate PSDs .

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Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs.


Decreased PSD density , but not PSD , thickness , or perforation ( % ) , in Ptprd - / - SLM ( P21 ; ) , revealed by EM . Arrows in panel K indicate PSDs . ( n = 18 from 3 mice [ WT KO ] , * * * p < 0 . 001 , ns , not significant , Student ' s t - for L , O - R , Mann - Whitney U for M , N ) . Scale bar , 500 nm .

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Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs. (n = 18 images from 3 mice [WT and KO], ***p < 0.001, ns, not significant, Student's t-test for L, O-R, Mann-Whitney U test for M, N). Scale bar, 500 nm.


Decreased density of presynaptic nerve terminals but normal of nerve terminals normal density of presynaptic vesicles in Ptprd - / - SLM ( P21 ; ) , revealed by EM . ( n = 3 mice [ WT KO ] , mean ± , * * * p < 0 . 001 , ns , not significant , Student ' s t - ) .

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Decreased density of presynaptic nerve terminals but normal area of nerve terminals and normal density of presynaptic vesicles in the Ptprd-/- SLM (P21; male), revealed by EM. (n = 3 mice [WT and KO], mean ± SEM, ***p < 0.001, ns, not significant, Student's t-test).


A volcano plot of that showed significant up - or downregulation of pTyr ( absolute fold change > 1 . 5 , p < 0 . 05 ) in whole brain of Ptprd - / - mice ( P21 ) . Note that IL1RAPL1 is with strongest decrease in pTyr . ( n = 3 for WT 3 for KO ) . See Dataset EV1 , ' Phosphoscan Results ' tab for a full list of significantly changed .

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A volcano plot of proteins that showed significant up- or downregulation of pTyr levels (absolute fold change > 1.5, p < 0.05) in the whole brain of Ptprd-/- mice (P21). Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels. (n = 3 for WT and 3 for KO). See Dataset EV1, 'Phosphoscan Results' tab for a full list of significantly changed proteins.


A volcano plot of presynaptic included in SynGO list to show that they are significantly changed in pTyr ( p < 0 . 05 ) .

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A volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).


A volcano plot of postsynaptic included in SynGO list to show that they are significantly changed in pTyr ( p < 0 . 05 ) .

A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).


Decreased pTyr , but normal total of IL1RAPL1 , in Ptprd - / - brain ( P21 - 27 ) , as revealed by immunoprecipitation of IL1RAPL1 from whole - brain lysates followed by immunoblotting for total IL1RAPL1 pTyr ( 4G10 antibody ) .

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Decreased pTyr levels, but normal total levels of IL1RAPL1 protein, in the Ptprd-/- brain (P21-27), as revealed by immunoprecipitation of IL1RAPL1 from whole-brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels (4G10 antibody).


Decreased of synaptic IL1RAPL1 in Ptprd - / - brain ( P21 - 27 ) , as revealed by immunoblotting of crude synaptosomal ( P2 ) , synaptic plasma ( SPM ) , PSD ( PSD II ) fractions . of immunoblots reveal significant decrease of IL1RAPL1 in SPM PSD fractions of Ptprd - / - brain samples . ( n = 3 mice [ WT KO ] for each fraction [ P2 , SPM , PSD ] , mean ± , * * p < 0 . 01 , Student ' s t - ) .

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Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-/- brain (P21-27), as revealed by immunoblotting of crude synaptosomal (P2), synaptic plasma membrane (SPM), and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-/- brain samples. (n = 3 mice [WT and KO] for each fraction [P2, SPM, and PSD], mean ± SEM, **p<0.01, Student's t-test).


Normal of PTPδ in whole - brain lysates of Ptprd - meA - / - mice ( P21 - 27 ) , which contrasts with Ptprd - / - brain , where PTPδ is undetectable .

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Normal levels of the PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), which contrasts with the Ptprd-/- brain, where PTPδ protein is undetectable.


Strongly decreased of tyrosine - phosphorylated PTPδ in whole - brain lysates of Ptprd - meA - / - mice ( P21 - 27 ) , as shown by immunoprecipitation of IL1RAPL1 from whole - brain lysates followed by immunoblotting for total tyrosine - phosphorylated PTPδ .

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Strongly decreased levels of tyrosine-phosphorylated PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), as shown by the immunoprecipitation of IL1RAPL1 protein from whole-brain lysates followed by immunoblotting for total and tyrosine-phosphorylated PTPδ protein.


Decreased of synaptic IL1RAPL1 in Ptprd - meA - / - brain ( P21 - 27 ) , as revealed by immunoblotting P2 , SPM , PSD ( PSD II ) fractions . of immunoblots reveal significant decrease of IL1RAPL1 in SPM PSD fractions of Ptprd - meA - / - brain samples . ( n = 3 mice [ Ptprd - meA + / + Ptprd - meA - / - ] [ P2 , SPM , PSD ] , mean ± , * p < 0 . 05 , * * * p < 0 . 001 , Student ' s t - ) . Note that , while unchanged in P2 SPM fractions , PTPδ is significantly decreased in PSD of Ptprd - meA - / - samples .

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Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-meA-/- brain (P21-27), as revealed by immunoblotting P2, SPM, and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-meA-/- brain samples. (n = 3 mice [Ptprd-meA+/+ and Ptprd-meA-/-] and [P2, SPM, and PSD], mean ± SEM, *p<0.05, ***p < 0.001, Student's t-test). Note that, while unchanged in the P2 and SPM fractions, PTPδ is significantly decreased in the PSD of Ptprd-meA-/- samples.


Decreased basal excitatory synaptic transmission in hippocampal SLM region of Ptprd - meA - / - mice , as shown by I / O at TA - SLM pathway ( P20 - 24 ) . ( n = 15 slice from 5 mice [ Ptprd - meA + / + ] , 12 , 4 [ Ptprd - meA - / - ] , mean ± , * p < 0 . 05 ; * * * p < 0 . 001 , two - way RM ANOVA with Holm - Sidak ) .

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Decreased basal excitatory synaptic transmission in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the I/O ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+], 12, 4 [Ptprd-meA-/-], mean ± SEM, *p < 0.05; ***p<0.001, two-way RM ANOVA with Holm-Sidak test).


Normal presynaptic release in hippocampal SLM region of Ptprd - meA - / - mice , as shown by PPF at TA - SLM pathway ( P20 - 24 ) . ( n = 15 slice from 5 mice [ Ptprd - meA + / + ] 13 , 4 [ Ptprd - meA - / - ] , mean ± , ns , not significant , two - way RM ANOVA ) .

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Normal presynaptic release in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the PPF ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+] and 13, 4 [Ptprd-meA-/-], mean ± SEM, ns, not significant, two-way RM ANOVA).


Hyperactivity of Ptprd - / - mice ( 2 . 5 - 4 months ) in Laboras cages observed during - off - on periods were strongly mimicked by Emx1 - cKO mice , but more weakly by Viaat - cKO mice not at by Camk2a - cKO mice . Note also that immobility , a measure that better reflects sleep behavior , showed similar changes among different mouse lines . ( n = 14 mice [ WT ] , 14 [ global KO ] , 16 [ Emx1 - cWT ] , 16 [ Emx1 - cKO ] , 11 [ Viaat - cWT ] , 13 [ Viaat - cKO ] , 18 [ Camk2a - cWT ] 13 [ Camk2a - cKO ] , mean ± , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; ns , not significant , two - way RM ANOVA with Holm - Sidak ) .

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Hyperactivity of Ptprd-/- mice (2.5-4 months) in Laboras cages observed during light-off and light-on periods were strongly mimicked by Emx1-cKO mice, but more weakly by Viaat-cKO mice and not at all by Camk2a-cKO mice. Note also that immobility, a measure that better reflects sleep behavior, showed similar changes among different mouse lines. (n = 14 mice [WT], 14 [global KO], 16 [Emx1-cWT], 16 [Emx1-cKO], 11 [Viaat-cWT], 13 [Viaat-cKO], 18 [Camk2a-cWT] and 13 [Camk2a-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Hyperactive behavior decreased immobility of Ptprd - meA - / - mice ( 2 . 5 - 4 months ) in Laboras cages observed during - off periods - on periods , similar to Ptprd - / - mice . ( n = 7 mice for Ptprd - meA + / + 7 mice for Ptprd - meA - / - , mean ± , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak ) .

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Hyperactive behavior and decreased immobility of Ptprd-meA-/- mice (2.5-4 months) in Laboras cages observed during light-off periods and light-on periods, similar to Ptprd-/- mice. (n = 7 mice for Ptprd-meA+/+ and 7 mice for Ptprd-meA-/-, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Increased WAKE duration decreased NREM duration during first three hours of - on period in Emx1 - Cre ; Ptprdfl / fl mice ( 2 . 5 - 4 months ) . ( n = 9 mice [ Emx1 - cWT ] 9 mice [ Emx1 - cKO ] , mean ± , * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , two - way RM ANOVA with Holm - Sidak ) .

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Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). (n = 9 mice [Emx1-cWT] and 9 mice [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Decreased delta power ( normalized to total power ) in NREM , but not WAKE , oscillations in Emx1 - Cre ; Ptprdfl / fl mice ( 2 . 5 - 4 months ) . Note also normal theta power ( normalized to total power ) in REM oscillations in Emx1 - Cre ; Ptprdfl / fl mice . ( n = 9 [ Emx1 - cWT ] 9 [ Emx1 - cKO ] , mean ± , * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , two - way RM ANOVA with Holm - Sidak ) .

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Decreased delta power (normalized to total power) in NREM, but not WAKE, oscillations in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). Note also the normal theta power (normalized to total power) in REM oscillations in Emx1-Cre;Ptprdfl/fl mice. (n = 9 [Emx1-cWT] and 9 [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


( D ) Increased WAKE duration decreased NREM duration during first three hours of - on period decreased REM duration in first half of - on period in Ptprd - meA - / - mice ( 2 . 5 - 4 months ) . Note also that Ptprd - meA - / - mice also display increased WAKE duration decreased NREM duration during - off period . ( n = 8 [ Ptprd - meA + / + ] 8 [ Ptprd - meA - / - ] , mean ± , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak ) .

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(D) Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period and decreased REM duration in the first half of the light-on period in Ptprd-meA-/- mice (2.5-4 months). Note also that Ptprd-meA-/- mice also display increased WAKE duration and decreased NREM duration during the light-off period. (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Decreased delta power ( normalized to total power ) in NREM ( both - off - on periods ) WAKE ( - on period ) oscillations , decreased theta power in REM ( mainly - on period ) oscillations in Ptprd - meA - / - mice ( 2 . 5 - 4 months ) . ( n = 8 [ Ptprd - meA + / + ] 8 [ Ptprd - meA - / - ] , mean ± , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak ) .

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Decreased delta power (normalized to total power) in NREM (both light-off and light-on periods) and WAKE (light-on period) oscillations, and decreased theta power in REM (mainly light-on period) oscillations in Ptprd-meA-/- mice (2.5-4 months). (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Lethality was followed - up . LPS - treated mice ( n = 5 ) are depicted as black circles DEX / LPS - treated mice ( n = 5 ) are depicted as circles . P - values for survival curves were analyzed with a Fisher ' s exact .

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WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Lethality was followed-up. LPS-treated mice (n=5) are depicted as black circles and DEX/LPS-treated mice (n=5) are depicted as white circles. P-values for survival curves were analyzed with a Fisher's exact test.


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Blood samples were taken 2 hours after LPS TNF were measured in serum ( n = 4 - 5 ) .

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WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Blood samples were taken 2 hours after LPS and TNF levels were measured in serum (n=4-5).


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Three hours after LPS challenge , dextran labeled with FITC was administered by oral gavage . Five hours after gavaging , blood plasma was collected was measured as a degree of intestinal permeability ( λexc / λem = 488 / 520 nm ) ( PBS : n = 3 , LPS DEX / LPS : n = 5 ) .

O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O

WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Three hours after LPS challenge, dextran labeled with FITC was administered by oral gavage. Five hours after gavaging, blood plasma was collected and fluorescence was measured as a degree of intestinal permeability (λexc/λem = 488/520 nm) (PBS: n=3, LPS and DEX/LPS: n=5).


sequencing ( RNAseq ) results of IECs of WT mice ( n = 3 ) treated with PBS or 12 . 5 mg / kg LPS for 6 hours . with selection of LPS - induced genes . Color key shows log2 of normalized .

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RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. Heatmap with selection of LPS-induced genes. Color key shows the log2 of normalized counts.


WT mice ( n = 5 ) were treated with PBS or 12 . 5 mg / kg LPS with or without 10 mg / kg DEX pre - treatment . Ileum was isolated gene expression was measured via qPCR .

O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O

WT mice (n=5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated and gene expression was measured via qPCR.


WT mice ( n = 3 - 5 ) were treated with PBS or 12 . 5 mg / kg LPS with or without 10 mg / kg DEX pre - treatment . Ileum was isolated 6 hours after LPS challenge . ( A ) p - MLKL MLKL ( B ) RIPK3 were analyzed via western blot using ACTIN as a loading control . Expression were quantified using FIJI normalized to ACTIN . P - values were calculated using 1 - way ANOVA .

O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O

WT mice (n=3-5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated 6 hours after LPS challenge. (A) p-MLKL and MLKL protein levels and (B) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control. Expression levels were quantified using FIJI and normalized to ACTIN levels. P-values were calculated using 1-way ANOVA.


Gene expression ( Defa6 , lysozyme ) was measured via qPCR . P - values were calculated using 1 - way ANOVA . bars represent mean ± . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P ≤ 0 . 01 ; * P ≤ 0 . 05 .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Gene expression (Defa6, lysozyme) was measured via qPCR. P-values were calculated using 1-way ANOVA. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.


H & E was performed on ileum samples . Scale bar : 50µm .

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H&E staining was performed on ileum samples. Scale bar: 50µm.


Lysozyme expression ( in red ) was detected via , with Hoechst counterstaining . Scale bars : 50µm . of lysozyme expression . For every picture ( n = 3 - 4 per treatment ) , a selection of 10 crypts was made . Lysozyme positive signal was quantified in this region of interest ( ROI ) using an automated script in QuPath . Values are positive relative to total of ROI .

B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysozyme expression (in red) was detected via IHC, with Hoechst counterstaining. Scale bars: 50µm. Quantification of lysozyme expression. For every picture (n=3-4 per treatment), a selection of 10 crypts was made. Lysozyme positive signal was quantified in this region of interest (ROI) using an automated script in QuPath. Values are the positive area relative to total area of ROI.


TNFR1 + / + ( n = 9 ) TNFR1 - / - mice ( n = 11 ) were treated with 12 . 5 mg / kg LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

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TNFR1+/+ (n=9) and TNFR1-/- mice (n=11) were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TLR4fl / fl mice ( n = 7 ) TLR4VillinKO ( n = 9 ) mice were treated with 12 . 5 mg / kg LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

TLR4fl/fl mice (n=7) and TLR4VillinKO (n=9) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNFR1fl / fl mice ( n = 13 ) TNFR1VillinKO ( n = 10 ) mice were treated with 12 . 5 mg / kg LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

TNFR1fl/fl mice (n=13) and TNFR1VillinKO (n=10) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNFR1 + / + TNFR1 - / - mice ( n = 4 - 6 ) were treated with 12 . 5 mg / kg LPS . Ileum was isolated gene expression was measured via qPCR . Fold changes are shown on graph .

B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O

TNFR1+/+ and TNFR1-/- mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.


TNFR1fl / fl TNFR1VillinKO mice ( n = 4 - 6 ) were treated with 12 . 5 mg / kg LPS . Ileum was isolated gene expression was measured via qPCR . Fold changes are shown on graph .

B-GENEPROD O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O

TNFR1fl/fl and TNFR1VillinKO mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.


GRfl / fl ( n = 8 , ) GRLysMKO mice ( n = 4 ) were treated with 2 . 5 mg / kg LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

B-GENEPROD O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

GRfl/fl (n=8,) and GRLysMKO mice (n=4) were treated with 2.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


GRLysMKO mice were treated with PBS ( n = 12 ) or 10 mg / kg DEX ( n = 13 ) before a lethal dose of LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

GRLysMKO mice were treated with PBS (n=12) or 10 mg/kg DEX (n=13) before a lethal dose of LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in serum of GRfl / fl ( n = 4 ) GRLysMKO mice ( n = 3 ) . Mice were treated with 2 . 5 mg / kg LPS blood was drawn 2 hours later . TNF was measured with ELISA .

B-GENEPROD O O B-TISSUE O B-GENEPROD O O O O O O O B-GENEPROD B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE B-TISSUE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O

TNF measurement in the serum of GRfl/fl (n=4) and GRLysMKO mice (n=3). Mice were treated with 2.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


TNF measurement in serum of GRfl / fl GRLysMKO ( n = 4 ) . Mice were treated with PBS or a lethal dose of LPS ( 12 . 5 2 . 5 mg / kg for GRfl / fl GRLysMKO mice respectively ) , with or without pre - treatment with 10 mg / kg DEX blood was drawn 2 hours later . TNF was measured with ELISA .

B-GENEPROD O O B-TISSUE O B-GENEPROD O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE B-TISSUE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O

TNF measurement in the serum of GRfl/fl and GRLysMKO (n=4). Mice were treated with PBS or a lethal dose of LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) , with or without pre-treatment with 10 mg/kg DEX and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


GRfl / fl ( n = 4 ) GRLysMKO mice ( n = 3 - 4 ) were treated with 2 . 5 mg / kg LPS . Ileum was isolated after 6 hours gene expression was measured via qPCR . Fold changes are depicted on graphs .

B-GENEPROD O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O

GRfl/fl (n=4) and GRLysMKO mice (n=3-4) were treated with 2.5 mg/kg LPS. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRfl / fl GRLysMKO mice ( n = 4 ) were treated with a PBS or LPS ( 12 . 5 2 . 5 mg / kg for GRfl / fl GRLysMKO mice respectively ) with or without 10 mg / kg DEX pre - treatment of 30 minutes . Ileum was isolated after 6 hours gene expression was measured via qPCR . Fold changes are depicted on graphs .

B-GENEPROD O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O

GRfl/fl and GRLysMKO mice (n=4) were treated with a PBS or LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) with or without 10 mg/kg DEX pre-treatment of 30 minutes. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRfl / fl ( n = 8 ) GRVillinKO mice ( n = 10 ) were treated with 5 mg / kg LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

B-GENEPROD O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

GRfl/fl (n=8) and GRVillinKO mice (n=10) were treated with 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


GRVillinKO mice were treated with PBS ( n = 9 ) or 10 mg / kg DEX ( n = 10 ) before 5 mg / kg LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

GRVillinKO mice were treated with PBS (n=9) or 10 mg/kg DEX (n=10) before 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in serum of GRfl / fl ( n = 4 ) GRVillinKO mice ( n = 4 ) . Mice were treated with 5 mg / kg LPS blood was drawn 2 hours later .

B-GENEPROD O O B-TISSUE O B-GENEPROD O O O O O O O B-GENEPROD B-ORGANISM O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE B-TISSUE O O O O O O

TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n= 4). Mice were treated with 5 mg/kg LPS and blood was drawn 2 hours later.


TNF measurement in serum of GRfl / fl ( n = 4 ) GRVillinKO mice ( n = 4 ) . Mice were treated with PBS or LPS ( 12 . 5 5 mg / kg for GRfl / fl GRVillinKO mice respectively ) with or without pre - treatment with 10 mg / kg DEX for 30 minutes . Blood was taken 2 hours after LPS challenge TNF concentrations were measured with ELISA .

B-GENEPROD O O B-TISSUE O B-GENEPROD O O O O O O O B-GENEPROD B-ORGANISM O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-EXP_ASSAY O

TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n=4). Mice were treated with PBS or LPS (12.5 and 5 mg/kg for GRfl/fl and GRVillinKO mice respectively) with or without pre-treatment with 10 mg/kg DEX for 30 minutes. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.


GRVillinKO mice were treated with PBS ( n = 8 ) or a neutralizing P55 ( TNFR1 ) antibody ( n = 9 ) 2 hours before 8 hours after 5 mg / kg LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

B-GENEPROD B-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

GRVillinKO mice were treated with PBS (n=8) or a neutralizing P55 (TNFR1) antibody (n=9) 2 hours before and 8 hours after 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


Ileum was isolated from non - stimulated GRfl / fl ( n = 4 ) GRVillinKO mice ( n = 4 ) gene expression was measured via qPCR . Expression are shown as fold induction with GRfl / fl expression values set at 1 . P - values were calculated using a student ' s t - .

B-TISSUE O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

Ileum was isolated from non-stimulated GRfl/fl (n=4) and GRVillinKO mice (n=4) and gene expression was measured via qPCR. Expression levels are shown as fold induction with GRfl/fl expression values set at 1. P-values were calculated using a student's t-test.


GRfl / fl ( n = 4 ) GRVillinKO mice ( n = 4 ) were treated with 5 mg / kg LPS . Ileum was isolated gene expression was measured via qPCR .

B-GENEPROD O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O

GRfl/fl (n=4) and GRVillinKO mice (n=4) were treated with 5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR.


Left : LPS dose - response curves of GRwt / wt ( n = 3 - 7 per LPS dose ) GRdim / dim mice ( n = 3 - 7 per LPS dose ) treated with 1 . 5 mg / kg LPS . Right : LD50 values of LPS are depicted on top of each bar for each group .

O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O

Left: LPS dose-response curves of GRwt/wt (n=3-7 per LPS dose) and GRdim/dim mice (n=3-7 per LPS dose) treated with 1.5 mg/kg LPS. Right: LD50 values of LPS are depicted on top of each bar for each group.


GRdim / dim mice were treated with PBS ( n = 5 ) or 10 mg / kg DEX ( n = 5 ) before 1 . 5 mg / kg LPS survival was monitored . P - values were analyzed with a Fisher ' s exact .

B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

GRdim/dim mice were treated with PBS (n=5) or 10 mg/kg DEX (n=5) before 1.5 mg/kg LPS and survival was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in serum of GRwt / wt ( n = 5 ) GRdim / dim mice ( n = 5 ) . Mice were treated with 1 . 5 mg / kg LPS blood was drawn 2 hours later . TNF was measured with ELISA .

B-GENEPROD O O B-TISSUE O B-GENEPROD O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE B-TISSUE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O

TNF measurement in the serum of GRwt/wt (n=5) and GRdim/dim mice (n= 5). Mice were treated with 1.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


TNF measurement in serum of GRwt / wt GRdim / dim mice ( n = 4 ) treated with PBS or LPS ( 12 . 5 1 . 5 mg / kg in GRwt / wt GRdim / dim mice respectively ) with or without pretreatment with 10 mg / kg DEX . Blood was taken 2 hours after LPS challenge TNF concentrations were measured with ELISA .

B-GENEPROD O O B-TISSUE O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-EXP_ASSAY O

TNF measurement in the serum of GRwt/wt and GRdim/dim mice (n=4) treated with PBS or LPS (12.5 and 1.5 mg/kg in GRwt/wt and GRdim/dim mice respectively) with or without pretreatment with 10 mg/kg DEX. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.


GRdim / dim mice were treated with PBS ( n = 6 ) or a neutralizing P55 ( TNFR1 ) antibody ( n = 8 ) 2 hours before 8 hours after 1 . 5 mg / kg LPS lethality was monitored . P - values were analyzed with a Fisher ' s exact .

B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

GRdim/dim mice were treated with PBS (n=6) or a neutralizing P55 (TNFR1) antibody (n=8) 2 hours before and 8 hours after 1.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


Tofacitinib ( n = 6 ) or vehicle ( n = 7 ) was given orally to GRdim / dim mice . After 1 . 5 mg / kg LPS injection survival was monitored p - values were analyzed with a Fisher ' s exact .

B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O

Tofacitinib (n=6) or vehicle (n=7) was given orally to GRdim/dim mice. After 1.5 mg/kg LPS injection survival was monitored and p-values were analyzed with a Fisher's exact test.


GRwt / wt ( n = 4 ) GRdim / dim ( n = 4 ) mice were treated with 1 . 5 mg / kg LPS . Ileum was isolated gene expression was measured via qPCR . Fold changes are depicted on graphs .

B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O

GRwt/wt (n=4) and GRdim/dim (n=4) mice were treated with 1.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRdim / dim mice ( n = 4 ) were treated with PBS or 1 . 5 mg / kg LPS with or without 10 mg / kg DEX pretreatment . Ileum was isolated gene expression was measured via qPCR . Fold changes are depicted on graphs .

B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O

GRdim/dim mice (n=4) were treated with PBS or 1.5 mg/kg LPS with or without 10 mg/kg DEX pretreatment. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


For SPR measurements , IL - 27Rα or GP130 were immobilized on chip surface by biotin - streptavidin interaction IL - 27 were flowed across chip in solution . SPR data are representative of three biological replicates . Kinetic charts for IL - 27Rα ( A ) GP130 ( B ) . Concentrations used are shown on curves . Data traces were fitted using a 1 : 1 interaction model ( black ) to quantify kinetics ( kon , koff ) binding affinity ( KD ) of interactions .

O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O

For SPR measurements, IL-27Rα or GP130 were immobilized on the chip surface by biotin-streptavidin interaction and IL-27 were flowed across the chip in solution. SPR data are representative of three biological replicates. Kinetic charts for IL-27Rα (A) and GP130 (B). Concentrations used are shown on the curves. Data traces were fitted using a 1:1 interaction model (black) to quantify the kinetics (kon, koff) and binding affinity (KD) of the interactions.


For SPR measurements , IL - 27Rα were immobilized on chip surface by biotin - streptavidin interaction p28 were flowed across chip in solution . SPR data are representative of three biological replicates . ( C , Equilibrium chart for p28 binding to IL - 27Rα ( C , left panel ) curve - fitting to data points generated at various concentrations of p28 ( C , right panel ) .

O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

For SPR measurements, IL-27Rα were immobilized on the chip surface by biotin-streptavidin interaction and p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. (C, Equilibrium chart for p28 binding to IL-27Rα (C, left panel) and curve-fitting to data points generated at various concentrations of p28 (C, right panel).


For SPR measurements , GP130 were immobilized on chip surface by biotin - streptavidin interaction p28 were flowed across chip in solution . SPR data are representative of three biological replicates . ( D ) Equilibrium chart for p28 binding GP130 .

O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY B-GENEPROD O

For SPR measurements, GP130 were immobilized on the chip surface by biotin-streptavidin interaction and p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. (D) Equilibrium chart for p28 binding GP130.


( F , G ) Dose response curves for pSTAT1 ( F ) pSTAT3 ( G ) in resting mouse CD8 + T . were stimulated with mIL - 27 or p28 for 15 minutes with indicated doses . Data shown are mean of four biological replicates with error bars depicting standard error of mean .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD 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,G) Dose response curves for pSTAT1 (F) and pSTAT3 (G) in resting mouse CD8+ T cells. Cells were stimulated with mIL-27 or p28 for 15 minutes with the indicated doses. Data shown are the mean of four biological replicates with error bars depicting standard error of the mean.


( B - D ) Flow cytometric of DHE > Eth . positive wild type yeast ( allowing detection of both dead exhibiting sub - lethal oxidative stress ) after 42 h ( B ) or 66 h ( C , D ) expressing Abeta constructs . Dot plots show data points along with mean ( bar ) ± s . d . . n = 5 - 12 biologically independent cultures . * * * , p < 0 . 001 , * * , p < 0 . 01 , * , p < 0 . 05 . ANOVA with Tukey ' s post - hoc .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Flow cytometric quantification of DHE>Eth. positive wild type yeast cells (allowing the detection of both dead cells and cells exhibiting sub-lethal oxidative stress) after 42 h (B) or 66 h (C, D) expressing Abeta constructs. Dot plots show all data points along with the mean (bar) ± s.d.. n = 5-12 biologically independent cultures. ***, p < 0.001, **, p < 0.01, *, p < 0.05. ANOVA with Tukey's post-hoc test.


( E ) Annexin V ( AV ) / PI co - to assess in yeast expressing A42 after 48 h . Data represent means ± s . d . n = 6 biologically independent cultures . * * * , p < 0 . 001 ( comparing PI positive populations ) . Unpaired , two - t - .

O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O

(E) Annexin V (AV)/PI co-staining to assess cell death in yeast expressing A42 after 48 h. Data represent means ± s.d. n = 6 biologically independent cultures. ***, p < 0.001 (comparing PI positive populations). Unpaired, two-tailed t-test.


( F ) Immunoblot of whole extract ( WCE ) of wild type yeast after 16 h expression of Abeta constructs . For western blot ( WB ) Abeta - specific antibody ( Abeta ) 6E10 was used showing long ( Long exp . ) short time exposure ( Short exp . ) . Ssc1 was used as a loading control .

O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O 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-GENEPROD O O O O O O O

(F) Immunoblot of whole cell extract (WCE) of wild type yeast cells after 16 h expression of Abeta constructs. For western blot (WB) Abeta-specific antibody (Abeta) 6E10 was used showing long (Long exp.) and short time exposure (Short exp.). Ssc1 was used as a loading control.


( G ) Confocal microscopy of wild type yeast after 18 h of Abeta expression . Colors indicate .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O

(G) Confocal microscopy of wild type yeast cells after 18 h of Abeta expression. Colors indicate fluorescence intensity.


( A ) Immunoblot of total post - supernatant ( PNS ) , mitochondrial , microsomal fractions of wild type yeast after 18 h expression of EGFP - Abeta42 ( A42 ) , EGFP - Abeta40 ( A40 ) EGFP empty vector ( ev ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) short time exposure ( Short exp . ) . Purity of fractions was tested with antibodies against Cyc1 ( mitochondria ) , Sec61 ( microsomes ) Pgk1 ( cytosol ) .

O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-ORGANISM O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O

(A) Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial, microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP-Abeta42 (A42), EGFP-Abeta40 (A40) and EGFP empty vector (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Purity of fractions was tested with antibodies against Cyc1 (mitochondria), Sec61 (microsomes) and Pgk1 (cytosol).


( B ) ATP content of wild type yeast after 42 h expression of EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) . Dot plots show data points along with mean ( bar ) ± s . d . . n = 16 biologically independent cultures . * * , p < 0 . 01 . Unpaired , two - t - .

O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP-A42 (A42) or EGFP empty vector (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 16 biologically independent cultures. **, p < 0.01. Unpaired, two-tailed t-test.


( C ) Relative abundance of EGFP - Abeta42 ( A42 ) versus empty vector ( ev ) expressing wild type yeast . significant subset of heat shock ( HSP ) detected in a proteomics of isolated mitochondria is depicted . Log2 SILAC ratios of A42 / ev of two independent proteome measurements are shown . Dot plots show data points along with mean ( ) . Significance was determined using an outlier ( Significance A , p < 0 . 003 ) .

O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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) Relative protein abundance of EGFP-Abeta42 (A42) versus empty vector (ev) expressing wild type yeast cells. The significant subset of cytosolic heat shock proteins (HSP) detected in a proteomics analysis of isolated mitochondria is depicted. Log2 SILAC ratios of A42/ev of two independent proteome measurements are shown. Dot plots show all data points along with the mean (line). Significance was determined using an outlier test (Significance A, p <0.003).


( D ) Screen of 123 deletion strains assessing Abeta42 ( A42 ) - induced toxicity ( assessed by DHE > Eth . positive indicative of sum of sub - lethal oxidative stress ) in yeast . Relative A42 - toxicity normalized to wild type ( WT ) is depicted . Potential hits ( mutants that reduce A42 toxicity ) are shown in green . Data represent means ± s . d . . n ≥ 3 biologically independent cultures .

O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

(D) Screen of 123 deletion strains assessing Abeta42 (A42)-induced toxicity (assessed by DHE>Eth. positive cells indicative of the sum of sub-lethal oxidative stress and cell death) in yeast. Relative A42-toxicity normalized to wild type (WT) cells is depicted. Potential hits (mutants that reduce A42 toxicity) are shown in green. Data represent means ± s.d.. n ≥ 3 biologically independent cultures.


of positive for PI indicating at indicated time points after start of expression of EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) of wild type ( WT ) ( A ) YDJ1 deleted ( Δydj1 ) ( B ) . Mean ± s . d . . n = 5 biologically independent cultures . Comparisons by two - way ANOVA ( mixed - design ) followed by simple main effects ( * * * , p < 0 . 001 , vs . control ) .

O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP-A42 (A42) or EGFP empty vector (ev) of wild type (WT) (A) and YDJ1 deleted (Δydj1) cells (B). Mean ± s.d.. n = 5 biologically independent cultures. Comparisons by two-way ANOVA (mixed-design) followed by simple main effects (***, p < 0.001, vs. control).


Immunoblot of whole extract ( Input ) eluate of FLAG - tagged Ydj1 . Immunoprecipitation ( IP : FLAG ) of YDJ1 deletion ( Δydj1 ) expressing EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) co - overexpressing Ydj1 - FLAG using Abeta - specific antibody ( 6E10 ) FLAG antibody ( FLAG ) .

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Immunoblot of whole cell extract (Input) and eluate of FLAG-tagged Ydj1. Immunoprecipitation (IP: FLAG) of YDJ1 deletion strain (Δydj1) cells expressing EGFP-A42 (A42) or EGFP empty vector (ev) and co-overexpressing Ydj1-FLAG using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).


Confocal microscopy of wild type ( WT ) yeast expressing EGFP - A42 ( A42 ) only co - overexpressing Ydj1 - FLAG or with corresponding vector control after 18 h of expression . Colors indicate .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

Confocal microscopy of wild type (WT) yeast cells expressing EGFP-A42 (A42) only and co-overexpressing Ydj1-FLAG or with the corresponding vector control after 18 h of expression. Colors indicate fluorescence intensity.


Confocal microscopy of wild type ( WT ) yeast expression of EGFP - A42 ( A42 ) co - overexpressing Ydj1 - FLAG after 18 h of expression . Mitochondria were visualized with MitoTracker Red ( magenta ) . See also Fig EV2J , EV1C .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O

Confocal microscopy of wild type (WT) yeast cells expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG after 18 h of expression. Mitochondria were visualized with MitoTracker Red (magenta). See also Fig EV2J, EV1C.


Immunoblot of total post - supernatant ( PNS ) , mitochondrial ( Mito . ) , microsomal ( Micro . ) ( Cyt . ) fractions of wild type ( WT ) YDJ1 deletion ( Δydj1 ) after 18 h of expression of EGFP - A42 ( A42 ) co - overexpressing Ydj1 - FLAG or corresponding empty vector controls ( ev ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) short time exposure ( Short exp . ) . Tom22 - specific antibody is a marker for mitochondria , Sec61 for microsomes Ssa1 was used to verify fraction .

B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O 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-GENEPROD O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR B-GENEPROD O O O O O O

Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.) and cytosolic (Cyt.) fractions of wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Tom22-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.


of full EGFP - A42 in mitochondrial fraction / fraction , using densitometry of immunoblot representatively shown in Fig 3F Appendix Fig S1C from three / 4 independent experiments . Dot plots show data points along with mean ( / bar ) ± s . d . . n = 3 - 4 biologically independent cultures . * , p < 0 . 05 . Unpaired , two - t - .

O O B-GENEPROD O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ratio of full length EGFP-A42 in the mitochondrial fraction / cytosolic fraction, using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three/4 independent experiments. Dot plots show all data points along with the mean (line/bar) ± s.d.. n = 3-4 biologically independent cultures. *, p < 0.05. Unpaired, two-tailed t-test.


for degradation using cycloheximide ( CH ) to stall translation . EGFP was measured at two time points ( t0 t2 , 2 h after CH administration ) normalized to t0 in wild type ( WT ) YDJ1 deletion ( Δydj1 ) after 18 h of expression of EGFP - A42 ( A42 ) co - overexpressing Ydj1 - FLAG or corresponding empty vector controls ( ev ) . Dot plots show data points along with mean ( bar ) ± s . d . . n = 8 biologically independent cultures . * * * , p < 0 . 001 . ANOVA with Tukey ' s post - hoc . See also Appendix Fig S2D .

O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity was measured at two time points (t0 and t2, 2 h after CH administration) and normalized to t0 in wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 8 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc. See also Appendix Fig S2D.


of between EGFP - A42 tetramer monomer in wild type ( WT ) YDJ1 deletion ( Δydj1 ) expressing EGFP - A42 ( J ) as well as between WT expressing EGFP - A42 only co - overexpressing Ydj1 - FLAG ( K ) at indicated time points . Dot plots show data points along with mean ( bar ) ± s . d . . n = 8 ( J ) n = 12 ( K ) biologically independent cultures . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 . ANOVA with Tukey ' s post - hoc . See also Appendix Fig S2A - B .

O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the ratio between EGFP-A42 tetramer and monomer in wild type (WT) and YDJ1 deletion strain (Δydj1) expressing EGFP-A42 (J) as well as between WT expressing EGFP-A42 only and co-overexpressing Ydj1-FLAG (K) at indicated time points. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8(J) n=12(K) biologically independent cultures. ***, p < 0.001; **, p < 0.01; *, p < 0.05. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2A-B.


of propidium iodide ( PI ) - positive indicating at indicated time points of expression of EGFP - A42 ( A42 ) Δydj1 with co - expression of DnaJA1 - FLAG ( DnaJA1 ) ( B ) or corresponding empty vector controls ( ev ) ( A ) . Mean ± SD n = 4 - 6 biologically independent cultures . Comparisons by two - way ANOVA ( mixed design ) followed by simple main effects ( * P < 0 . 05 , versus control ) .

O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of propidium iodide (PI)-staining positive cells indicating cell death at indicated time points of expression of EGFP-A42 (A42) Δydj1 cells with co-expression of DnaJA1-FLAG (DnaJA1) (B) or corresponding empty vector controls (ev) (A). Mean ± SD n = 4-6 biologically independent cultures. Comparisons by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus control).


( C ) Immunoblot ( WB ) of whole - extract ( Input ) eluate of FLAG - tagged DnaJA1 immunoprecipitation ( IP : FLAG ) of wild - type ( WT ) expressing EGFP - A42 ( A42 ) co - expressing DnaJA1 - FLAG ( A1 ) using Abeta - specific antibody ( 6E10 ) FLAG antibody ( FLAG ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O

(C)Immunoblot (WB) of whole-cell extract (Input) and eluate of FLAG-tagged DnaJA1 immunoprecipitation (IP: FLAG) of wild-type strain (WT) expressing EGFP-A42 (A42) and co-expressing DnaJA1-FLAG (A1) using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).


for degradation using cycloheximide ( CH ) to stall translation . EGFP of YDJ1 deletion ( Δydj1 ) wild type ( WT ) expressing EGFP - A42 ( A42 ) only or co - expressing DnaJA1 was measured at two time points ( t0 t2 ) normalized to t0 . Dot plots show data points along with mean ( bar ) ± s . d . . n = 6 biologically independent cultures . * * * , p < 0 . 001 . ANOVA with Tukey ' s post - hoc . See also Appendix Fig S2C .

O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity of YDJ1 deletion strain (Δydj1) and wild type (WT) cells expressing EGFP-A42 (A42) only or co-expressing DnaJA1 was measured at two time points (t0 and t2) and normalized to t0. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2C.


Immunoblot ( WB ) of total post - supernatant ( PNS ) , mitochondrial ( Mito . ) , microsomal ( Micro . ) , ( Cyt . ) fractions of YDJ1 - deleted ( Δydj1 ) after 18 h expression of EGFP - A42 ( A42 ) only or co - expressing DnaJA1 - FLAG ( DnaJA1 ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) short time ( Short exp . ) exposure ( two sections from one immunoblot ) . Cox4 - specific antibody is a marker for mitochondria , Sec61 for microsomes Ssa1 was used to verify fraction .

B-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O 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-EXP_ASSAY O O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O O O O

Immunoblot (WB) of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.), and cytosolic (Cyt.) fractions of YDJ1-deleted cells (Δydj1) after 18 h expression of EGFP-A42 (A42) only or co-expressing DnaJA1-FLAG (DnaJA1) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time (Short exp.) exposure (two sections from one immunoblot). Cox4-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.


Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over indicated time course with or without DnaJA1 ( A1 ) quantified by densitometry in ( G ) .

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Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 (A1) and quantified by densitometry in (G).


of oligomer - to - monomer of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in ( F ) . Dot plots show data points along with mean ( ) ± SD n = 3 biologically independent cultures . P - values by two - way ANOVA ( mixed design ) followed by simple main effects ( * P < 0 . 05 , versus A42 control ) . See also Appendix Fig S2D .

O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O

Quantification of the oligomer-to-monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in (F). Dot plots show all data points along with the mean (line) ± SD n = 3 biologically independent cultures. P-values by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus A42 control). See also Appendix Fig S2D.


A42 beta - sheet - rich assembly formation monitored by increase in ThT over time with BSA or with DnaJ . Data represent means of at least eight measurements . See also Appendix Fig S1E .

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A42 beta-sheet-rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ. Data represent means of at least eight measurements. See also Appendix Fig S1E.


( A ) Immunoblot of cytosol - enriched cerebral tissue homogenate ( Input ) eluate of Abeta42 or DnaJA1 using Abeta - specific antibody ( 6E10 ) DnaJA1 - specific antibody ( DnaJA1 ) , respectively , showing optimal long exposure ( Long exp . ) . Immunoprecipitation using magnetic beads without antibody , DnaJA1 or 6E10 antibody of ( 15 months old ) wild type ( WT ) or 3xTg ( PS1M146V / APPSwe / tauP301L ) mice . Sections showing monomer low - n oligomers , dodecamer full - amyloid precursor ( APP ) are from 1 immunoblot .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O

(A) Immunoblot of cytosol-enriched cerebral tissue homogenate (Input) and eluate of Abeta42 or DnaJA1 using Abeta-specific antibody (6E10) and DnaJA1-specific antibody (DnaJA1), respectively, showing optimal and long exposure (Long exp.). Immunoprecipitation using magnetic beads without antibody, DnaJA1 or 6E10 antibody of female (15 months old) wild type (WT) or 3xTg (PS1M146V/APPSwe/tauP301L) mice. Sections showing monomer and low-n oligomers, dodecamer and full-length amyloid precursor protein (APP) are from 1 immunoblot.


( B ) Representative immunoblot of DnaJA1 from hippocampi of AD ( AD ) aged non - demented controls ( ctrl ) . PonceauS serves as a loading control . ( C ) by densitometry of DnaJA1 normalized to PonceauS from hippocampi of AD ( AD ) aged non - demented controls ( ctrl ) . Representative immunoblot is shown in ( B ) . Dot plots show data points along with mean ( bar ) ± s . d . . n = 4 - 5 independent . * , p < 0 . 05 . Unpaired , two - t - .

O O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O B-DISEASE O B-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O B-TISSUE O B-DISEASE O B-DISEASE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). PonceauS serves as a loading control. (C) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). Representative immunoblot is shown in (B). Dot plots show all data points along with the mean (bar) ± s.d.. n = 4-5 independent patients. *, p < 0.05. Unpaired, two-tailed t-test.


( A ) Representative confocal microscopy of 10 - day - old fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) reference nuclei with DAPI ( blue ) of Droj2 knockdown flies ( Droj2 + / - ) flies corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O O O O O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O

(A) Representative confocal microscopy of 10-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( B ) Representative confocal gSTED deconvolved ( decon ) of Kenyon in 18 - day - old fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) reference nuclei with DAPI ( blue ) of Droj2 knockdown flies ( Droj2 + / - ) flies corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O B-ORGANISM B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O O B-ORGANISM O O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O

(B) Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 18-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( C - D ) Average ( C ) total ( D ) of Abeta ( 6E10 ) signal from confocal representatively shown in ( A ) from 12 brains of w1118 wild type ( Droj2 + / + ) knockdown ( Droj2 + / - ) 10 - day - old flies . Dot plots show data points along with mean ( bar ) ± s . d . . n = 12 . * , p < 0 . 05 . Unpaired , two - t - .

O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O 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-D) Average intensity (C) and total area (D) of Abeta (6E10) signal from confocal images representatively shown in (A) from 12 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 10-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 12. *, p < 0.05. Unpaired, two-tailed t-test.


( E - F ) qPCR of Droj2 - mRNA of 3 - 6 days old ( E ) ( F ) flies expressing human Abeta42 ( UAS - A42 ) of w1118 wild type flies ( Droj2 + / + ) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression ( Droj2 + / + ctrl ) . Reference gene is Rpl32 . Dot plots show data points along with mean ( ) ± s . d . . n = 3 . * * , p < 0 . 01 . One sample t - against 1 .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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-F) qPCR analysis of Droj2-mRNA levels of 3-6 days old female (E) and male (F) flies expressing human Abeta42 (UAS-A42) of w1118 wild type flies (Droj2+/+) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression (Droj2+/+ ctrl). Reference gene is Rpl32. Dot plots show all data points along with the mean (line) ± s.d.. n = 3. **, p < 0.01. One sample t-test against 1.


( G ) Immunoprecipitation ( IP : 6E10 ) of w1118 wild type flies ( Droj2 + / + ) using synthetic Abeta42 added to fly head extract ( Input ) . Abeta - specific antibody 6E10 was used for Abeta42 DnaJA1 - specific antibody for Droj2 immunoblot detection . Showing input , supernatant , supernatant after washing ( WS ) eluate .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O

(G) Immunoprecipitation (IP: 6E10) of w1118 wild type flies (Droj2+/+) using synthetic Abeta42 added to the fly head extract (Input). Abeta-specific antibody 6E10 was used for Abeta42 and DnaJA1-specific antibody for Droj2 immunoblot detection. Showing input, supernatant, supernatant after washing (WS) and eluate.


( A - B ) Survival of ( A ) ( B ) w1118 wild type flies ( Droj2 + / + ) Droj2 knockdown flies ( Droj2 + / - ) with expression of human Abeta42 ( UAS - A42 ) or control flies without expression ( ctrl ) , upon supplementation of sugar ( 10 % sucrose ) with 20 mM MnCl2 . Survival was determined at indicated time points . n = 6 with 100 - 120 flies per experiment . indicated p - value refers to interaction ( int . ) term of a Cox Proportional Hazards model comparing Abeta42 toxicity ( UAS - A42 vs . ctrl ) Droj2 expression ( Droj2 + / + vs . Droj2 + / - ) as main factors . following pairwise comparisons of indicated groups survival were done by Log Rank ( * * * * , p < 0 . 0001 ; ns , p > 0 . 05 ) .

O O O O O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O B-ORGANISM B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O 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-B) Survival of female (A) and male (B) w1118 wild type flies (Droj2+/+) and Droj2 knockdown flies (Droj2+/-) with expression of human Abeta42 (UAS-A42) or control flies without expression (ctrl), upon supplementation of sugar (10% sucrose) with 20 mM MnCl2. Survival was determined at indicated time points. n = 6 with 100-120 flies per experiment. The indicated p-value refers to the interaction (int.) term of a Cox Proportional Hazards model comparing Abeta42 toxicity (UAS-A42 vs. ctrl) and Droj2 expression (Droj2+/+ vs. Droj2+/-) as main factors. The following pairwise comparisons of the indicated groups survival were done by Log Rank test (****, p < 0.0001; ns, p > 0.05).


( C ) Aversive associative memory performance 2 min after training of aged ( 18 days old ) Droj2 knockdown flies ( Droj2 + / - ) corresponding isogenic w1118 wild type flies ( Droj2 + / + ) both expressing human Abeta42 ( UAS - A42 ) of six independent biological replicates . Dot plots show data points along with mean ( bar ) ± s . d . . n = 6 . * , p < 0 . 05 . Unpaired , two - t - .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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) Aversive associative memory performance 2 min after training of aged (18 days old) male Droj2 knockdown flies (Droj2+/-) and corresponding isogenic w1118 wild type flies (Droj2+/+) both expressing human Abeta42 (UAS-A42) of six independent biological replicates. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6. *, p < 0.05. Unpaired, two-tailed t-test.


( D ) Representative confocal gSTED deconvolved of Kenyon in 15 - day - old fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) mitochondrial marker ATP5A - specific antibody ( ATP5A , green ) of Droj2 knockdown flies ( Droj2 + / - ) flies corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-ORGANISM B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O O O O O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O

(D) Representative confocal and gSTED deconvolved images of Kenyon cells in 15-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A-specific antibody (ATP5A, green) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( E - F ) Solidity ( E ) circularity ( F ) of ATP5A - stained mitochondria from fly brain gSTED deconvolved representatively shown in ( D ) from 8 - 10 brains of w1118 wild type ( Droj2 + / + ) knockdown ( Droj2 + / - ) 15 - day - old flies . Dot plots show data points along with mean ( bar ) ± s . d . . n = 8 - 10 . * , p < 0 . 05 . Unpaired , two - t - .

O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-SUBCELLULAR O B-ORGANISM B-TISSUE B-EXP_ASSAY O O O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O 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-F) Solidity (E) and circularity (F) of ATP5A-stained mitochondria from fly brain gSTED deconvolved images representatively shown in (D) from 8-10 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 15-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8-10. *, p < 0.05. Unpaired, two-tailed t-test.


SUMO conjugation assays performed under multiple turnover conditions using wild - type or mutant forms of RPA , SUMO Siz2 . Only 80 - minute time points are presented to highlight formation of Rfa1 - SUMO Siz2 - SUMO species . complete time series are presented in Appendix Fig S1 . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions using wild-type or mutant forms of RPA, SUMO and Siz2. Only the 80-minute time points are presented to highlight the formation of Rfa1-SUMO and Siz2-SUMO species. The complete time series are presented in Appendix Fig S1. Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions using Siz2 ∆ CT different RPA - complexes . SDS - PAGE gels were stained with Coomassie . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions using Siz2∆CT and different RPA-DNA complexes. SDS-PAGE gels were stained with Coomassie. Bands for Mw marker are fully annotated in Figure 1B.


Plot showing initial rate of SUMO conjugation versus RPA - as obtained from single - turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA - complexes . Representative gels are shown in Appendix Fig S4 .

O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O

Plot showing the initial rate of SUMO conjugation versus RPA-DNA concentration as obtained from single-turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA-DNA complexes. Representative gels are shown in Appendix Fig S4.


SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without WH domain of Rfa2 in presence of Siz2 ∆ CT lacking ( A ) or including ( B ) its SAP domain . In both cases , reactions were done in technical triplicate SDS - PAGE gels were stained with SYPRO . Only one representative gel is shown . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-GENEPROD O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2∆CT lacking (A) or including (B) its SAP domain. In both cases, reactions were done in technical triplicate and SDS-PAGE gels were stained with SYPRO. Only one representative gel is shown. Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 . Experiments were performed in technical triplicates ( six times for WT ) . structural models highlight position of mutations using a homology model of SAP domain of Siz2 obtained using structure of Siz1 ( PDB 2RNN ) as a template . A dashed box indicates relative position of residues that undergo chemical shift perturbation upon binding according to ( Suzuki et al , 2009 ) . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions as in Fig 4. Experiments were performed in technical triplicates (six times for WT). The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.


SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 using different Siz2 ∆ CT variants . structural models highlight position of mutations using a homology model of SAP domain of Siz2 obtained using structure of Siz1 ( PDB 2RNN ) as a template . A dashed box indicates relative position of residues that undergo chemical shift perturbation upon binding according to ( Suzuki et al , 2009 ) . Bands for Mw marker are fully annotated in Figure 1B .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2∆CT variants. The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.


Electrophoretic mobility shift assays ( EMSA ) to assess binding of RPA or SUMO - RPA to 25nM single - stranded 6 - FAM - labeled 32dT displacement of SUMO - RPA from 25nM SUMO - RPA bound to single - stranded 6 - FAM - labeled 32dT by RPA vice versa . titrations were done in technical triplicates . One representative gel is shown .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

Electrophoretic mobility shift assays (EMSA) to assess binding of RPA or SUMO-RPA to 25nM single-stranded 6-FAM-labeled 32dT DNA and displacement of SUMO-RPA from 25nM SUMO-RPA bound to single-stranded 6-FAM-labeled 32dT DNA by RPA and vice versa. All titrations were done in technical triplicates. One representative gel is shown.


Electrophoretic mobility shift assays ( EMSA ) performed with 10 nM 6 - FAM - ds20ss32 , a 6 - FAM - labeled 20 - mer dsDNA with a 32 nucleotide 3 ' overhang its complexes with RPA , RPA with K170 - SUMO modified Rfa1 or RPA reconstituted with ∆ WH - Rfa2 serially diluted Siz2 . titrations were done in technical triplicates . One representative gel is shown .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O

Electrophoretic mobility shift assays (EMSA) performed with 10 nM 6-FAM-ds20ss32, a 6-FAM-labeled 20-mer dsDNA with a 32 nucleotide 3' overhang and its complexes with RPA, RPA with K170-SUMO modified Rfa1 or RPA reconstituted with ∆WH-Rfa2 and serially diluted Siz2. All titrations were done in technical triplicates. One representative gel is shown.


A Representative 3 - color STED of synapses of cultured hippocampal neurons fixed stained for Synapsin1 / 2 ( presynaptic marker ) , PI ( 3 ) P Homer ( postsynaptic marker ) . Scale bar , 0 . 5 µm . PI ( 3 ) P - positive structures appear in both pre - postsynaptic compartments . B scan profiles of single synapses from in A , normalized to maximum value for each channel . Data information : Data presented as mean ± n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 of cultured hippocampal neurons fixed stained for VGlut1 , VGAT PI ( 3 ) P . Arrows indicate PI ( 3 ) P in VGlut1 + or VGAT + presynaptic terminals . Scale bar , 10 µm . D PI ( 3 ) P in VGlut1 + VGAT + presynaptic terminals . PI ( 3 ) P is equally abundant in excitatory inhibitory presynaptic terminals . N = 3 independent experiments ( ≥ 15 per condition ) ; Student ' s t - . Data information : Data presented as mean ± n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 in MAP2 + ( Soma ) Syp + ( Synapse ) areas in DMSO VPS34IN1 - treated neurons . N = 3 independent experiments ( ≥ 35 per condition ) ; Student ' s t - . Data information : Data presented as mean ± ; n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 stained for MAP2 , Synaptophysin ( Syp ) , PI ( 3 ) P . Arrows indicate PI ( 3 ) P in synapses . Scale bar , 10 µm . PI ( 3 ) P that do not overlap with neuronal soma synapses belong to astrocytes in culture are disregarded from .

O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE O O O O O O

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 of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) from in F . N = 5 independent experiments ; ( ≥ 40 per condition ) ; Student ' s t - . Data information : Data presented as mean ± n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 per condition ) ; Student ' s t - . Data information : Data presented as mean ± n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 stained for MAP2 , Syp PI ( 3 ) P . Arrows indicate PI ( 3 ) P in synapses . Scale bar , 10 µm .

O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR 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 of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) from in I . N ≥ 4 independent experiments ( ≥ 60 per condition ) ; Student ' s t - . Data information : Data presented as mean ± n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of PI ( 3 ) P in neuronal somata ( MAP2 + ) or synapses ( Syp + ) of cultured hippocampal neurons were treated with Roscovitine ( 10 µM ) for 1 h 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 per condition ) ; Two - way ANOVA ; Fisher ' s Least Significant Difference . Data information : Data presented as mean ± n . s . not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 VPS34 inhibitors , VPS34IN1 ( 3 µM ) or SAR405 ( 20 µM ) . Representative fEPSP traces ( above ) relative slope of fEPSPs ( below ) are shown . VPS34 inhibition causes reduced excitatory synaptic transmission down to 46 . 0 ± 4 . 1 % 55 . 0 ± 4 . 4 % for VPS34IN1 SAR405 at end of 60 - min treatment . N = 6 slices per condition from ≥ 4 mice ; Student ' s t - . Data information : Data presented as mean ± ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

O O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE O B-TISSUE O O O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O 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