- infiltrating immune from tumors were analyzed by FACS : MDSCs CD25 + Foxp3 + Tregs ( G ) Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

O O O O B-DISEASE O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Tumor-infiltrating immune cells from tumors were analyzed by FACS: MDSCs and CD25+ Foxp3+ Tregs (G) 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+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


- infiltrating immune from tumors were analyzed by FACS NK ( H ) Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

O O O O B-DISEASE O O O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Tumor-infiltrating immune cells from tumors were analyzed by FACS NK cells (H) 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+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


- infiltrating immune from tumors were analyzed by FACS : CD8 + T ( I ) . Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

O O O O B-DISEASE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Tumor-infiltrating immune cells from tumors were analyzed by FACS: CD8+ T cells (I). 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+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


BJcl2 with or without hTRF2 overexpression were inoculated into backs of immunodeficient Nude mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) NK recruitment , degranulation MDSC infiltration were monitored by FACS Density plot of CD11bHi GR1Hi MDSC in control or anti - GR1 - treated Matrigel plugs ( K ) .

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

BJcl2 cells with or without hTRF2 overexpression were inoculated into the backs of immunodeficient Nude mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) and NK cells recruitment, degranulation and MDSC infiltration were monitored by FACS Density plot of CD11bHi GR1Hi MDSC staining in control or anti-GR1-treated Matrigel plugs (K).


BJcl2 with or without hTRF2 overexpression were inoculated into backs of immunodeficient Nude mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) NK recruitment , degranulation MDSC infiltration were monitored by FACS Box plots of percentages of infiltrating or activated NK ( CD107a or CD69 ) ( L ) Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

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

BJcl2 cells with or without hTRF2 overexpression were inoculated into the backs of immunodeficient Nude mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) and NK cells recruitment, degranulation and MDSC infiltration were monitored by FACS Box plots of the percentages of infiltrating or activated NK cells (CD107a or CD69) (L) 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+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


BJcl2 with or without hTRF2 overexpression were inoculated into backs of immunodeficient Nude mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) NK recruitment , degranulation MDSC infiltration were monitored by FACS Box plots of percentages of CD11bHi GR1Hi MDSC or NKp46 + NK ( M ) infiltrating Matrigel plugs among CD45 + . Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

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

BJcl2 cells with or without hTRF2 overexpression were inoculated into the backs of immunodeficient Nude mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) and NK cells recruitment, degranulation and MDSC infiltration were monitored by FACS Box plots of the percentages of CD11bHi GR1Hi MDSC or NKp46+ NK cells (M) infiltrating the Matrigel plugs assay among the CD45+ cells. 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+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


of STAT3 phosphorylation by MSC2 after 18 hours ' co - culture with BJcl2 overexpressing or compromised for TRF2 Histograms of pSTAT3 mean ( MFI ) ( B ) .

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Analysis of STAT3 phosphorylation by MSC2 cells after 18 hours' co-culture with BJcl2 overexpressing or compromised for TRF2 Histograms of the pSTAT3 mean fluorescence intensity (MFI) (B).


+ / - inhibitors : anti - MyD88 peptide inhibitor , anti - mouse TLR2 antibody , or lipopolysaccharide ( LPS ) - RS , an antagonist of TLR4 in ( D )

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+/- inhibitors: anti-MyD88 peptide inhibitor, anti-mouse TLR2 antibody, or lipopolysaccharide (LPS)-RS, an antagonist of TLR4 in (D)


+ / - inhibitors : anti - mouse IL - 6 or JAK1 / 2 inhibitor ( Ruxolitinib ) in ( E ) .

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+/- inhibitors: anti-mouse IL-6 or JAK1/2 inhibitor (Ruxolitinib) in (E).


TRF2 - overexpressing or - knockdown BJcl2 were co - cultured with MSC2 for 18h + / - mouse IL6 blocking antibody or Ruxolitinib . After FACS sorting , MSC2 were co - cultured for 18h with primed purified NK before a 4h challenge with YAK - 1 . NK degranulation IFN - γ production were analyzed by FACS ( G ) . Data information : Panels show points with mean ± corresponding to percentages of each population among NK ( G ) ( n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - ) .

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

TRF2-overexpressing or -knockdown BJcl2 cells were co-cultured with MSC2 cells for 18h +/- mouse IL6 blocking antibody or Ruxolitinib. After FACS sorting, MSC2 cells were co-cultured for 18h with primed and purified NK cells before a 4h challenge with YAK-1. NK cell degranulation and IFN-γ production were analyzed by FACS (G). Data information: Panels show all points with mean ± SEM corresponding to the percentages of each cell population among NK cells (G) (n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001; Student's t-test).


TRF2 - overexpressing or - knockdown BJcl2 were co - cultured with MSC2 for 18h + / - mouse IL6 blocking antibody or Ruxolitinib . Alternatively , a 4h challenge with NIH3T3 was performed ability of NK to kill NIH3T3 were determined by colorimetric ( AlamarBlue ) ( H ) . Data information : Panels show points with mean ± corresponding to of specific target lysis ( H ) ( n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - ) .

B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-CELL_LINE O O O O B-CELL_TYPE O O B-CELL_LINE O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TRF2-overexpressing or -knockdown BJcl2 cells were co-cultured with MSC2 cells for 18h +/- mouse IL6 blocking antibody or Ruxolitinib. Alternatively, a 4h challenge with NIH3T3 cells was performed and the ability of NK cells to kill NIH3T3 cells were determined by colorimetric analysis (AlamarBlue) (H). Data information: Panels show all points with mean ± SEM corresponding to the percentage of specific target lysis (H) (n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001; Student's t-test).


Gene expression 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 ±

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

Gene 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 ± SEM


B , C of H3K27ac ( B ) or H3K23me3 ( C ) peaks on 4 kilobase ( Kb ) region around genomic ITSs ( ENCODE data for K562 ) . mean of region is depicted in histograms . ITSs are clustered depending of peak mean obtained from H3K27ac or H3K27me3 immunoprecipitation sequencing ( ChIP - seq ) of 4 Kb region . ITSs detected by ChIP - seq using an anti - TRF2 antibody are shown on left .

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

B, 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 , VCAN ITSs after ChIP using an anti - H3K27ac antibody in TRF2 - overexpressing or - knockdown BJcl2 . Data represent fold enrichment of H3K27ac level compared with total H3 level . Data information : data represent n = 3 independent experiments with mean ±

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

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


B - E of CD11bHi GR1Hi MDSCs ( B ) , CD3 - NKp46 + NK ( C ) , NKp46 + CD107a + NK ( D ) or CD69 + NK ( E ) Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or NK ( n = 8 mice per group ; * p < 0 . 05 * * * p < 0 . 001 ; Mann - Whitney ) .

O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

B-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 pSTAT3 mean ( MFI ) of MSC2 when TRF2 was overexpressed target gene knocked down ( G ) Data information MFI of n = 3 independent experiment is normalized to controls represented as fold change of MFI ( n = 3 independent experiments ; * p < 0 . 05 * * p < 0 . 01 ; Student ' s t - ) .

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

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 pSTAT3 mean ( MFI ) of MSC2 when both TRF2 its target gene were knocked down ( H ) . Data information : MFI of n = 3 independent experiment is normalized to controls represented as fold change of MFI ( n = 3 independent experiments ; * p < 0 . 05 * * p < 0 . 01 ; Student ' s t - ) .

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

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 weights on day 19 after implantation of subcutaneous xenografts containing BJcl2 overexpressing or knocked down for HS3ST4 or GPC6 Data information : data are represented as mean + / - ( n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 ; Mann - Whitney ) .

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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).


A of Heparan Sulfate ( HS ) expression by FACS using 10E4 antibody . Data information histograms representative ( left panels ) MFIs from three independent experiments ( * p < 0 . 05 * * p < 0 . 01 ; Student ' s t - ) are shown + / - .

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A 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 ) of glycocalyx stiffness glycocalyx . Data information : In ( B ) , Box plots Min to Max showing points with median represent four independent experiments , each performed using at least 20 ( * p < 0 . 05 ; Mann - Whitney ) .

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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 showing Modulus ( stiffness ) in GFP - expressing or TRF2 - overexpressing cancer . At each of 128x128 force curve points , corresponding to an of 10 μm2 , AFM software automatically displays Sneddon modulus while scanning sample . color bar on left of shows a qualitative representation of elastic map .

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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 of pSTAT3 in MDSCs after co - culture with GFP - expressing or TRF2 - overexpressing cancer treated with or without sodium chlorate ( NaClO3 ) . Data information : In ( D ) , data represents mean of n = 3 independent experiments + / - ( * * * p < 0 . 001 ; Student ' s t - ) .

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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).


A Kaplan - Meier curves for overall survival of chemotherapy - treated according to TRF2 expression level Data information : optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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

A 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 with or without TRF2 overexpression treated with three intraperitoneal injections of 50 mg / kg 5 - FU . volume was determined every 2 days using a caliper , growth curves over time ( C ) Data information : , data represent mean values + / - ( n = 8 mice per group ; * p < 0 . 05 * * p < 0 . 01 ; Mann - Whitney ) .

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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 with or without TRF2 overexpression treated with three intraperitoneal injections of 50 mg / kg 5 - FU . volume were determined on day 19 represented using Box plots Min to Max showing points with median ( D ) . Data information : ( D ) , data are represented by Box plots Min to Max showing points with median ( n = 8 mice per group ; * p < 0 . 05 * * p < 0 . 01 ; Mann - Whitney )

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

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 with or without TRF2 overexpression treated with three intraperitoneal injections of 50 mg / kg 5 - FU . Overall survival was also determined ( E ) .

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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).


A Kaplan - Meier curves for overall survival of at stages of cancers ( breast , gastric , ovarian , lung cancer ) depending on TRF2 expression level . Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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A 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.


overall survival of gastric cancer ( stages ) was analyzed depending on TRF2 its targets genes expression level ( HS3ST4 , GPC6 VCAN collectively named HSPG genes ) Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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

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.


overall survival of gastric cancer ( stages ) was analyzed depending on TRF2 with MDSC level ( Lo or Hi ) , determine as mean expression level of CD33 C5AR , using KMplot Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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

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 depending on TRF2 MDSC classification . Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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

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 ( stages ) analyzed depending on TRF2 , HSPG genes ( HS3ST4 , GPC6 VCAN ) MDSC level ( Lo or Hi ) . Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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

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.


Testing an for SARS - CoV - 2 using synthetic targets . We show data from both ( a ) 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 on n = 3 technical replicates .

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

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.


mRNA expression of ANG in colonic tissues from normal controls ( n = 25 ) UC ( n = 27 ) or CD ( n = 46 ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O O O O O O O B-DISEASE O O O O O O B-DISEASE O O O O O O

mRNA 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 UC or CD .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O O B-DISEASE O B-DISEASE O

Representative western blotting of ANG in colonic tissues from normal controls and UC or CD patients.


Representative showing ANG immunohistochemical ( ) in normal control UC or CD sample .

O O B-GENEPROD B-EXP_ASSAY O O O O O B-DISEASE O B-DISEASE O O

Representative images showing ANG immunohistochemical (IHC) staining in normal control and UC or CD sample.


Corresponding statistical of ANG expression score in normal controls ( n = 10 ) UC ( n = 10 ) or CD ( n = 15 ) .

O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-DISEASE O O O O O O B-DISEASE O O O O O O

Corresponding statistical analysis of ANG expression score in normal controls (n = 10) and UC (n = 10) or CD (n = 15) patients.


Representative showing hematoxylin - eosin ( HE ) ANG in mild severe IBD samples .

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

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 ) severe ( n = 19 ) UC ( F ) or mild ( n = 20 ) severe ( n = 20 ) CD ( G ) .

O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O B-DISEASE O O O O

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.


Ang mRNA expression in colonic tissue from 2 . 5 % DSS - treated WT mice at indicated time point ( n = 4 mice / time point ) . DSS : dextran sulfate

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

Ang mRNA expression in colonic tissue from 2.5% DSS-treated WT mice at indicated time point (n = 4 mice/time point). DSS: dextran sulfate sodium


Kaplan - Meier curve of 3 . 5 % DSS - treated Ang - deficient mice ( Ang - / - , n = 14 ) or littermate controls ( WT , n = 13 ) .

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Kaplan-Meier curve of 3.5% DSS-treated Ang-deficient mice (Ang-/-, n = 14) or littermate controls (WT, n = 13).


loss of WT Ang - / - mice with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment .

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

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 Ang - / - mice with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment .

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

disease activity index (DAI) of WT and Ang-/- mice with (n = 9) or without (n = 6) 2.5% DSS treatment.


Colon of colonic section from WT Ang - / - mice on day 9 with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment .

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

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 WT Ang - / - mice on day 9 with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment . FITC - Dextran : fluorescein isothiocyanate - labeled dextran .

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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 image , ( H ) histopathological score of colonic section from WT Ang - / - mice on day 9 with ( n = 9 ) or without ( n = 6 ) 2 . 5 % DSS treatment .

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

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 WT Ang - / - mice on day 0 ( n = 3 ) or day 9 ( n = 9 ) during 2 . 5 % DSS treatment .

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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 Ang - / - mice on day 0 ( n = 3 ) or day 9 ( n = 7 ) during 2 . 5 % DSS treatment .

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

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.


loss of WT → WT , Ang - / - → WT WT → Ang - / - chimera mice with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

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

Body 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 WT → Ang - / - chimera mice with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

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

DAI of WT→WT, Ang-/-→WT and WT→Ang-/- chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment.


Colon of colonic section from these chimera colitis mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

B-EXP_ASSAY O B-TISSUE O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O

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 .

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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 image , ( 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 .

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

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 myeloid ; data represent two independent experiments ( n = 2 ) .

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

Ang mRNA expression in mouse lymphoid and myeloid cells; data represent two independent experiments (n = 2).


loss of Angfl / fl LyzMcre ; Angfl / fl mice with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment .

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

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 LyzMcre ; Angfl / fl mice with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment .

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

DAI of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment.


Colon of colonic section from Angfl / fl LyzMcre ; Angfl / fl mice on day 8 with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment .

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

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 Angfl / fl LyzMcre ; Angfl / fl mice on day 8 with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment .

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

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 image , ( M ) histopathological score of colonic section from Angfl / fl LyzMcre ; Angfl / fl mice on day 8 with ( n = 7 ) or without ( n = 3 ) 2 . 5 % DSS treatment .

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

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.


Quantitative mRNA expression of ANG PLXNB2 in isolated IECs or colonic laminar propria ( cLP ) from WT mice ( n = 9 ) .

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

Quantitative mRNA expression of ANG and PLXNB2 in isolated IECs or colonic laminar propria (cLP) cells from WT mice (n = 9).


western blotting of ANG PLXNB2 in isolated IECs or colonic laminar propria ( cLP ) from WT mice ( n = 9 ) .

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

western blotting of ANG and PLXNB2 in isolated IECs or colonic laminar propria (cLP) cells from WT mice (n = 9).


Representative showing immunofluorescence of ANG , PLXNB2 macrophage surface marker F4 / 80 in frozen colonic section from WT mice .

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

Representative images showing immunofluorescence staining of ANG, PLXNB2 and macrophage surface marker F4/80 in frozen colonic section from WT mice.


Representative showing immunofluorescence of GFP PLXNB2 in frozen colonic section from WT mice infected with adeno - associated ( AAV9 ) .

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

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 AAV9 - infected WT mice ( n = 3 ) .

B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-ORGANISM O O O B-ORGANISM O O O O O O

PLXNB2 expression in IECs from the AAV9-infected WT mice (n = 3).


loss of AA9 - infected WT mice with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

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

Body weight loss of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment.


DAI of AA9 - infected WT mice with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

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

DAI of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment.


Colon of colonic section from AAV9 - infected WT mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

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

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 AAV9 - infected WT mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

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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 image , ( K ) histopathological score of colonic section from AAV9 - infected WT mice on day 8 with ( n = 7 ) or without ( n = 5 ) 2 . 5 % DSS treatment .

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

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.


Representative showing - caspase 3 in colonic section from WT or Ang - / - colitis mice ( n = 3 ) at indicated time point ; arrows indicate - caspase 3 positive ; two fields per colon section are quantified .

O O O B-GENEPROD I-GENEPROD O B-TISSUE O O O O B-GENEPROD O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O

Representative 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.


number of apoptotic in colonic section from WT or Ang - / - colitis mice ( n = 3 ) at indicated time

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

the number of apoptotic cells in colonic section from WT or Ang-/- colitis mice (n = 3) at indicated time point


apoptotic status of IECs from WT or Ang - / - colitis mice ( n = 5 ) on day 4 .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD O O O B-DISEASE B-ORGANISM O O O O O O O O O

Cell apoptotic status of IECs from WT or Ang-/- colitis mice (n = 5) on day 4.


Representative showing Ki67 in colonic section from WT or Ang - / - colitis mice ( n = 3 ) at indicated time point ; five representative crypts per colon section are quantified .

O O B-GENEPROD O B-TISSUE O O O O B-GENEPROD O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O

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.


number of proliferating 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 .

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

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.


- cycle status of IECs from WT or Ang - / - colitis mice ( n = 5 ) on day 4 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD O O O B-DISEASE B-ORGANISM O O O O O O O O O

Cell-cycle status of IECs from WT or Ang-/- colitis mice (n = 5) on day 4.


Representative showing - caspase 3 in colonic section from Angfl / fl LyzMCre ; Angfl / fl , or AAV - shControl - AAV - shPlxnb2 - treated colitis mice on day 4 ( n = 3 ) ; arrows indicate - caspase 3 positive ; two fields per colon section are quantified .

O O O B-GENEPROD I-GENEPROD O B-TISSUE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O B-ORGANISM O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O

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.


number of apoptotic in colonic section from Angfl / fl LyzMCre ; Angfl / fl , or AAV - shControl - AAV - shPlxnb2 - treated colitis mice on day 4 ( n = 3 )

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

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 ( L ) number of proliferating per crypt in representative colonic section from Angfl / fl LyzMCre ; Angfl / fl , or AAV - shControl - AAV - shPlxnb2 - treated colitis mice on day 4 ( n = 3 ) ; five representative crypts per colon section are quantified .

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

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.


PLXNB2 expressions in HCT116 ( including wild type HCT116 ( HCT116WT ) ANG knockout HCT116 ( HCT116KO ) ) THP - 1 as detected by flow cytometry . upper three dash lines represent of unstained negative control of these , while solid lines represent of PLXNB2 antibody .

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

PLXNB2 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 ) .

O B-GENEPROD O O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O

Secreted ANG level in conditioned medium from THP1, HCT116WT or HCT116KO (n = 3).


interaction between ANGWT , ANGR66A or ANGK40I with PLXNB2 in HCT116KO detected by co - immunoprecipitation . ANGR66A , ANG receptor binding site variant ; ANGK40I , ANG enzymatic activity null

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

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 variant


THP - 1 - derived ANG translocating into HCT116KO through PLXNB2 in co - cultured system as showed by western blotting . Neamine , a blocker of ANG PLXNB2 interaction ; siCtrl : control siRNA ; siPLXNB2 - 1 siPLXNB2 -

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

THP-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 siPLXNB2-2


Flow cytometry of HCT116KO stained with Annexin V 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 )

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

Flow 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) phosphorylation


tiRNA 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 )

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tiRNA 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) phosphorylation


effect of 5 ' - tiRNA knockdown or overexpression on TNF - α - induced apoptosis in ANGWT - or ANGK40I - treated HCT116KO . si5 ' - tiRNA : tiRNA - targeting

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

The effect of 5'-tiRNA knockdown or overexpression on TNF-α-induced apoptosis in ANGWT- or ANGK40I-treated HCT116KO. si5'-tiRNA: tiRNA-targeting siRNA


interaction between 5 ' - tiRNA cytochrome c ( Cyt c ) in HCT116 as detected by RIP .

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

The 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 )

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

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) phosphorylation


viability of HCT116KO following different treatments as indicated . siCtrl : control siRNA ; siPLXNB2 - 1 siPLXNB2 - 2 : PLXNB2

B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD

Cell viability of HCT116KO following different treatments as indicated. siCtrl: control siRNA; siPLXNB2-1 and siPLXNB2-2: PLXNB2 siRNAs


Detection of 47S pre - rRNA transcription level with northern blotting in HCT116KO following different treatments as indicated . siPLXNB2 - 1 siPLXNB2 - 2 : PLXNB2

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

Detection of 47S pre-rRNA transcription level with northern blotting in HCT116KO following different treatments as indicated. siPLXNB2-1 and siPLXNB2-2: PLXNB2 siRNAs


tiRNA 5 ' - tiRNAAla production in IECs isolated from WT Ang - / - colitis mice at indicated time point .

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tiRNA 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 Ang - / - colitis mice at indicated time point .

B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O O O B-DISEASE B-ORGANISM O O O O O

47S pre-rRNA transcription level in IECs from WT and Ang-/- colitis mice at indicated time point.


Western blotting of ANG in colonic tissue from WT mice ( n = 2 ) at indicated time point in ANG prevention model . grayscale of ANG band in WT mice with no recombinant ANG DSS treatment ( untreated ) is arbitrarily set as 1 .

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

Western 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.


loss of pre - treated WT colitis mice ( n = 8 ) .

O O O O O O B-DISEASE B-ORGANISM O O O O O O

Body weight loss of the pre-treated WT colitis mice (n = 8).


DAI of pre - treated WT colitis mice ( n = 8 ) .

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DAI of the pre-treated WT colitis mice (n = 8).


Colon of colon section from pre - treated WT colitis mice on day 8 ( n = 8 ) .

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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 pre - treated WT colitis mice on day 8 ( n = 8 ) .

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serum FITC-dextran level of colon section from the pre-treated WT colitis mice on day 8 (n = 8).


representative HE image , ( H ) histopathological score of colon section from pre - treated WT colitis mice on day 8 ( n = 8 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-TISSUE O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O

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 ANG treatment model . grayscale of ANG band in WT mice with no recombinant ANG DSS treatment ( untreated ) is arbitrarily set as 1 .

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

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.


loss of treated WT colitis mice ( n = 8 ) .

O O O O B-DISEASE B-ORGANISM O O O O O O

Body weight loss of the treated WT colitis mice (n = 8).


DAI of treated WT colitis mice ( n = 8 ) .

B-EXP_ASSAY O O O B-DISEASE B-ORGANISM O O O O O O

DAI of the treated WT colitis mice (n = 8).


Colon of colon sections from treated WT colitis mice on day 8 ( n = 8 ) .

B-EXP_ASSAY O B-TISSUE O O O O B-DISEASE B-ORGANISM O O O O O O O O O

Colon length of colon sections from the treated WT colitis mice on day 8 (n = 8).


serum FITC - dextran level of colon sections from treated WT colitis mice on day 8 ( n = 8 ) .

B-TISSUE B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-TISSUE O O O O B-DISEASE B-ORGANISM O O O O O O O O O

serum FITC-dextran level of colon sections from the treated WT colitis mice on day 8 (n = 8).


representative HE image , ( P ) histopathological score of colon sections from treated WT colitis mice on day 8 ( n = 8 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-TISSUE O O O O B-DISEASE B-ORGANISM O O O O O O O O O

representative HE staining image, and (P) histopathological score of colon sections from the treated WT colitis mice on day 8 (n = 8).


B . Top : representative Western blots assessing mTOR phosphorylation at S2448 p70S6K phosphorylation at T389 as indexes of mTOR activity in PFC of adult WT mice are illustrated . Bottom : data represent ratios of immunoreactive signals of anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to immunoreactive signal of anti - β - actin antibody are expressed in % of values in vehicle - injected WT mice . They are 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 .

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

B. 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 behavioral tasks examined in WT mice . Bottom : plots represent discrimination index for novel object recognition task ( Vehicle N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) , sociability index ( Vehicle : N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) discrimination index for social discrimination task ( N = 8 for each group ) , measured in each condition . * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA followed by Bonferroni ( error bars correspond to mean ± , dotted to a discrimination index ( exploration of novel object - exploration of familiar object / total object exploration ) equal to zero .

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

C. 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 p70S6K phosphorylation at T389 in PFC of adult 5 - HT6 - / - mice are illustrated . Bottom : data represent ratios of immunoreactive signals of anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to immunoreactive signal of anti - β - actin antibody are expressed in % of values in vehicle - injected 5 - HT6 - / - mice . They are means ± S . E . M . of results obtained in four mice per group . n . s . p > 0 . 05 , one - way ANOVA followed by Newman - Keuls .

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

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 behavioral tasks in 5 - HT6 - / - mice . Bottom : plots represent discrimination index for novel object recognition ( discrimination index : 0 . 30 ± 0 . 05 , N = 10 0 . 31 ± 0 . 03 , N = 11 , for THC + Vehicle Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , 3 - chamber social preference ( sociability index : 0 . 49 ± 0 . 04 , N = 10 0 . 48 ± 0 . 07 , N = 11 for THC + Vehicle Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) social discrimination ( discrimination index : 0 . 15 ± 0 . 06 , N = 8 0 . 21 ± 0 . 06 , N = 8 for THC + Vehicle Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , measured in each condition . One - way ANOVA followed by Bonferroni ( error bars correspond to mean ± , dotted corresponds to a discrimination index equal to zero ) .

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

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).


A - E . PFC was stained with CB1 receptor ( A1 - D1 , green ) or 5HT6 receptor ( E1 - H1 , green ) , Bassoon ( A2 E2 , red ) , GAD65 ( B2 F2 , red ) , PSD - 95 ( C2 G2 , red ) , or SERT ( D2 H2 , red ) antibodies . Merge are also depicted ( A3 - H3 ) . CB1 receptors were mainly co - localized with presynaptic marker Bassoon ( arrows , A3 ) , mostly within GABAergic boutons ( arrows , B3 ) to a lesser extend with PSD - 95 ( arrows , C3 ) SERT ( D3 ) , while 5 - HT6 receptors were mainly co - localized with PSD - 95 ( arrows , G3 ) . No co - localization of CB1 5 - HT6 receptors was detected ( arrows indicate 5 - HT6 , H3 ) . Scale bar : 10 μm . scatter plot represents co - localization for CB1 5 - HT6 receptor immunostainings . Graph on top , Mander ' s split coefficient was used to identify 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 bottom . Mander ' s split coefficient was used to identify 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 mean ± .

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

A-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.


B . Top : representative Western blots assessing mTOR activity in PFC are illustrated . Bottom : data represent ratios of immunoreactive signals of anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to immunoreactive signal of anti - β - actin antibody are expressed in % of values in vehicle - injected mice . They are 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 .

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

B. 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 . plots represent discrimination index measured in each condition . * * * p < 0 . 001 , one - way ANOVA followed by Bonferroni . discrimination index for novel object recognition task is 0 . 34 ± 0 . 06 ( N = 14 ) - 0 . 08 ± 0 . 07 ( N = 20 ) , for mice injected with Vehicle THC , respectively , p < 0 . 001 - 0 . 09 ± 0 . 08 ( N = 21 ) 0 . 00 ± 0 . 06 ( N = 18 ) , for mice treated with THC + SB258285 THC + Rapa , respectively , p > 0 . 05 vs . THC - injected mice ( error bars correspond to mean ± , dotted to a discrimination index equal to zero ) .

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

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).


B . Left : representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated . Right : histograms represent means ± S . E . M . of GABA mIPSC frequency amplitude measured during last minute of recording . mIPSC frequency is : 3 . 2 ± 0 . 4 Hz 1 . 3 ± 0 . 2 Hz for Vehicle ( n = 9 from N = 4 ) THC ( n = 7 from N = 4 ) conditions , respectively , p < 0 . 001 2 . 5 ± 0 . 2 Hz 4 . 1 ± 0 . 4 Hz for THC + SB258585 ( n = 8 from N = 4 ) THC + Rapa ( n = 8 from N = 4 ) conditions , respectively , p < 0 . 01 p < 0 . 001 vs . THC - injected mice .

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

B. 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 : histograms represent means ± S . E . M . of AMPA mEPSC frequency amplitude measured during last minute of recording , n = neurons from 3 to 5 mice per group . mEPSC frequency is : 4 . 7 ± 0 . 4 Hz for Vehicle ( n = 9 from N = 7 ) 6 . 8 ± 0 . 4 Hz for THC ( n = 9 from N = 5 ) conditions , respectively , p < 0 . 01and 5 . 3 ± 0 . 5 Hz 4 . 6 ± 0 , 3 Hz for THC + SB258585 ( n = 10 from N = 6 ) THC + Rapa ( n = 9 from N = 5 ) conditions , respectively , p < 0 . 05 p < 0 . 01 vs . THC - injected mice . In B C , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , one - way ANOVA followed by Newman - Keuls .

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

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.


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 . A . resting potential ( RMP ) was determined immediately after whole - formation : - 71 . 8 ± 1 . 2 mV - 60 . 1 ± 1 . 5 mV for Vehicle THC conditions , respectively , p < 0 . 001 .

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

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. 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 only first action potential ( AP ) were used to estimate AP threshold : - 34 . 1 ± 0 . 7 mV - 39 . 7 ± 0 . 8 mV for Vehicle - THC - injected mice , respectively , p < 0 . 001 .

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

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 . rheobase represents minimal current required to induce neuronal firing , it was lowered in THC condition : 670 ± 43 pA 376 ± 43 pA for Vehicle THC conditions , respectively , p < 0 . 001 .

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

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 . firing rate induced by a 150 pA current injection during 250 ms is expressed as number of APs .

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

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 , vs . THC - injected mice . Errors bars correspond to mean ± . Voltage sag is indicated by arrowheads .

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

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 vehicle conditions are similar to those on panel A - D . ( F ) resting potential : 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 ,

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O

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 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

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE

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 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 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

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE

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. (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 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 vehicle conditions are similar to those on panel A - D . ( I ) firing rate . Note that Vehicle - THC - injected mice are 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 .

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

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. (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.


A . In left , normalized peak amplitudes of isolated AMPA EPSCs recorded at - 60 mV , before after pairing protocol , are illustrated . Representative traces of AMPA EPSCs before ( 1 ) or after ( 2 ) pairing protocol are also illustrated ( right panel ) for each experimental condition : Vehicle - injected mice ( circles , n = 3 from N = 3 ) , THC - injected mice ( green circles , n = 3 from N = 3 ) THC - injected mice treated with SB258585 during adolescence ( black circles , n = 3 from N = 3 ) . Errors bars correspond to mean ± .

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

A. 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 . histogram represents means ± of AMPA EPSCs in % of baseline , measured during last 5 min of baseline or last 5 min of recording , for each experimental condition : vehicle - injected mice ( bar , n = 3 from N = 3 ) , THC - injected mice ( green bar , n = 3 from N = 3 ) 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 .

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

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.


( a , b ) Isolated acini from wild - type ( WT ) Trpml1 - / - mice ( ML1 - / - ) were used to determine frequency of Ca2 + oscillations in response to stimulation with 3 10 pM CCK ( a ) response to high concentrations of CCK ( b ) . average frequency from multiple experiments is given in columns as mean ± s . e . m in ( a ) . traces columns in ( b ) show mean ± s . e . m of 3 experiments each includes 4 - 6 acini composed of 6 - 10 .

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

(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 indicated CCK concentrations . ( c ) shows exocytosis during first 5 min of stimulation , ( d ) shows two phases , first rapid second sustained phase , of exocytosis ( e ) shows exocytosis at indicated CCK concentrations measured during 30 min of stimulation . results are averages of 3 - 4 experiments with each mouse . denotes p < 0 . 01 or better # denotes p < 0 . 05 or better .

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

(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.


Panel ( a ) shows example ultrastructure obtained from mice pancreas columns shows granules distribution . Granules was analyzed in at least 10 from 3 wild - type 5 Trpml1 - / - mice .

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Panel (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 ) LAMP1 ( lysosomes ) fold overlap was calculated from 14 11 obtained from 3 wild - type 3 Trpml1 - / - mice , respectively .

O O O O B-TISSUE O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O

(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 Trpml1 - / - acini were labeled with mitotracker ( green ) lysotracker ( red ) . panels show merged green , red DIC . large vesicles are marked by arrows shown at higher magnification in inserts . overlap between lysosomes granules was determined by ImageJ ( see methods ) . results are from 14 wild - type 30 Trpml1 - / - acini each comprising 3 - 7 obtained from 3 wild - type 4 Trpml1 - / - mice .

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

(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 granules VAMP8 Rab27b . Note increased lysosomal / granules interaction .

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

(d) Co-IP of lysosomal LAMP1 and granules VAMP8 and Rab27b. Note the increased lysosomal/granules interaction.


( e ) Enhanced exocytosis of lysosomal acid phosphatase by Trpml1 - / - acini stimulated with 100 pM CCK . results are mean ± s . e . m of 3 experiments with acini obtained from 3 wild - type 3 Trpml1 - / - mice .

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

(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.


( a , b ) Plasmaamylase ( a ) pancreaticMPO ( b ) in 3 mice of each .

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(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 . Shown are examples of average damage in columns , which show mean ± s . e . m of 4 experiments with each mouse .

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(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 after stimulation with 10 nM CCK for 30 min . Shown are example averages .

O O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(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 after stimulation with 10 nM CCK for 30 min were stained for lipid ceramide . figure shows example before stimulation averaged .

O O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O

(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 after stimulation with 10 nM CCK for 30 min were stained with autophagy marker LC3 . figure shows example before stimulation columns depict number of autophagosomes as determined by ImageJ . Results in ( c - f ) are given as mean ± s . e . m of 4 experiments from 4 mice of each at least 10 acini in each experiment .

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

(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.