Figure 4Gene expression analysis by quantitative polymerase chain reaction ( qPCR ) of TRF2 target genes following TRF2 overexpression or knockdown . Data information : data represent n = 3 independent experiments with mean ± SEMB , C Heatmap of H3K27ac ( B ) or H3K23me3 ( C ) peaks on the 4 kilobase ( Kb ) region around genomic ITSs ( ENCODE data for the K562 cell line ) . The mean intensity of the region is depicted in the histograms . ITSs are clustered depending of the peak mean intensity obtained from H3K27ac or H3K27me3 chromatin immunoprecipitation sequencing ( ChIP - seq ) of the 4 Kb region . ITSs detected by ChIP - seq using an anti - TRF2 antibody are shown on the left . D Specific qPCR for HS3SH4 , GPC6 , and VCAN ITSs after ChIP using an anti - H3K27ac antibody in TRF2 - overexpressing or - knockdown BJcl2 cells . Data represent fold enrichment of the H3K27ac level compared with the total H3 level . Data information : data represent n = 3 independent experiments with mean ±

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

Figure 4Gene expression analysis by quantitative polymerase chain reaction (qPCR) of TRF2 target genes following TRF2 overexpression or knockdown. Data information: data represent n = 3 independent experiments with mean ± SEMB, C Heatmap of H3K27ac (B) or H3K23me3 (C) peaks on the 4 kilobase (Kb) region around genomic ITSs (ENCODE data for the K562 cell line). The mean intensity of the region is depicted in the histograms. ITSs are clustered depending of the peak mean intensity obtained from H3K27ac or H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) of the 4 Kb region. ITSs detected by ChIP-seq using an anti-TRF2 antibody are shown on the left.D Specific qPCR for HS3SH4, GPC6, and VCAN ITSs after ChIP using an anti-H3K27ac antibody in TRF2-overexpressing or -knockdown BJcl2 cells. Data represent fold enrichment of the H3K27ac level compared with the total H3 level. Data information: data represent n = 3 independent experiments with mean ± SEM


Figure 5B - E Percentage of CD11bHi GR1Hi MDSCs ( B ) , CD3 - NKp46 + NK cells ( C ) , NKp46 + CD107a + NK cells ( D ) or CD69 + NK cells ( E ) Data information : data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45 + or NK cells ( n = 8 mice per group ; * p < 0 . 05 and * * * p < 0 . 001 ; Mann - Whitney test ) . Histograms of the pSTAT3 mean fluorescence intensity ( MFI ) of MSC2 cells when TRF2 was overexpressed and the target gene knocked down ( G ) Data information MFI of n = 3 independent experiment is normalized to controls and represented as fold change of MFI ( n = 3 independent experiments ; * p < 0 . 05 and * * p < 0 . 01 ; Student ' s t - test ) . Histograms of the pSTAT3 mean fluorescence intensity ( MFI ) of MSC2 cells when both TRF2 and its target gene were knocked down ( H ) . Data information : MFI of n = 3 independent experiment is normalized to controls and represented as fold change of MFI ( n = 3 independent experiments ; * p < 0 . 05 and * * p < 0 . 01 ; Student ' s t - test ) . I Histograms of tumor weights on day 19 after implantation of subcutaneous xenografts containing BJcl2 cells overexpressing or knocked down for HS3ST4 or GPC6 Data information : data are represented as mean + / - SEM ( n = 8 mice per group ; * p < 0 . 05 , and * * p < 0 . 01 ; Mann - Whitney test ) .

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

Figure 5B-E Percentage of CD11bHi GR1Hi MDSCs (B), CD3- NKp46+ NK cells (C), NKp46+ CD107a+ NK cells (D) or CD69+ NK cells (E) Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ or NK cells (n = 8 mice per group; *p < 0.05 and ***p < 0.001; Mann-Whitney test).Histograms of the pSTAT3 mean fluorescence intensity (MFI) of MSC2 cells when TRF2 was overexpressed and the target gene knocked down (G) Data information MFI of n=3 independent experiment is normalized to controls and represented as fold change of MFI (n = 3 independent experiments; *p < 0.05 and **p < 0.01; Student's t-test).Histograms of the pSTAT3 mean fluorescence intensity (MFI) of MSC2 cells when both TRF2 and its target gene were knocked down (H). Data information: MFI of n=3 independent experiment is normalized to controls and represented as fold change of MFI (n = 3 independent experiments; *p < 0.05 and **p < 0.01; Student's t-test).I Histograms of tumor weights on day 19 after implantation of subcutaneous xenografts containing BJcl2 cells overexpressing or knocked down for HS3ST4 or GPC6 Data information: data are represented as mean +/- SEM (n = 8 mice per group; *p < 0.05, and **p < 0.01; Mann-Whitney test).


Figure 6A Analysis of Heparan Sulfate ( HS ) expression by FACS using the 10E4 antibody . Data information histograms representative ( left panels ) and MFIs from three independent experiments ( * p < 0 . 05 and * * p < 0 . 01 ; Student ' s t - test ) are shown + / - SEM . B Atomic Force Microscopic ( AFM ) analysis of glycocalyx and cell stiffness and glycocalyx length . Data information : In ( B ) , Box plots Min to Max showing all points with median represent four independent experiments , each performed using at least 20 cells ( * p < 0 . 05 ; Mann - Whitney test ) . C AFM images showing the Modulus heatmap ( stiffness ) in GFP - expressing or TRF2 - overexpressing cancer cells . At each of the 128x128 force curve points , corresponding to an area of 10 μm2 , the AFM software automatically displays the Sneddon modulus analysis while scanning the sample . The color bar on the left of the images shows a qualitative representation of the elastic map . D Analysis of pSTAT3 levels in MDSCs after co - culture with GFP - expressing or TRF2 - overexpressing cancer cells treated with or without sodium chlorate ( NaClO3 ) . Data information : In ( D ) , data represents mean of n = 3 independent experiments + / - SEM ( * * * p < 0 . 001 ; Student ' s t - test ) .

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

Figure 6A Analysis of Heparan Sulfate (HS) expression by FACS using the 10E4 antibody. Data information histograms representative (left panels) and MFIs from three independent experiments (*p < 0.05 and **p < 0.01; Student's t-test) are shown +/- SEM.B Atomic Force Microscopic (AFM) analysis of glycocalyx and cell stiffness and glycocalyx length. Data information: In (B), Box plots Min to Max showing all points with median represent four independent experiments, each performed using at least 20 cells (*p< 0.05; Mann-Whitney test).C AFM images showing the Modulus heatmap (stiffness) in GFP-expressing or TRF2-overexpressing cancer cells. At each of the 128x128 force curve points, corresponding to an area of 10 μm2, the AFM software automatically displays the Sneddon modulus analysis while scanning the sample. The color bar on the left of the images shows a qualitative representation of the elastic map.D Analysis of pSTAT3 levels in MDSCs after co-culture with GFP-expressing or TRF2-overexpressing cancer cells treated with or without sodium chlorate (NaClO3). Data information: In (D), data represents mean of n = 3 independent experiments +/- SEM (***p < 0.001; Student's t-test).


Figure 7A Kaplan - Meier curves for overall survival of chemotherapy - treated patients according to the TRF2 expression level Data information : the optimal cut - off is determined on KMplot . The p value ( log Rank test ) , the Hazard Ratio and number of patients are indicated . Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg / kg 5 - FU . The tumor volume was determined every 2 days using a caliper , and growth curves over time ( C ) Data information : , data represent mean values + / - SEM ( n = 8 mice per group ; * p < 0 . 05 and * * p < 0 . 01 ; Mann - Whitney test ) . Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg / kg 5 - FU . tumor volume were determined on day 19 and represented using Box plots Min to Max showing all points with median ( D ) . Data information : ( D ) , data are represented by Box plots Min to Max showing all points with median ( n = 8 mice per group ; * p < 0 . 05 and * * p < 0 . 01 ; Mann - Whitney test ) Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg / kg 5 - FU . Overall survival was also determined ( E ) .

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

Figure 7A Kaplan-Meier curves for overall survival of chemotherapy-treated patients according to the TRF2 expression level Data information: the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. The tumor volume was determined every 2 days using a caliper, and growth curves over time (C) Data information: , data represent mean values +/- SEM (n = 8 mice per group; *p < 0.05 and **p < 0.01; Mann-Whitney test).Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. tumor volume were determined on day 19 and represented using Box plots Min to Max showing all points with median (D). Data information: (D), data are represented by Box plots Min to Max showing all points with median (n = 8 mice per group; *p < 0.05 and **p < 0.01; Mann-Whitney test)Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. Overall survival was also determined (E).


Figure 8A Kaplan - Meier curves for overall survival of patients at all stages of cancers ( breast , gastric , ovarian , and lung cancer ) depending on TRF2 expression level . Data information : For all panels , the optimal cut - off is determined on KMplot . The p value ( log Rank test ) , the Hazard Ratio and number of patients are indicated . The overall survival of gastric cancer patients ( all stages ) was analyzed depending on TRF2 and its targets genes expression level ( HS3ST4 , GPC6 and VCAN collectively named the HSPG genes ) Data information : For all panels , the optimal cut - off is determined on KMplot . The p value ( log Rank test ) , the Hazard Ratio and number of patients are indicated . The overall survival of gastric cancer patients ( all stages ) was analyzed depending on TRF2 and with MDSC level ( Lo or Hi ) , determine as the mean expression level of CD33 and C5AR , using KMplot Data information : For all panels , the optimal cut - off is determined on KMplot . The p value ( log Rank test ) , the Hazard Ratio and number of patients are indicated . E Repartition of number of gastric cancer patients depending on TRF2 and MDSC classification . Data information : For all panels , the optimal cut - off is determined on KMplot . The p value ( log Rank test ) , the Hazard Ratio and number of patients are indicated . F Overall survival of gastric cancer patients ( all stages ) analyzed depending on TRF2 , HSPG genes ( HS3ST4 , GPC6 and VCAN ) and MDSC level ( Lo or Hi ) . Data information : For all panels , the optimal cut - off is determined on KMplot . The p value ( log Rank test ) , the Hazard Ratio and number of patients are indicated .

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

Figure 8A Kaplan-Meier curves for overall survival of patients at all stages of cancers (breast, gastric, ovarian, and lung cancer) depending on TRF2 expression level. Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.The overall survival of gastric cancer patients (all stages) was analyzed depending on TRF2 and its targets genes expression level (HS3ST4, GPC6 and VCAN collectively named the HSPG genes) Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.The overall survival of gastric cancer patients (all stages) was analyzed depending on TRF2 and with MDSC level (Lo or Hi), determine as the mean expression level of CD33 and C5AR, using KMplot Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.E Repartition of number of gastric cancer patients depending on TRF2 and MDSC classification. Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.F Overall survival of gastric cancer patients (all stages) analyzed depending on TRF2, HSPG genes (HS3ST4, GPC6 and VCAN) and MDSC level (Lo or Hi). Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.


Figure 1 . Testing an assay for SARS - CoV - 2 using synthetic RNA targets . We show data from both fluorescent ( a ) and lateral flow detection ( b ) . Target concentrations ( in cp / μl ) are indicated . RPA NTC : water input into RPA ; Det NTC : water input into Cas13 detection reaction . In ( a ) , error bars indicate one standard deviation based on n = 3 technical replicates .

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

Figure 1.Testing an assay for SARS-CoV-2 using synthetic RNA targets. We show data from both fluorescent (a) and lateral flow detection (b). Target concentrations (in cp/μl) are indicated. RPA NTC: water input into RPA; Det NTC: water input into Cas13 detection reaction. In (a), error bars indicate one standard deviation based on n=3 technical replicates.


Figure 1mRNA expression of ANG in colonic tissues from normal controls ( n = 25 ) and UC ( n = 27 ) or CD ( n = 46 ) patients . Representative western blotting of ANG in colonic tissues from normal controls and UC or CD patients . Representative images showing ANG immunohistochemical ( IHC ) staining in normal control and UC or CD sample . Corresponding statistical analysis of ANG expression score in normal controls ( n = 10 ) and UC ( n = 10 ) or CD ( n = 15 ) patients . Representative images showing hematoxylin - eosin ( HE ) staining and ANG IHC staining in mild and severe IBD samples . Statistical analyses of ANG expression scores in mild ( n = 18 ) and severe ( n = 19 ) UC ( F ) or mild ( n = 20 ) and severe ( n = 20 ) CD ( G ) patients .

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

Figure 1mRNA expression of ANG in colonic tissues from normal controls (n = 25) and UC (n = 27) or CD (n = 46) patients.Representative western blotting of ANG in colonic tissues from normal controls and UC or CD patients.Representative images showing ANG immunohistochemical (IHC) staining in normal control and UC or CD sample.Corresponding statistical analysis of ANG expression score in normal controls (n = 10) and UC (n = 10) or CD (n = 15) patients.Representative images showing hematoxylin-eosin (HE) staining and ANG IHC staining in mild and severe IBD samples.Statistical analyses of ANG expression scores in mild (n = 18) and severe (n = 19) UC (F) or mild (n = 20) and severe (n = 20) CD (G) patients.


Figure 2Ang mRNA expression in colonic tissue from 2 . 5 % DSS - treated WT mice at indicated time point ( n = 4 mice / time point ) . DSS : dextran sulfate sodiumKaplan - Meier curve of 3 . 5 % DSS - treated Ang - deficient mice ( Ang - / - , n = 14 ) or littermate controls ( WT , n = 13 ) . Body weight loss of WT and Ang - / - mice with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment . disease activity index ( DAI ) of WT and Ang - / - mice with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment . Colon length of colonic section from the WT and Ang - / - mice on day 9 with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment . serum FITC - Dextran level of colonic section from the WT and Ang - / - mice on day 9 with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment . FITC - Dextran : fluorescein isothiocyanate - labeled dextran . representative HE staining image , and ( H ) histopathological score of colonic section from the WT and Ang - / - mice on day 9 with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment . Quantitative mRNA expression of cytokine genes in colonic tissue from the WT and Ang - / - mice on day 0 ( n = 3 ) or day 9 ( n = 9 ) during 2 . 5 % DSS treatment . Soluble cytokine level in supernatant of cultured colonic tissue isolated from WT and Ang - / - mice on day 0 ( n = 3 ) or day 9 ( n = 7 ) during 2 . 5 % DSS treatment .

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

Figure 2Ang mRNA expression in colonic tissue from 2.5% DSS-treated WT mice at indicated time point (n = 4 mice/time point). DSS: dextran sulfate sodiumKaplan-Meier curve of 3.5% DSS-treated Ang-deficient mice (Ang-/-, n = 14) or littermate controls (WT, n = 13).Body weight loss of WT and Ang-/- mice with (n = 9) or without (n = 6) 2.5% DSS treatment.disease activity index (DAI) of WT and Ang-/- mice with (n = 9) or without (n = 6) 2.5% DSS treatment.Colon length of colonic section from the WT and Ang-/- mice on day 9 with (n = 9) or without (n = 6) 2.5% DSS treatment.serum FITC-Dextran level of colonic section from the WT and Ang-/- mice on day 9 with (n = 9) or without (n = 6) 2.5% DSS treatment. FITC-Dextran: fluorescein isothiocyanate-labeled dextran.representative HE staining image, and (H) histopathological score of colonic section from the WT and Ang-/- mice on day 9 with (n = 9) or without (n = 6) 2.5% DSS treatment.Quantitative mRNA expression of cytokine genes in colonic tissue from the WT and Ang-/- mice on day 0 (n = 3) or day 9 (n = 9) during 2.5% DSS treatment.Soluble cytokine level in supernatant of cultured colonic tissue isolated from WT and Ang-/- mice on day 0 (n = 3) or day 9 (n = 7) during 2.5% DSS treatment.


Figure 3Body weight loss of WT → WT , Ang - / - → WT and WT → Ang - / - chimera mice with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . DAI of WT → WT , Ang - / - → WT and WT → Ang - / - chimera mice with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . Colon length of colonic section from these chimera colitis mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . serum FITC - Dextran level , of colonic section from these chimera colitis mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . representative HE staining image , and ( F ) histopathological score of colonic section from these chimera colitis mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . Ang mRNA expression in mouse lymphoid and myeloid cells ; data represent two independent experiments ( n = 2 ) . Body weight loss of Angfl / fl and LyzMcre ; Angfl / fl mice with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment . DAI of Angfl / fl and LyzMcre ; Angfl / fl mice with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment . Colon length of colonic section from the Angfl / fl and LyzMcre ; Angfl / fl mice on day 8 with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment . serum FITC - Dextran level , of colonic section from the Angfl / fl and LyzMcre ; Angfl / fl mice on day 8 with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment . representative HE staining image , and ( M ) histopathological score of colonic section from the Angfl / fl and LyzMcre ; Angfl / fl mice on day 8 with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment .

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

Figure 3Body weight loss of WT→WT, Ang-/-→WT and WT→Ang-/- chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment.DAI of WT→WT, Ang-/-→WT and WT→Ang-/- chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment.Colon length of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.serum FITC-Dextran level, of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.representative HE staining image, and (F) histopathological score of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.Ang mRNA expression in mouse lymphoid and myeloid cells; data represent two independent experiments (n = 2).Body weight loss of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment.DAI of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment.Colon length of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment.serum FITC-Dextran level, of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment.representative HE staining image, and (M) histopathological score of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment.


Figure 4Quantitative mRNA expression of ANG and PLXNB2 in isolated IECs or colonic laminar propria ( cLP ) cells from WT mice ( n = 9 ) . western blotting of ANG and PLXNB2 in isolated IECs or colonic laminar propria ( cLP ) cells from WT mice ( n = 9 ) . Representative images showing immunofluorescence staining of ANG , PLXNB2 and macrophage surface marker F4 / 80 in frozen colonic section from WT mice . Representative images showing immunofluorescence staining of GFP and PLXNB2 in frozen colonic section from WT mice infected with adeno - associated virus ( AAV9 ) . PLXNB2 expression in IECs from the AAV9 - infected WT mice ( n = 3 ) . Body weight loss of the AA9 - infected WT mice with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . DAI of the AA9 - infected WT mice with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . Colon length of colonic section from the AAV9 - infected WT mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . serum FITC - Dextran level of colonic section from the AAV9 - infected WT mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment . representative HE staining image , and ( K ) histopathological score of colonic section from the AAV9 - infected WT mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

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

Figure 4Quantitative mRNA expression of ANG and PLXNB2 in isolated IECs or colonic laminar propria (cLP) cells from WT mice (n = 9).western blotting of ANG and PLXNB2 in isolated IECs or colonic laminar propria (cLP) cells from WT mice (n = 9).Representative images showing immunofluorescence staining of ANG, PLXNB2 and macrophage surface marker F4/80 in frozen colonic section from WT mice.Representative images showing immunofluorescence staining of GFP and PLXNB2 in frozen colonic section from WT mice infected with adeno-associated virus (AAV9).PLXNB2 expression in IECs from the AAV9-infected WT mice (n = 3).Body weight loss of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment.DAI of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment.Colon length of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.serum FITC-Dextran level of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.representative HE staining image, and (K) histopathological score of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.


Figure 5Representative images showing cleaved - caspase 3 staining in colonic section from WT or Ang - / - colitis mice ( n = 3 ) at indicated time point ; arrows indicate cleaved - caspase 3 positive cells ; two fields per colon section are quantified . the number of apoptotic cells in colonic section from WT or Ang - / - colitis mice ( n = 3 ) at indicated time pointCell apoptotic status of IECs from WT or Ang - / - colitis mice ( n = 5 ) on day 4 . Cell apoptotic status of IECs from WT or Ang - / - colitis mice ( n = 5 ) on day 4 . Representative images showing Ki67 staining in colonic section from WT or Ang - / - colitis mice ( n = 3 ) at indicated time point ; five representative crypts per colon section are quantified . the number of proliferating cells per crypt in colonic section from WT or Ang - / - colitis mice ( n = 3 ) at indicated time point ; five representative crypts per colon section are quantified . Cell - cycle status of IECs from WT or Ang - / - colitis mice ( n = 5 ) on day 4 . Representative images showing cleaved - caspase 3 staining in colonic section from Angfl / fl and LyzMCre ; Angfl / fl , or AAV - shControl - and AAV - shPlxnb2 - treated colitis mice on day 4 ( n = 3 ) ; arrows indicate cleaved - caspase 3 positive cells ; two fields per colon section are quantified . the number of apoptotic cells in colonic section from Angfl / fl and LyzMCre ; Angfl / fl , or AAV - shControl - and AAV - shPlxnb2 - treated colitis mice on day 4 ( n = 3 ) Ki67 staining and ( L ) the number of proliferating cells per crypt in representative colonic section from Angfl / fl and LyzMCre ; Angfl / fl , or AAV - shControl - and AAV - shPlxnb2 - treated colitis mice on day 4 ( n = 3 ) ; five representative crypts per colon section are quantified .

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

Figure 5Representative images showing cleaved-caspase 3 staining in colonic section from WT or Ang-/- colitis mice (n = 3) at indicated time point; arrows indicate cleaved-caspase 3 positive cells; two fields per colon section are quantified.the number of apoptotic cells in colonic section from WT or Ang-/- colitis mice (n = 3) at indicated time pointCell apoptotic status of IECs from WT or Ang-/- colitis mice (n = 5) on day 4.Cell apoptotic status of IECs from WT or Ang-/- colitis mice (n = 5) on day 4.Representative images showing Ki67 staining in colonic section from WT or Ang-/- colitis mice (n = 3) at indicated time point; five representative crypts per colon section are quantified.the number of proliferating cells per crypt in colonic section from WT or Ang-/- colitis mice (n = 3) at indicated time point; five representative crypts per colon section are quantified.Cell-cycle status of IECs from WT or Ang-/- colitis mice (n = 5) on day 4.Representative images showing cleaved-caspase 3 staining in colonic section from Angfl/fl and LyzMCre;Angfl/fl, or AAV-shControl- and AAV-shPlxnb2- treated colitis mice on day 4 (n = 3); arrows indicate cleaved-caspase 3 positive cells; two fields per colon section are quantified.the number of apoptotic cells in colonic section from Angfl/fl and LyzMCre;Angfl/fl, or AAV-shControl- and AAV-shPlxnb2- treated colitis mice on day 4 (n = 3)Ki67 staining and (L) the number of proliferating cells per crypt in representative colonic section from Angfl/fl and LyzMCre;Angfl/fl, or AAV-shControl- and AAV-shPlxnb2- treated colitis mice on day 4 (n = 3); five representative crypts per colon section are quantified.


Figure 6PLXNB2 expressions in HCT116 ( including wild type HCT116 ( HCT116WT ) and ANG knockout HCT116 ( HCT116KO ) ) and THP - 1 as detected by flow cytometry analysis . The upper three dash lines represent the intensity of unstained negative control of these cells , while the solid lines represent the intensity of PLXNB2 antibody staining . Secreted ANG level in conditioned medium from THP1 , HCT116WT or HCT116KO ( n = 3 ) . The interaction between ANGWT , ANGR66A or ANGK40I with PLXNB2 in HCT116KO detected by co - immunoprecipitation assay . ANGR66A , ANG receptor binding site variant ; ANGK40I , ANG enzymatic activity null variantTHP - 1 - derived ANG translocating into HCT116KO through PLXNB2 in co - cultured system as showed by western blotting . Neamine , a blocker of ANG and PLXNB2 interaction ; siCtrl : control siRNA ; siPLXNB2 - 1 and siPLXNB2 - 2Flow cytometry analysis of HCT116KO stained with Annexin V and PI following different treatments as indicated . ANGR66A , ANG receptor binding site variant ; ANGK40I , ANG enzymatic activity null variant ; BAY , inhibitor of TNF - α - induced κB kinase ( IKK ) phosphorylationtiRNA and 5 ' - tiRNAAla production in HCT116KO following different treatments as indicated . ANGR66A , ANG receptor binding site variant ; ANGK40I , ANG enzymatic activity null variant ; BAY , inhibitor of TNF - α - induced κB kinase ( IKK ) phosphorylationThe effect of 5 ' - tiRNA knockdown or overexpression on TNF - α - induced apoptosis in ANGWT - or ANGK40I - treated HCT116KO . si5 ' - tiRNA : tiRNA - targeting siRNAThe interaction between 5 ' - tiRNA and cytochrome c ( Cyt c ) in HCT116 cells as detected by RIP . Caspase activity of HCT116KO following different treatments as indicated . ANGR66A , ANG receptor binding site variant ; ANGK40I , ANG enzymatic activity null variant ; BAY , inhibitor of TNF - α - induced κB kinase ( IKK ) phosphorylationCell viability of HCT116KO following different treatments as indicated . siCtrl : control siRNA ; siPLXNB2 - 1 and siPLXNB2 - 2 : PLXNB2 siRNAsDetection of 47S pre - rRNA transcription level with northern blotting in HCT116KO following different treatments as indicated . siPLXNB2 - 1 and siPLXNB2 - 2 : PLXNB2 siRNAstiRNA and 5 ' - tiRNAAla production in IECs isolated from WT and Ang - / - colitis mice at indicated time point . 47S pre - rRNA transcription level in IECs from WT and Ang - / - colitis mice at indicated time point .

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

Figure 6PLXNB2 expressions in HCT116 (including wild type HCT116 (HCT116WT) and ANG knockout HCT116 (HCT116KO)) and THP-1 as detected by flow cytometry analysis. The upper three dash lines represent the intensity of unstained negative control of these cells, while the solid lines represent the intensity of PLXNB2 antibody staining.Secreted ANG level in conditioned medium from THP1, HCT116WT or HCT116KO (n = 3).The interaction between ANGWT, ANGR66A or ANGK40I with PLXNB2 in HCT116KO detected by co-immunoprecipitation assay. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variantTHP-1-derived ANG translocating into HCT116KO through PLXNB2 in co-cultured system as showed by western blotting. Neamine, a blocker of ANG and PLXNB2 interaction; siCtrl: control siRNA; siPLXNB2-1 and siPLXNB2-2Flow cytometry analysis of HCT116KO stained with Annexin V and PI following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylationtiRNA and 5'-tiRNAAla production in HCT116KO following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylationThe effect of 5'-tiRNA knockdown or overexpression on TNF-α-induced apoptosis in ANGWT- or ANGK40I-treated HCT116KO. si5'-tiRNA: tiRNA-targeting siRNAThe interaction between 5'-tiRNA and cytochrome c (Cyt c) in HCT116 cells as detected by RIP.Caspase activity of HCT116KO following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylationCell viability of HCT116KO following different treatments as indicated. siCtrl: control siRNA; siPLXNB2-1 and siPLXNB2-2: PLXNB2 siRNAsDetection of 47S pre-rRNA transcription level with northern blotting in HCT116KO following different treatments as indicated. siPLXNB2-1 and siPLXNB2-2: PLXNB2 siRNAstiRNA and 5'-tiRNAAla production in IECs isolated from WT and Ang-/- colitis mice at indicated time point.47S pre-rRNA transcription level in IECs from WT and Ang-/- colitis mice at indicated time point.


Figure 7Western blotting of ANG in colonic tissue from WT mice ( n = 2 ) at indicated time point in the ANG prevention model . The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment ( untreated ) is arbitrarily set as 1 . Body weight loss of the pre - treated WT colitis mice ( n = 8 ) . DAI of the pre - treated WT colitis mice ( n = 8 ) . Colon length of colon section from the pre - treated WT colitis mice on day 8 ( n = 8 ) . serum FITC - dextran level of colon section from the pre - treated WT colitis mice on day 8 ( n = 8 ) . representative HE staining image , and ( H ) histopathological score of colon section from the pre - treated WT colitis mice on day 8 ( n = 8 ) . Western blotting of ANG in colonic tissue from WT ( n = 2 ) mice at indicated time point in the ANG treatment model . The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment ( untreated ) is arbitrarily set as 1 . Body weight loss of the treated WT colitis mice ( n = 8 ) . DAI of the treated WT colitis mice ( n = 8 ) . Colon length of colon sections from the treated WT colitis mice on day 8 ( n = 8 ) . serum FITC - dextran level of colon sections from the treated WT colitis mice on day 8 ( n = 8 ) . representative HE staining image , and ( P ) histopathological score of colon sections from the treated WT colitis mice on day 8 ( n = 8 ) .

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

Figure 7Western blotting of ANG in colonic tissue from WT mice (n = 2) at indicated time point in the ANG prevention model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1.Body weight loss of the pre-treated WT colitis mice (n = 8).DAI of the pre-treated WT colitis mice (n = 8).Colon length of colon section from the pre-treated WT colitis mice on day 8 (n = 8).serum FITC-dextran level of colon section from the pre-treated WT colitis mice on day 8 (n = 8).representative HE staining image, and (H) histopathological score of colon section from the pre-treated WT colitis mice on day 8 (n = 8).Western blotting of ANG in colonic tissue from WT (n = 2) mice at indicated time point in the ANG treatment model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1.Body weight loss of the treated WT colitis mice (n = 8).DAI of the treated WT colitis mice (n = 8).Colon length of colon sections from the treated WT colitis mice on day 8 (n = 8).serum FITC-dextran level of colon sections from the treated WT colitis mice on day 8 (n = 8).representative HE staining image, and (P) histopathological score of colon sections from the treated WT colitis mice on day 8 (n = 8).


Figure 1B . Top : representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 as indexes of mTOR activity in PFC of adult WT mice are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected WT mice . They are the means ± S . E . M . of results obtained in five mice per group . * p < 0 . 05 ; * * p < 0 . 01 , one - way ANOVA followed by Newman - Keuls test . C . Top : schemas illustrating the behavioral tasks examined in WT mice . Bottom : the plots represent the discrimination index for the novel object recognition task ( Vehicle N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) , the sociability index ( Vehicle : N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) and the discrimination index for the social discrimination task ( N = 8 for each group ) , measured in each condition . * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA followed by Bonferroni test ( error bars correspond to the mean ± SEM , the dotted line to a discrimination index ( exploration of novel object - exploration of familiar object / total object exploration ) equal to zero . D . Top : representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5 - HT6 - / - mice are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected 5 - HT6 - / - mice . They are the means ± S . E . M . of results obtained in four mice per group . n . s . p > 0 . 05 , one - way ANOVA followed by Newman - Keuls test . E . Top : schemas illustrating the behavioral tasks in 5 - HT6 - / - mice . Bottom : the plots represent the discrimination index for the novel object recognition test ( discrimination index : 0 . 30 ± 0 . 05 , N = 10 and 0 . 31 ± 0 . 03 , N = 11 , for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , the 3 - chamber social preference test ( sociability index : 0 . 49 ± 0 . 04 , N = 10 and 0 . 48 ± 0 . 07 , N = 11 for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) and the social discrimination test ( discrimination index : 0 . 15 ± 0 . 06 , N = 8 and 0 . 21 ± 0 . 06 , N = 8 for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , measured in each condition . One - way ANOVA followed by Bonferroni test ( error bars correspond to the mean ± SEM , the dotted line corresponds to a discrimination index equal to zero ) .

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

Figure 1B. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 as indexes of mTOR activity in PFC of adult WT mice are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected WT mice. They are the means ± S.E.M. of results obtained in five mice per group. * p < 0.05; ** p < 0.01, one-way ANOVA followed by Newman-Keuls test.C. Top: schemas illustrating the behavioral tasks examined in WT mice. Bottom: the plots represent the discrimination index for the novel object recognition task (Vehicle N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12), the sociability index (Vehicle: N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12) and the discrimination index for the social discrimination task (N=8 for each group), measured in each condition. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line to a discrimination index (exploration of novel object - exploration of familiar object/total object exploration) equal to zero.D. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5-HT6-/- mice are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected 5-HT6-/- mice. They are the means ± S.E.M. of results obtained in four mice per group. n.s. p > 0.05, one-way ANOVA followed by Newman-Keuls test.E. Top: schemas illustrating the behavioral tasks in 5-HT6-/- mice. Bottom: the plots represent the discrimination index for the novel object recognition test (discrimination index: 0.30 ± 0.05, N=10 and 0.31 ± 0.03, N=11, for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), the 3-chamber social preference test (sociability index: 0.49 ± 0.04, N=10 and 0.48 ± 0.07, N=11 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05) and the social discrimination test (discrimination index: 0.15 ± 0.06, N=8 and 0.21 ± 0.06, N=8 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), measured in each condition. One-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line corresponds to a discrimination index equal to zero).


Figure 2A - E . The PFC was stained with CB1 receptor ( A1 - D1 , green ) or 5HT6 receptor ( E1 - H1 , green ) , Bassoon ( A2 and E2 , red ) , GAD65 ( B2 and F2 , red ) , PSD - 95 ( C2 and G2 , red ) , or SERT ( D2 and H2 , red ) antibodies . Merge images are also depicted ( A3 - H3 ) . CB1 receptors were mainly co - localized with the presynaptic marker Bassoon ( white arrows , A3 ) , mostly within GABAergic boutons ( white arrows , B3 ) and to a lesser extend with PSD - 95 ( white arrows , C3 ) and SERT ( D3 ) , while 5 - HT6 receptors were mainly co - localized with PSD - 95 ( white arrows , G3 ) . No co - localization of CB1 and 5 - HT6 receptors was detected ( white arrows indicate the 5 - HT6 staining , H3 ) . Scale bar : 10 μm . The scatter plot represents the co - localization analysis for CB1 and 5 - HT6 receptor immunostainings . Graph on the top , Mander ' s split coefficient was used to identify the fraction of CB1 receptors that co - localizes with Bassoon ( Mander ' s coefficient : 0 . 66 ± 0 . 02 , n = 4 ) , GAD65 ( Mander ' s coefficient : 0 . 56 ± 0 . 01 , n = 4 ) , PSD95 ( Mander ' s coefficient : 0 . 15 ± 0 . 01 , n = 4 ) or SERT ( Mander ' s coefficient : 0 . 082 ± 0 . 004 , n = 4 ) . Graph on the bottom . Mander ' s split coefficient was used to identify the fraction of 5 - HT6 receptors that co - localizes with Bassoon ( Mander ' s coefficient : 0 . 09 ± 0 . 01 , n = 4 ) , GAD65 ( Mander ' s coefficient : 0 . 059 ± 0 . 004 , n = 4 ) , PSD95 ( Mander ' s coefficient : 0 . 58 ± 0 . 01 , n = 4 ) or CB1 receptors ( Mander ' s coefficient : 0 . 0020 ± 0 . 0004 , n = 4 ) . Error bars correspond to the mean ± SEM .

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

Figure 2A-E. The PFC was stained with CB1 receptor (A1-D1, green) or 5HT6 receptor (E1-H1, green), Bassoon (A2 and E2, red), GAD65 (B2 and F2, red), PSD-95 (C2 and G2, red), or SERT (D2 and H2, red) antibodies. Merge images are also depicted (A3-H3). CB1 receptors were mainly co-localized with the presynaptic marker Bassoon (white arrows, A3), mostly within GABAergic boutons (white arrows, B3) and to a lesser extend with PSD-95 (white arrows, C3) and SERT (D3), while 5-HT6 receptors were mainly co-localized with PSD-95 (white arrows, G3). No co-localization of CB1 and 5-HT6 receptors was detected (white arrows indicate the 5-HT6 staining, H3). Scale bar: 10 μm. The scatter plot represents the co-localization analysis for CB1 and 5-HT6 receptor immunostainings. Graph on the top, Mander's split coefficient was used to identify the fraction of CB1 receptors that co-localizes with Bassoon (Mander's coefficient: 0.66 ± 0.02, n=4), GAD65 (Mander's coefficient: 0.56 ± 0.01, n=4), PSD95 (Mander's coefficient: 0.15 ± 0.01, n=4) or SERT (Mander's coefficient: 0.082 ± 0.004, n=4). Graph on the bottom. Mander's split coefficient was used to identify the fraction of 5-HT6 receptors that co-localizes with Bassoon (Mander's coefficient: 0.09 ± 0.01, n=4), GAD65 (Mander's coefficient: 0.059 ± 0.004, n=4), PSD95 (Mander's coefficient: 0.58 ± 0.01, n=4) or CB1 receptors (Mander's coefficient: 0.0020 ± 0.0004, n=4). Error bars correspond to the mean ± SEM.


Figure 3B . Top : representative Western blots assessing mTOR activity in PFC are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected mice . They are the means ± S . E . M . of results obtained in six mice per group . * p < 0 . 05 ; * * p < 0 . 01 , one - way ANOVA followed by Newman - Keuls test . C . The plots represent the discrimination index measured in each condition . * * * p < 0 . 001 , one - way ANOVA followed by Bonferroni test . The discrimination index for the novel object recognition task is 0 . 34 ± 0 . 06 ( N = 14 ) and - 0 . 08 ± 0 . 07 ( N = 20 ) , for mice injected with Vehicle and THC , respectively , p < 0 . 001 and - 0 . 09 ± 0 . 08 ( N = 21 ) and 0 . 00 ± 0 . 06 ( N = 18 ) , for mice treated with THC + SB258285 and THC + Rapa , respectively , p > 0 . 05 vs . THC - injected mice ( error bars correspond to the mean ± SEM , the dotted line to a discrimination index equal to zero ) .

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

Figure 3B. Top: representative Western blots assessing mTOR activity in PFC are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected mice. They are the means ± S.E.M. of results obtained in six mice per group. * p < 0.05; ** p < 0.01, one-way ANOVA followed by Newman-Keuls test.C. The plots represent the discrimination index measured in each condition. *** p < 0.001, one-way ANOVA followed by Bonferroni test. The discrimination index for the novel object recognition task is 0.34 ± 0.06 (N=14) and -0.08 ± 0.07 (N= 20), for mice injected with Vehicle and THC, respectively, p < 0.001 and -0.09 ± 0.08 (N=21) and 0.00 ± 0.06 (N=18), for mice treated with THC+SB258285 and THC+Rapa, respectively, p > 0.05 vs. THC-injected mice (error bars correspond to the mean ± SEM, the dotted line to a discrimination index equal to zero).


Figure 4B . Left : representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated . Right : the histograms represent means ± S . E . M . of GABA mIPSC frequency and amplitude measured during the last minute of recording . The mIPSC frequency is : 3 . 2 ± 0 . 4 Hz and 1 . 3 ± 0 . 2 Hz for Vehicle ( n = 9 from N = 4 ) and THC ( n = 7 from N = 4 ) conditions , respectively , p < 0 . 001 and 2 . 5 ± 0 . 2 Hz and 4 . 1 ± 0 . 4 Hz for THC + SB258585 ( n = 8 from N = 4 ) and THC + Rapa ( n = 8 from N = 4 ) conditions , respectively , p < 0 . 01 and p < 0 . 001 vs . THC - injected mice . C . Left : representative traces of AMPA mEPSCs recorded in layer V pyramidal neurons are illustrated . Right : the histograms represent means ± S . E . M . of AMPA mEPSC frequency and amplitude measured during the last minute of recording , n = neurons from 3 to 5 mice per group . The mEPSC frequency is : 4 . 7 ± 0 . 4 Hz for Vehicle ( n = 9 from N = 7 ) and 6 . 8 ± 0 . 4 Hz for THC ( n = 9 from N = 5 ) conditions , respectively , p < 0 . 01and 5 . 3 ± 0 . 5 Hz and 4 . 6 ± 0 , 3 Hz for THC + SB258585 ( n = 10 from N = 6 ) and THC + Rapa ( n = 9 from N = 5 ) conditions , respectively , p < 0 . 05 and p < 0 . 01 vs . THC - injected mice . In B and C , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , one - way ANOVA followed by Newman - Keuls test .

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

Figure 4B. Left: representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of GABA mIPSC frequency and amplitude measured during the last minute of recording. The mIPSC frequency is: 3.2 ± 0.4 Hz and 1.3 ± 0.2 Hz for Vehicle (n=9 from N= 4) and THC (n=7 from N= 4) conditions, respectively, p < 0.001 and 2.5 ± 0.2 Hz and 4.1 ± 0.4 Hz for THC+SB258585 (n=8 from N= 4) and THC+Rapa (n=8 from N= 4) conditions, respectively, p < 0.01 and p < 0.001 vs. THC-injected mice.C. Left: representative traces of AMPA mEPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of AMPA mEPSC frequency and amplitude measured during the last minute of recording, n = neurons from 3 to 5 mice per group. The mEPSC frequency is: 4.7 ± 0.4 Hz for Vehicle (n=9 from N= 7) and 6.8 ± 0.4 Hz for THC (n=9 from N= 5) conditions, respectively, p < 0.01and 5.3 ± 0.5 Hz and 4.6 ± 0,3 Hz for THC+SB258585 (n=10 from N= 6) and THC+Rapa (n=9 from N= 5) conditions, respectively, p < 0.05 and p < 0.01 vs. THC-injected mice. In B and C, * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA followed by Newman-Keuls test.


Figure 5A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . A . The resting membrane potential ( RMP ) was determined immediately after whole - cell formation : - 71 . 8 ± 1 . 2 mV and - 60 . 1 ± 1 . 5 mV for Vehicle and THC conditions , respectively , p < 0 . 001 . A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . B . Action potentials were evoked by a ramp current injection with a 10 pA - step for 2 ms and only the first action potential ( AP ) were used to estimate the AP threshold : - 34 . 1 ± 0 . 7 mV and - 39 . 7 ± 0 . 8 mV for Vehicle - and THC - injected mice , respectively , p < 0 . 001 . A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . C . The rheobase represents the minimal current required to induce neuronal firing , it was lowered in THC condition : 670 ± 43 pA and 376 ± 43 pA for Vehicle and THC conditions , respectively , p < 0 . 001 . A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . D . The firing rate induced by a 150 pA current injection during 250 ms is expressed as the number of APs . E . top : Voltage sag in response to hyperpolarizing current injection ( 50 pA - increments , from - 400 to 0 pA ) in PFC pyramidal neurons from mice injected with vehicle ( Veh , n = 10 from N = 5 ) , THC ( n = 11 from N = 5 ) , THC + SB258585 ( THC / SB , n = 7 from N = 4 ) or THC + Rapa ( THC / Rapa , n = 7 from N = 4 ) during adolescence . * * * p < 0 . 001 , one - way ANOVA followed by Bonferroni test , vs . THC - injected mice . Errors bars correspond to the mean ± SEM . Voltage sag is indicated by arrowheads . F - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( F ) resting potential membrane : THC + ZD : - 74 . 14 ± 1 . 70 mV , p < 0 . 001 vs . THC condition , THC + DDOA : - 70 . 47 ± 1 . 19 mV , p < 0 . 001 vs . THC condition , THC + ODQ : - 63 . 10 ± 0 . 81 mV , p < 0 . 001 vs . THC condition , F - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( G ) AP threshold : THC + ZD : - 33 . 59 ± 2 . 96 mV , p < 0 . 01 vs . THC condition , THC + DDOA : - 34 . 28 ± 0 . 98mV , p < 0 . 001 vs . THC condition , THC + ODQ : - 36 . 84 ± 0 . 79 mV , p < 0 . 001 vs . THC conditionF - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( H ) rheobase : THC + ZD : 806 ± 63 pA , p < 0 . 001 vs . THC condition , THC + DDOA : 607 ± 43 pA , p < 0 . 001 vs . THC condition , THC + ODQ : 422 ± 34 pA , p < 0 . 001 vs . THC conditionF - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( I ) firing rate . Note that Vehicle - and THC - injected mice are the same as those used in experiments illustrated on Figure 5A - D . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , n . s . p > 0 . 05 one - way ANOVA followed by Bonferroni test .

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

Figure 5A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. A. The resting membrane potential (RMP) was determined immediately after whole-cell formation: -71.8 ± 1.2 mV and -60.1 ± 1.5 mV for Vehicle and THC conditions, respectively, p < 0.001.A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. B. Action potentials were evoked by a ramp current injection with a 10 pA-step for 2 ms and only the first action potential (AP) were used to estimate the AP threshold: -34.1 ± 0.7 mV and -39.7 ± 0.8 mV for Vehicle- and THC-injected mice, respectively, p < 0.001.A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. C. The rheobase represents the minimal current required to induce neuronal firing, it was lowered in THC condition: 670 ± 43 pA and 376 ± 43 pA for Vehicle and THC conditions, respectively, p < 0.001.A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. D. The firing rate induced by a 150 pA current injection during 250 ms is expressed as the number of APs.E. top: Voltage sag in response to hyperpolarizing current injection (50 pA-increments, from -400 to 0 pA) in PFC pyramidal neurons from mice injected with vehicle (Veh, n= 10 from N=5), THC (n = 11 from N=5), THC+SB258585 (THC/SB, n=7 from N=4) or THC+Rapa (THC/Rapa, n=7 from N=4) during adolescence. *** p < 0.001, one-way ANOVA followed by Bonferroni test, vs. THC-injected mice. Errors bars correspond to the mean ± SEM. Voltage sag is indicated by arrowheads.F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (F) resting potential membrane: THC + ZD: -74.14 ± 1.70 mV, p < 0.001 vs. THC condition, THC + DDOA: -70.47 ± 1.19 mV, p < 0.001 vs. THC condition, THC + ODQ: -63.10 ± 0.81 mV, p < 0.001 vs. THC condition,F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (G) AP threshold: THC + ZD: -33.59 ± 2.96 mV, p < 0.01 vs. THC condition, THC + DDOA: -34.28 ± 0.98mV, p < 0.001 vs. THC condition, THC + ODQ: -36.84 ± 0.79 mV, p < 0.001 vs. THC conditionF-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (H) rheobase: THC + ZD: 806 ± 63 pA, p < 0.001 vs. THC condition, THC + DDOA: 607 ± 43 pA, p < 0.001 vs. THC condition, THC + ODQ: 422 ± 34 pA, p < 0.001 vs. THC conditionF-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (I) firing rate. Note that Vehicle- and THC-injected mice are the same as those used in experiments illustrated on Figure 5A-D. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s. p > 0.05 one-way ANOVA followed by Bonferroni test.


Figure 6A . In left , normalized peak amplitudes of isolated AMPA EPSCs recorded at - 60 mV , before and after pairing protocol , are illustrated . Representative traces of AMPA EPSCs before ( 1 ) or after ( 2 ) the pairing protocol are also illustrated ( right panel ) for each experimental condition : Vehicle - injected mice ( white circles , n = 3 from N = 3 ) , THC - injected mice ( green circles , n = 3 from N = 3 ) and THC - injected mice treated with SB258585 during adolescence ( black circles , n = 3 from N = 3 ) . Errors bars correspond to mean ± SEM . B . The histogram represents the means ± SEM of AMPA EPSCs in % of baseline , measured during the last 5 min of baseline or the last 5 min of recording , for each experimental condition : vehicle - injected mice ( white bar , n = 3 from N = 3 ) , THC - injected mice ( green bar , n = 3 from N = 3 ) and THC - injected mice treated with SB258585 during adolescence ( black bar , n = 3 from N = 3 ) , n . s . p > 0 . 05 , * p < 0 . 05 , one - way ANOVA followed by Tukey test .

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

Figure 6A. In left, normalized peak amplitudes of isolated AMPA EPSCs recorded at -60 mV, before and after pairing protocol, are illustrated. Representative traces of AMPA EPSCs before (1) or after (2) the pairing protocol are also illustrated (right panel) for each experimental condition: Vehicle-injected mice (white circles, n=3 from N=3), THC-injected mice (green circles, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black circles, n=3 from N=3). Errors bars correspond to mean ± SEM.B. The histogram represents the means ± SEM of AMPA EPSCs in % of baseline, measured during the last 5 min of baseline or the last 5 min of recording, for each experimental condition: vehicle-injected mice (white bar, n=3 from N=3), THC-injected mice (green bar, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black bar, n=3 from N=3), n.s. p > 0.05, * p< 0.05, one-way ANOVA followed by Tukey test.


Figure 1 ( a , b ) Isolated acini from wild - type ( WT ) and Trpml1 - / - mice ( ML1 - / - ) were used to determine the frequency of Ca2 + oscillations in response to stimulation with 3 and 10 pM CCK ( a ) and the response to high concentrations of CCK ( b ) . The average frequency from multiple experiments is given in the columns as mean ± s . e . m in ( a ) . The traces and columns in ( b ) show the mean ± s . e . m of 3 experiments each includes 4 - 6 acini composed of 6 - 10 cells . ( c - e ) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations . ( c ) shows exocytosis during the first 5 min of stimulation , ( d ) shows the two phases , first rapid and second sustained phase , of exocytosis and ( e ) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation . The results are averages of 3 - 4 experiments with each mouse line . denotes p < 0 . 01 or better and # denotes p < 0 . 05 or better .

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

Figure 1(a, b) Isolated acini from wild-type (WT) and Trpml1-/- mice (ML1-/-) were used to determine the frequency of Ca2+ oscillations in response to stimulation with 3 and 10 pM CCK (a) and the response to high concentrations of CCK (b). The average frequency from multiple experiments is given in the columns as mean±s.e.m in (a). The traces and columns in (b) show the mean±s.e.m of 3 experiments each includes 4-6 acini composed of 6-10 cells.(c-e) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations. (c) shows exocytosis during the first 5 min of stimulation, (d) shows the two phases, first rapid and second sustained phase, of exocytosis and (e) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation. The results are averages of 3-4 experiments with each mouse line. denotes p<0.01 or better and # denotes p<0.05 or better.


Figure 2Panel ( a ) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution . Granules size was analyzed in at least 10 images from 3 wild - type and 5 Trpml1 - / - mice . ( b ) Fixed acini were co - stained with Amylase ( granules ) and LAMP1 ( lysosomes ) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively . ( c ) Live wild - type and Trpml1 - / - acini were labeled with mitotracker ( green ) and lysotracker ( red ) . The panels show the merged green , red and DIC images . The large vesicles are marked by white arrows and shown at higher magnification in the inserts . The overlap between lysosomes and granules was determined by ImageJ ( see methods ) . The results are from 14 wild - type and 30 Trpml1 - / - acini each comprising 3 - 7 cells and obtained from 3 wild - type and 4 Trpml1 - / - mice . ( d ) Co - IP of lysosomal LAMP1 and granules VAMP8 and Rab27b . Note the increased lysosomal / granules interaction . ( e ) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1 - / - acini stimulated with 100 pM CCK . The results are the mean ± s . e . m of 3 experiments with acini obtained from 3 wild - type and 3 Trpml1 - / - mice .

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

Figure 2Panel (a) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution. Granules size was analyzed in at least 10 images from 3 wild-type and 5 Trpml1-/- mice.(b) Fixed acini were co-stained with Amylase (granules) and LAMP1 (lysosomes) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild-type and 3 Trpml1-/- mice, respectively.(c) Live wild-type and Trpml1-/- acini were labeled with mitotracker (green) and lysotracker (red). The panels show the merged green, red and DIC images. The large vesicles are marked by white arrows and shown at higher magnification in the inserts. The overlap between lysosomes and granules was determined by ImageJ (see methods). The results are from 14 wild-type and 30 Trpml1-/- acini each comprising 3-7 cells and obtained from 3 wild-type and 4 Trpml1-/- mice.(d) Co-IP of lysosomal LAMP1 and granules VAMP8 and Rab27b. Note the increased lysosomal/granules interaction.(e) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1-/-acini stimulated with 100 pM CCK. The results are the mean±s.e.m of 3 experiments with acini obtained from 3 wild-type and 3 Trpml1-/-mice.


Figure 3 ( a , b ) Plasmaamylase ( a ) and pancreaticMPO ( b ) in 3 mice of each line . ( c ) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H & amp ; amp ; E staining . Shown are examples of images and the average damage in the columns , which show the mean ± s . e . m of 4 experiments with each mouse line . ( d ) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min . Shown are example images and the averages . ( e ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide . The figure shows example images before stimulation and the averaged fluorescence intensity . ( f ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3 . The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ . Results in ( c - f ) are given as the mean ± s . e . m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment .

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

Figure 3(a, b) Plasmaamylase(a) and pancreaticMPO(b) in 3 mice of each line.(c) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H&amp;E staining. Shown are examples of images and the average damage in the columns, which show the mean±s.e.m of 4 experiments with each mouse line.(d) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min. Shown are example images and the averages.(e) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide. The figure shows example images before stimulation and the averaged fluorescence intensity.(f) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3. The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ. Results in (c-f) are given as the mean±s.e.m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment.


Figure 4 ( a ) Isolated acini from wild - type ( black ) and Trpml1 - / - mice ( red ) loaded with Fura2 were used to determine [ Ca2 + ] i in response to stimulation with 100 µM carbachol . The traces are the mean ± s . e . m . of 6 acinar clusters . Similar results were obtained in 3 experiments . ( b , c ) Saliva was collected from wild - type ( black ) and Trpml1 - / - mice ( red ) injected I . P . with 10 mg / kg pilocarpine ( b ) or 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol ( c ) over 20 - 40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol . Each condition used 4 - 5 mice from each like and the results are given as the mean ± s . e . m . ( d ) the saliva collected in ( c ) was used to determine the amount of secreted amylase . ( e , f ) Isolated acini from the parotid glands of wild - type ( black ) or Trpml1 - / - mice ( red ) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times ( e ) and the indicated isoproterenol concentrations during 30 min stimulation ( f ) . The results are the mean ± s . e . m obtained from two mice of each line .

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

Figure 4(a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments.(b, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m.(d) the saliva collected in (c) was used to determine the amount of secreted amylase.(e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.


Figure 5 ( a ) shows example TEM images and ( b ) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild - type mice and 14 images obtained from 4 Trpml1 - / - mice . ( c ) Co - IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b . Input control is actin . ( d ) Acini isolated from the parotid glands of wild - type and Trpml1 - / - mice were incubated with mitotracker ( green ) and lysotracker . The large vesicles in the secretory granules / lysosomal area are marked by white arrows . The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line . ( e ) Shown is the time course ( left panel ) and averaged 10 min secretion ( columns ) of the lysosomal marker acid phosphatase in the saliva collected from wild - type ( black ) and Trpml1 - / - mice ( red ) stimulated with 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol . The mean ± s . e . m is from 4 mice of each line .

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

Figure 5(a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice.(c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin.(d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line.(e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.


Figure 6 ( a , b ) example traces of mEPSCs recorded from wild - type ( a ) and Trpml1 - / - ( b ) micebrain slices as detailed in Methods . The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale . ( c ) histogram distribution of the events at each mEPSC . The lower columns show the distribution of the higher amplitude mEPSCs . ( d ) the frequency of mEPSCs in each line . The results in ( c , b ) are the mean ± s . e . m of 8 experiments each obtained from 4 mice of each line . ( e ) the mean ± s . e . m of the Ca2 + signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild - type and Trpml1 - / - slices , respectively .

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

Figure 6(a, b) example traces of mEPSCs recorded from wild-type (a) and Trpml1-/-(b) micebrain slices as detailed in Methods. The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale. (c) histogram distribution of the events at each mEPSC. The lower columns show the distribution of the higher amplitude mEPSCs. (d) the frequency of mEPSCs in each line. The results in (c, b) are the mean±s.e.m of 8 experiments each obtained from 4 mice of each line.(e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.


Figure 7 ( a ) example TEM images of synapses in wild - type and Trpml1 - / - micebraincortex . ( b ) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively . ( c ) example TEM images at low magnification of wild - type and Trpml1 - / - micebraincortical slices with the dark PSD profiles marked by white arrows . ( d ) average number of PSD / µm2 analyzed in at least 10 images obtained from 3 mice of each line . ( e ) Glutamate levels and secretion by cultured cerebral cortical neurons . Total glutamate measured in non - stimulated wild - type ( black ) and Trpml1 - / - ( green ) neurons and neurons stimulated by depolarization with 30 mM KCl ( blue and red ) . ( f ) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons . Total glutamate measured in non - stimulated Trpml1 - / - neurons ( green ) and Trpml1 - / - neurons transfected with TRPML1 ( black ) and untransfected ( red ) and TRPML1 - transfected ( clue ) Trpml1 - / - neurons stimulated by depolarization with 30 mM KCl . ( g ) averaged Co - IP of SNAP25 and VAMP2 in three brain regions of wild - type and Trpml1 - / - mice . The images of all blots are given in Extended view Figure E4 .

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

Figure 7(a) example TEM images of synapses in wild-type and Trpml1-/-micebraincortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/-mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/-micebraincortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line.(e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red).(f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl.(g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.


Figure 1 ( B ) Assessment of exogenous DDX60 expression . HEK293T cells transfected with DDX60 wild type ( wt ) , or DDX60 mutants and analyzed by western blot for DDX60 , ß - actin and GAPDH ( loading controls ) , and RFP ( reporter ) . DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below . ( C ) HCV antiviral assays with DDX60 wt or mutant panel . Huh - 7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase ( Fluc , negative , and transfection control ) , IRF1 ( positive antiviral control ) , DDX60 wt , or DDX60 mutants and infected with HCV - Ypet , a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C , E1 , E2 , p7 , NS2 , NS3 , 4A , 4B , NS5A , and NS5B is driven by EMCV IRES . Data information : For ( C ) percent of Ypet + cells in RFP + cells is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against Fluc . ( D ) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs . Huh - 7 cells transfected as in ( C ) and infected with either bicistronic HCV - Ypet ( left ) or monocistronic HCV J6 / JFH - 5AB - YPet . Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B . Data information : For ( D ) , percent of Ypet + cells in RFP + cells is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against Fluc .

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

Figure 1(B) Assessment of exogenous DDX60 expression. HEK293T cells transfected with DDX60 wild type (wt), or DDX60 mutants and analyzed by western blot for DDX60, ß-actin and GAPDH (loading controls), and RFP (reporter). DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below.(C) HCV antiviral assays with DDX60 wt or mutant panel. Huh-7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase (Fluc, negative, and transfection control), IRF1 (positive antiviral control), DDX60 wt, or DDX60 mutants and infected with HCV-Ypet, a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C, E1, E2, p7, NS2, NS3, 4A, 4B, NS5A, and NS5B is driven by EMCV IRES. Data information: For (C) percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.(D) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs. Huh-7 cells transfected as in (C) and infected with either bicistronic HCV-Ypet (left) or monocistronic HCV J6/JFH-5AB-YPet. Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B. Data information: For (D), percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.