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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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

mRNA expression of 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 .

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 .

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 O O O O O O O O O O O O O O O O O O O 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 .

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

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

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 ) .

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

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

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 .

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

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 .

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

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 .

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

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

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 .

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

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

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 .

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 .

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 .

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

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 .

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

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 .

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 ) .

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

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 .

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 .

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

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 .

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

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 O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O 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 ) .

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

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 .

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 .

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

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 .

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 .

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

O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O 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 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 )

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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

O O O O O O O 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 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 -

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

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 )

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

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 )

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

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 O O O O O O O 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 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 .

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 )

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

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

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

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 .

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

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 .

O O O O O O O O O O O O O O O O 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 .

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

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 ) .

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


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

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 ) .

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 ) .

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

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 O O O O O O O O O O O O O O O O O O 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 .

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 ) .

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


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

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


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

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


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

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

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 .

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

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

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

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

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

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

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

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

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

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

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 O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

(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 .

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

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 O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

(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 O O O O O O O O O O O O O O O O O O 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) 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 .

(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 .

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

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

(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 O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR 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 O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( a ) Isolated acini from wild - type ( black ) Trpml1 - / - mice ( red ) loaded with Fura2 were used to determine [ Ca2 + ] i in response to stimulation with 100 µM carbachol . traces are mean ± s . e . m . of 6 acinar clusters . Similar results were obtained in 3 experiments .

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

(a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments.


( b , c ) Saliva was collected from wild - type ( black ) Trpml1 - / - mice ( red ) injected I . P . with 10 mg / kg pilocarpine ( b ) or 1 mg / kg pilocarpine 0 . 6 mg / kg isoproterenol ( c ) over 20 - 40 min to stimulate fluid secretion by pilocarpine amylase secretion by isoproterenol . Each condition used 4 - 5 mice from each like results are given as mean ± s . e . m .

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

(b, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m.


( d ) saliva collected in ( c ) was used to determine amount of secreted amylase .

(d) the saliva collected in (c) was used to determine the amount of secreted amylase.


( e , f ) Isolated acini from parotid of wild - type ( black ) or Trpml1 - / - mice ( red ) were used to measure amylase secretion in response to 50 nM isoproterenol for indicated times ( e ) indicated isoproterenol concentrations during 30 min stimulation ( f ) . results are mean ± s . e . m obtained from two mice of each .

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

(e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.


( a ) shows example ( b ) shows granules distribution in parotid determined from 10 obtained from 3 wild - type mice 14 obtained from 4 Trpml1 - / - mice .

O O O O O O O O O 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) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice.


( c ) Co - IP of lysosomal LAMP1 secretory granules VAMP8 Rab27b . Input control is actin .

(c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin.


( d ) Acini isolated from parotid of wild - type Trpml1 - / - mice were incubated with mitotracker ( green ) lysotracker . large vesicles in secretory granules / lysosomal are marked by arrows . average number of enlarged profiles is given in columns obtained from 3 acinar preparations of each .

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

(d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line.


( e ) Shown is time course ( left panel ) averaged 10 min secretion ( columns ) of lysosomal marker acid phosphatase in saliva collected from wild - type ( black ) Trpml1 - / - mice ( red ) stimulated with 1 mg / kg pilocarpine 0 . 6 mg / kg isoproterenol . mean ± s . e . m is from 4 mice of each .

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

(e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.


( a , b ) example traces of mEPSCs recorded from wild - type ( a ) Trpml1 - / - ( b ) micebrain slices as detailed in Methods . lower traces in each panel show portion of traces marked by dotted boxes in an expanded time scale . ( c ) histogram distribution of events at each mEPSC . lower columns show distribution of higher amplitude mEPSCs . ( d ) frequency of mEPSCs in each . results in ( c , b ) are mean ± s . e . m of 8 experiments each obtained from 4 mice of each .

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

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


( e ) mean ± s . e . m of Ca2 + signal in response to depolarization with 30 mM KCl of 6 5 experiments with wild - type Trpml1 - / - slices , respectively .

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

(e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.


( a ) example of synapses in wild - type Trpml1 - / - micebraincortex . ( b ) distribution of synaptic vesicle determined in 10 14 obtained from 3 wild - type 3 Trpml1 - / - mice , respectively . ( c ) example at low magnification of wild - type Trpml1 - / - micebraincortical slices with PSD profiles marked by arrows . ( d ) average number of PSD / µm2 analyzed in at least 10 obtained from 3 mice of each .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) example TEM images of synapses in wild-type and Trpml1-/-micebraincortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/-mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/-micebraincortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line.


( e ) Glutamate secretion by cultured cerebral cortical neurons . Total glutamate measured in non - stimulated wild - type ( black ) Trpml1 - / - ( green ) neurons neurons stimulated by depolarization with 30 mM KCl ( blue red ) .

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

(e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red).


( f ) Rescue of glutamate secretion by expression of TRPML1 in cultured cerebral cortical neurons . Total glutamate measured in non - stimulated Trpml1 - / - neurons ( green ) Trpml1 - / - neurons transfected with TRPML1 ( black ) untransfected ( red ) TRPML1 - transfected ( clue ) Trpml1 - / - neurons stimulated by depolarization with 30 mM KCl .

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

(f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl.


( g ) averaged Co - IP of SNAP25 VAMP2 in three brain regions of wild - type Trpml1 - / - mice . of blots are given in Extended view Figure E4 .

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

(g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.


( B ) Assessment of exogenous DDX60 expression . HEK293T transfected with DDX60 wild type ( wt ) , or DDX60 mutants analyzed by western blot for DDX60 , ß - actin GAPDH ( loading controls ) , RFP ( reporter ) . DDX60 RFP relative to GAPDH from one representative blot are shown below .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Assessment of exogenous DDX60 expression. HEK293T cells transfected with DDX60 wild type (wt), or DDX60 mutants and analyzed by western blot for DDX60, ß-actin and GAPDH (loading controls), and RFP (reporter). DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below.


( C ) HCV antiviral assays with DDX60 wt or mutant panel . Huh - 7 transfected with an RFP containing plasmid backbone encoding either Firefly luciferase ( Fluc , negative , transfection control ) , IRF1 ( positive antiviral control ) , DDX60 wt , or DDX60 mutants infected with HCV - Ypet , a bicistronic reporter HCV where Ypet reporter is driven by HCV IRES HCV polyprotein consisting of C , E1 , E2 , p7 , NS2 , NS3 , 4A , 4B , NS5A , NS5B is driven by EMCV IRES . Data information : For ( C ) of Ypet + in RFP + is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against Fluc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HCV antiviral assays with DDX60 wt or mutant panel. Huh-7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase (Fluc, negative, and transfection control), IRF1 (positive antiviral control), DDX60 wt, or DDX60 mutants and infected with HCV-Ypet, a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C, E1, E2, p7, NS2, NS3, 4A, 4B, NS5A, and NS5B is driven by EMCV IRES. Data information: For (C) percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.


( D ) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs . Huh - 7 transfected as in ( C ) infected with either bicistronic HCV - Ypet ( left ) or monocistronic HCV J6 / JFH - 5AB - YPet . Ypet reporter in monocistronic HCV is placed in between NS5A NS5B . Data information : For ( D ) , of Ypet + in RFP + is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against Fluc .

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

(D) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs. Huh-7 cells transfected as in (C) and infected with either bicistronic HCV-Ypet (left) or monocistronic HCV J6/JFH-5AB-YPet. Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B. Data information: For (D), percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.


( A ) Plasmid - dual luciferase bicistronic reporter assays . HEK293T co - transfected with dual luciferase bicistronic reporter plasmid ( Renilla luciferase ( Rluc ) translationally driven by a 5 ' cap , Firefly luciferase ( Fluc ) translationally driven by different IRESs as indicated ) GFP plasmid ( negative control ) or increasing amounts of IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A mutant . Total amount of transfected was kept constant by supplementing transfection mixes with GFP plasmid . Luciferase units after lysis is plotted as a of GFP transfected ( left ) of IRES Fluc units over 5 ' cap Rluc units ( middle ) . Diagram to right of ratios plot depicts expected ratios of IRES - driven Fluc units to 5 ' cap - driven Rluc units given either : equal translation of 5 ' cap - driven Rluc IRES - driven Fluc ( top ) , greater translation of IRES - driven Fluc ( middle ) , or greater translation of 5 ' cap - driven Rluc ( bottom ) . Data information : Data shows mean ± SD for at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against GFP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid-based dual luciferase bicistronic reporter assays. HEK293T cells co-transfected with dual luciferase bicistronic reporter plasmid (Renilla luciferase (Rluc) translationally driven by a 5' cap, and Firefly luciferase (Fluc) translationally driven by different IRESs as indicated) and GFP plasmid (negative control) or increasing amounts of IRF1 (positive control), DDX60 wt, or DDX60 E890A mutant. Total amount of DNA transfected was kept constant by supplementing transfection mixes with GFP plasmid. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells (left) and ratio of IRES Fluc units over 5' cap Rluc units (middle). Diagram to the right of ratios plot depicts expected ratios of IRES-driven Fluc units to 5' cap-driven Rluc units given either: equal translation of 5' cap-driven Rluc and IRES-driven Fluc (top), greater translation of IRES-driven Fluc (middle), or greater translation of 5' cap-driven Rluc (bottom). Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.


( B ) - monocistronic luciferase reporter assays . HEK293T transfected with GFP ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A subsequently transfected with in vitro transcribed 5 ' cap or different IRES driven Fluc mRNA constructs as indicated . Luciferase units after lysis is plotted as a of GFP transfected . Data information : Data shows mean ± SD for at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against GFP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNA-based monocistronic luciferase reporter assays. HEK293T cells transfected with GFP (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and subsequently transfected with in vitro transcribed 5' cap or different IRES driven Fluc mRNA constructs as indicated. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells. Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.


( A - E ) ( A , B , E ) Multi - cycle infection assays with type I type II IRES containing . HeLa stably expressing Firefly luciferase ( Fluc ) ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A infected with ( A ) encephalomyocarditis , ( B ) poliovirus , or ( E ) a chimeric poliovirus with poliovirus IRES replaced with IRES of EMCV ( EMCV - IRES - PV ) at MOI 0 . 001 . Supernatants were collected 24 hours post infection ( hpi ) ( EMCV ) or 48 hpi ( poliovirus EMCV - IRES - PV ) titers determined via plaque on HeLa . ( C , D ) Single - cycle infection assays with type I type II IRES containing . BHK - J stably expressing empty vector ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A infected with ( C ) foot mouth disease , or ( D ) bovine enterovirus - 1 at MOI 1 . Supernatants were collected 5 hours post infection ( hpi ) titers determined via plaque on BHK - 21 clone 13 . Data information : Data shows mean ± infectious titers relative to Fluc from at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against DDX60 E890A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (A, B, E) Multi-cycle infection assays with type I and type II IRES containing viruses. HeLa cells stably expressing Firefly luciferase (Fluc) (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (A) encephalomyocarditis virus, (B) poliovirus, or (E) a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV (EMCV-IRES-PV) at MOI 0.001. Supernatants were collected 24 hours post infection (hpi) (EMCV) or 48 hpi (poliovirus and EMCV-IRES-PV) and titers determined via plaque assay on HeLa cells. (C, D) Single-cycle infection assays with type I and type II IRES containing viruses. BHK-J cells stably expressing empty vector (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (C) foot and mouth disease virus, or (D) bovine enterovirus-1 at MOI 1. Supernatants were collected 5 hours post infection (hpi) and titers determined via plaque assay on BHK-21 clone 13 cells. Data information: Data shows mean ± SEM percent infectious titers relative to Fluc from at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against DDX60 E890A.


B ) HEK293T transfected with DDX60 wt or DDX60 E890A ( negative control ) transfected with in vitro transcribed 5 ' cap - or IRES - driven Fluc mRNA constructs as indicated lysed 16 hours later . ( B ) IRES - or cap - driven translation from same samples assayed in parallel using luciferase . Data information : B ) Mean ± SD from at least n = 3 biological replicates ; * * * * FDR < 0 . 01 % ( p < 0 . 0001 ) using unpaired t - with Welch correction Benjamini Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected . ns , non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells transfected with DDX60 wt or DDX60 E890A (negative control) and transfected with in vitro transcribed 5' cap- or IRES-driven Fluc mRNA constructs as indicated and lysed 16 hours later. (B) IRES- or cap-driven translation from the same samples assayed in parallel using luciferase assay. Data information: B) Mean ± SD from at least n=3 biological replicates; ****FDR < 0.01% (p<0.0001) using unpaired t-test with Welch correction and Benjamini and Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected cells. ns, non-significant.


( C , HEK293T stably expressing a type I IFN sensitive response element ( ISRE ) driven Fluc gene were transfected with increasing amounts of RIG - I , RIG - I in combination with DDX60 wt , or RIG - I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize total amount of transfected . Transfected were then treated with either PBS ( negative control ) or transfected with LMW poly ( I : C ) . In parallel , untransfected were treated with either PBS ( mock ) , transfected with LMW poly ( I : C ) , or treated with IFN - ß ( positive control ) . were subsequently used for a luciferase to assess ISRE activity ( C ) Data information : ( C ) Mean ± SD from at least n = 3 biological replicates ; * p < 0 . 05 using unpaired t - with Welch ' s correction , ns , not significant using repeated measures one - way ANOVA with Geisser - Greenhouse correction comparing RIG - I transfected vs RIG - I + DDX60 wt or RIG - I + DDX60 E890A transfected .

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

(C, HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. Transfected cells were then treated with either PBS (negative control) or transfected with LMW poly(I:C). In parallel, untransfected cells were treated with either PBS (mock), transfected with LMW poly(I:C), or treated with IFN-ß (positive control). Cells were subsequently used for a luciferase assay to assess ISRE activity (C) Data information: (C) Mean ± SD from at least n=3 biological replicates; *p < 0.05 using unpaired t-test with Welch's correction, ns, not significant using repeated measures one-way ANOVA with Geisser-Greenhouse correction comparing RIG-I transfected vs RIG-I + DDX60 wt or RIG-I + DDX60 E890A transfected cells.


D ) HEK293T stably expressing a type I IFN sensitive response element ( ISRE ) driven Fluc gene were transfected with increasing amounts of RIG - I , RIG - I in combination with DDX60 wt , or RIG - I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize total amount of transfected . western blot for of DDX60 , RIG - I , ß - actin ( loading control ) , or GAPDH ( loading control ) products ( D ) . of DDX60 RIG - I band intensities relative to ß - actin are shown below each respective lane in ( D ) . Data information : ( D ) representative western blot from ( C ) .

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

D) HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. western blot for analysis of DDX60, RIG-I, ß-actin (loading control), or GAPDH (loading control) protein products (D). Quantification of DDX60 and RIG-I band intensities relative to ß-actin are shown below each respective lane in (D). Data information: (D) representative western blot from (C).


( B ) Effect of DDX60 on 5 ' cap ( left ) or EMCV IRES ( right ) driven Fluc mRNA polysomes . Data information : Mean ± of Fluc mRNA of each fraction relative to Fluc mRNA in fractions , from n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of DDX60 on 5' cap (left) or EMCV IRES (right) driven Fluc mRNA polysomes. Data information: Mean percent ± SEM of Fluc mRNA of each fraction relative to Fluc mRNA in all fractions, from n=3 biological replicates.


( B ) depicted in ( A ) were subjected to polysome profiling by lysing in presence of cycloheximide protease phosphatase inhibitors , applying lysates to 15 % - 50 % sucrose gradients , subjecting to ultracentrifugation . Centrifuged gradients were run through a fractionator total in each fraction was measured by UV absorbance ( 254 nm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells depicted in (A) were subjected to polysome profiling by lysing in the presence of cycloheximide and protease and phosphatase inhibitors, applying cell lysates to 15%-50% sucrose gradients, and subjecting to ultracentrifugation. Centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance (254 nm).


( C ) Representative results of electrophoresis of fractions . Shown are results from one replicate of DDX60 - transfected , EMCV - IRES - PV - infected . was analyzed by Bioanalyzer to visualize 28S , 18S ribosome subunit distribution . RIN , integrity number .

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

(C) Representative results of RNA electrophoresis analysis of individual fractions. Shown are results from one replicate of DDX60-transfected, EMCV-IRES-PV-infected cells. RNA was analyzed by Bioanalyzer to visualize 28S, and 18S ribosome subunit distribution. RIN, RNA integrity number.


( D ) Effect of DDX60 expression on parental poliovirus ( type I IRES , purple circles ) versus chimeric poliovirus ( type II IRES , red squares ) VP4 mRNA polysomes ( left ) GAPDH mRNA polysomes in same samples . Data information : Mean ± of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in fractions from n = 3 replicate plates per condition .

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

(D) Effect of DDX60 expression on parental poliovirus (type I IRES, purple circles) versus chimeric poliovirus (type II IRES, red squares) VP4 mRNA polysomes (left) and cellular GAPDH mRNA polysomes in the same samples. Data information: Mean percent ± SEM of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in all fractions from n=3 replicate plates per condition.


B Expression ( ) secretion ( media ) of VgrG1 from V . parahaemolyticus RIMD 2210633 derivative POR1 ( Vpara ; T6SS1 + ) its T6SS1 - mutant ( Vpara / ∆ hcp1 ) , from V . natriegens ( Vnat ) carrying indicated plasmids . Samples were treated ( + ) or not ( - ) with 20 µM phenamil to activate surface sensing in media containing 3 % NaCl at 30 ° C for 5 h . Loading control ( LC ) is shown for total lysates . Arrows denote bands corresponding to VgrG1 . Asterisks denote non - specific bands detected in Vnat samples .

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

B Expression (cells) and secretion (media) of VgrG1 from V. parahaemolyticus RIMD 2210633 derivative POR1 (Vpara; T6SS1+) and its T6SS1- mutant (Vpara/∆hcp1), and from V. natriegens (Vnat) carrying the indicated plasmids. Samples were treated (+) or not (-) with 20 µM phenamil to activate surface sensing in media containing 3% NaCl at 30 °C for 5 h. Loading control (LC) is shown for total protein lysates. Arrows denote bands corresponding to VgrG1. Asterisks denote non-specific bands detected in Vnat samples.


C Viability of V . natriegens prey before ( 0 h ) after ( 4 h ) co - incubation with indicated attackers , as described in B , on media containing 3 % NaCl at 30 ° C . Data are shown as mean ± SD , n = 3 technical replicates . Statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above . A significant difference was considered as P < 0 . 05 . DL , detection limit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with the indicated attackers, as described in B, on media containing 3% NaCl at 30 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit.


B Viability of V . natriegens prey before ( 0 h ) after ( 4 h ) co - incubation with wild type V . natriegens attackers harboring indicated plasmids , on media containing 3 % NaCl at 30 ° C . Data information : data are shown as mean ± SD , n = 3 technical replicates ; statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above is color coded to match relevant samples . A significant difference was considered as P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with wild type V. natriegens attackers harboring the indicated plasmids, on media containing 3% NaCl at 30 °C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


C Expression ( ) secretion ( media ) of VgrG1 from V . natriegens containing an arabinose - inducible vp1407 in chromosomal dns locus ( VnatReg ) harboring an empty plasmid ( pEmpty ) or plasmids containing VpT6SS1 that lacks vp1407 ( pT6SS1Ind ) or both vp1407 hcp1 ( pT6SS1Ind / ∆ hcp1 ; T6SS - ) . Samples were grown in media containing 3 % NaCl either supplemented ( + ) or not ( - ) with 0 . 1 % arabinose ( Ara ) at 30 ° C . Loading control ( LC ) is shown for total lysates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Expression (cells) and secretion (media) of VgrG1 from V. natriegens containing an arabinose-inducible vp1407 in the chromosomal dns locus (VnatReg) and harboring an empty plasmid (pEmpty) or plasmids containing VpT6SS1 that lacks vp1407 (pT6SS1Ind) or both vp1407 and hcp1 (pT6SS1Ind/∆hcp1; T6SS-). Samples were grown in media containing 3% NaCl and either supplemented (+) or not (-) with 0.1% arabinose (Ara) at 30 °C. Loading control (LC) is shown for total protein lysates.


E Viability of V . natriegens prey before ( 0 h ) after ( 4 h ) co - incubation with VnatReg attackers carrying indicated plasmids on solid media plates , as described in C . Data information : data are shown as mean ± SD , n = 3 technical replicates ; statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above is color coded to match relevant samples . A significant difference was considered as P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with VnatReg attackers carrying the indicated plasmids on solid media plates, as described in C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


A , B Viability of V . natriegens prey before ( 0 h ) after ( 4 or 24 h ) co - incubation with VnatReg attackers harboring indicated plasmids , on media containing 3 % NaCl 0 . 1 % arabinose ( A ) , or on media containing 0 . 1 % arabinose at 28 ° C ( B ) . Data information : Data are shown as mean ± SD , n = 3 technical replicates . Statistical significance between samples at 4 or 24 h timepoints by an unpaired , two - Student ' s t - is denoted above is color coded to match relevant samples . A significant difference was considered as P < 0 . 05 .

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

A, B Viability counts of V. natriegens prey before (0 h) and after (4 or 24 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose (A), or on media containing 0.1% arabinose at 28 °C (B). Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.