E . Quantification of PDI fluorescence intensity into Lamp1 positive lysosomes . Mean + / - standard error of the mean ( sem ) of N = 10 cells / experiment were analyzed . Analysis of variance one way ( ANOVA ) p < 0 . 001 ; Sidak ' s multiple comparison test * * * p < 0 . 0005 ; NS not 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

E. Quantification of PDI fluorescence intensity into Lamp1 positive lysosomes. Mean +/- standard error of the mean (sem) of N=10 cells/experiment were analyzed. Analysis of variance one way (ANOVA) p<0.001; Sidak's multiple comparison test ***p<0.0005; NS not significant.


F . Western blot analysis of type II procollagen from a pool of medaka fish embryos with indicated genotypes , showing procollagen accumulation in Fam134bmo fish . β - actin was used as loading control . Bar graph shows quantification of type II procollagen normalized to β - actin . N = 3 biological replicates . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot analysis of type II procollagen from a pool of medaka fish embryos with indicated genotypes, showing procollagen accumulation in Fam134bmo fish. β-actin was used as loading control. Bar graph shows quantification of type II procollagen normalized to β-actin. N=3 biological replicates. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.


G . Representative TEM images of medaka fish chondrocytes . Arrows indicated the ER . Insets show ER enlargement in Fam134bmo chondrocyte . N = 3 fish / genotype were analyzed . Bar graph shows number of ER enlargement per cell in medaka fish with indicated genotypes . N = 29 cells ( Fam134bwt ) and N = 37 cells ( Fam134bmo ) were analyzed . Mean + / - standard error of the mean ( sem ) . Student ' s un - paired T - test * * * p < 0 . 0005 . Scale bar 500 nm . N = nucleus , ER = Endoplasmic Reticulum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative TEM images of medaka fish chondrocytes. Arrows indicated the ER. Insets show ER enlargement in Fam134bmo chondrocyte. N=3 fish/genotype were analyzed. Bar graph shows number of ER enlargement per cell in medaka fish with indicated genotypes. N=29 cells (Fam134bwt) and N=37 cells (Fam134bmo) were analyzed. Mean +/- standard error of the mean (sem). Student's un-paired T-test ***p<0.0005. Scale bar 500 nm. N=nucleus, ER= Endoplasmic Reticulum.


H . Lateral ( top ) and ventral ( bottom ) projections of medaka fish embryos with indicated genotypes . Scale bar 1mm . Bar graphs show quantification of total length and head size of medaka fish model of Fam134bmo expressed as % relative to the scramble . Mean + / - standard error of the mean ( sem ) of at least n = 8 fish / genotype . Student un - paired T - Test * p < 0 . 05 .

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

H. Lateral (top) and ventral (bottom) projections of medaka fish embryos with indicated genotypes. Scale bar 1mm. Bar graphs show quantification of total length and head size of medaka fish model of Fam134bmo expressed as % relative to the scramble. Mean +/- standard error of the mean (sem) of at least n=8 fish/genotype. Student un-paired T-Test *p<0.05.


Alcian blue ( cartilage ) ( I ) staining of scramble and Fam134bmo medaka fish . Graph shows quantification of Ethmoid plate ( EP ) , Palatoquadrate ( PQ ) , Ceratohyal ( CH ) , Paired Prootics ( PO ) , Ceretobranchials 1 to 5 ( CB1 to CB5 ) cartilage length ( I ) in Fam134bmo and scramble fish . Values were expressed as % relative to the scramble ( 100 % red dotted line ) . Mean + / - standard error of the mean ( sem ) of n = 9 fish / genotype . Student unpaired T - Test * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 . NS not 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 O O O O O O O O O O O O O O O

Alcian blue (cartilage) (I) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) cartilage length (I) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.


Alizarin Red ( bone ) ( J ) staining of scramble and Fam134bmo medaka fish . Graph shows quantification of Ethmoid plate ( EP ) , Palatoquadrate ( PQ ) , Ceratohyal ( CH ) , Paired Prootics ( PO ) , Ceretobranchials 1 to 5 ( CB1 to CB5 ) bone mineralization ( J ) in Fam134bmo and scramble fish . Values were expressed as % relative to the scramble ( 100 % red dotted line ) . Mean + / - standard error of the mean ( sem ) of n = 9 fish / genotype . Student unpaired T - Test * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 0005 . NS not significant .

Alizarin Red (bone) (J) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) bone mineralization (J) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.


( D ) Immunofluorescence of neurons expressing BioID2 - tau or BioID2 ( myc ) or non - transduced control cells . Cells were incubated in biotin medium ( 50 μM ) or vehicle medium for 30 h prior to fixation and probed for biotin ( streptavidin , SA ) and cell nuclei ( DAPI ) ( n = 3 biological replicates ) . BioID2 - tau and incorporated biotin co - localise in axons and somata . Scale bar , 25 μm .

(D) Immunofluorescence of neurons expressing BioID2-tau or BioID2 (myc) or non-transduced control cells. Cells were incubated in biotin medium (50 μM) or vehicle medium for 30 h prior to fixation and probed for biotin (streptavidin, SA) and cell nuclei (DAPI) (n = 3 biological replicates). BioID2-tau and incorporated biotin co-localise in axons and somata. Scale bar, 25 μm.


( B ) Immunoblot of combined brain lysates from mice ( P35 ) expressing BioID2 - tau or BioID2 control ( n = 4 biological replicates ) probed for myc - tagged BioID2 and BioID2 - tau . Controls : lysates from ( 1 ) BioID2 - or ( 2 ) BioID2 - tau - expressing and ( 3 ) non - transduced mouse brain . Loading , glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) . * , unspecific band . Relative quantification confirmed similar expression levels of BioID2 and BioID2 - tau . Means ± S . E . M . ns , not significant ( Student ' s t - test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of combined brain lysates from mice (P35) expressing BioID2-tau or BioID2 control (n = 4 biological replicates) probed for myc-tagged BioID2 and BioID2-tau. Controls: lysates from (1) BioID2- or (2) BioID2-tau-expressing and (3) non-transduced mouse brain. Loading, glyceraldehyde-3-phosphate dehydrogenase (Gapdh). *, unspecific band. Relative quantification confirmed similar expression levels of BioID2 and BioID2-tau. Means ± S.E.M. ns, not significant (Student's t-test)


( C ) Immunofluorescence of P35 mouse brain expressing BioID2 - tau . Hippocampal Cornu ammonis 1 ( CA1 ) region was probed for BioID2 - tau ( myc ) , for incorporated biotin ( streptavidin , SA ) and for cell nuclei ( DAPI ) ( n = 3 - 5 biological replicates ) . Scale bar , 50 μm .

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

(C) Immunofluorescence of P35 mouse brain expressing BioID2-tau. Hippocampal Cornu ammonis 1 (CA1) region was probed for BioID2-tau (myc), for incorporated biotin (streptavidin, SA) and for cell nuclei (DAPI) (n = 3-5 biological replicates). Scale bar, 50 μm.


( A ) Confocal immunofluorescence for endogenous tau and NSF in primary murine hippocampal neurons ( DIV15 ) . Colocalization of tau and NSF is apparent in perinuclear compartments ( arrows ) and neurites ( insets i - iii ) . Scale bar , 25 μm . Inset of magnified neurite with partial overlap of tau and NSF signals ( arrowheads ) . Scale bar , 3 μm . Co - distribution ( R Ch1 - Ch2 ) are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal immunofluorescence for endogenous tau and NSF in primary murine hippocampal neurons (DIV15). Colocalization of tau and NSF is apparent in perinuclear compartments (arrows) and neurites (insets i-iii). Scale bar, 25 μm. Inset of magnified neurite with partial overlap of tau and NSF signals (arrowheads). Scale bar, 3 μm. Co-distribution (R Ch1-Ch2) are indicated.


( B - C ) Co - immunoprecipitation ( Co - IP ) of tau and NSF from mouse brain . ( B ) Endogenous and transgenic tau were immunoprecipitated from mouse brain lysates of tau - / - , ALZ17tg or ALZ17non - tg mice using anti - tau antibody followed by immunoblot probed for NSF , tau and α - actin . Endogenous NSF was co - immunoprecipitated with transgenic and endogenous tau from ALZ17tg or ALZ17non - tg but not from tau - / - with . GAPDH , input loading control ( n = 3 biological replicates ) . Recombinant tau and NSF served as controls . ( C ) Densitometric quantification of co - IP results in ( B ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Co-immunoprecipitation (Co-IP) of tau and NSF from mouse brain. (B) Endogenous and transgenic tau were immunoprecipitated from mouse brain lysates of tau-/-, ALZ17tg or ALZ17non-tg mice using anti-tau antibody followed by immunoblot probed for NSF, tau and α-actin. Endogenous NSF was co-immunoprecipitated with transgenic and endogenous tau from ALZ17tg or ALZ17non-tg but not from tau-/- with. GAPDH, input loading control (n = 3 biological replicates). Recombinant tau and NSF served as controls. (C) Densitometric quantification of co-IP results in (B). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


Co - IP of HA - tagged tau with NSF from 293T cells . ( E ) Co - IP of NSF with haemagglutinin ( HA ) - tagged tau variants from cultured cells . HA - tagged tau variants and NSF were expressed in 293T cells followed by immunoprecipitation using HA antibody and immunoblot probed for NSf , tau and GAPDH . ( 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 O O O O O O O O O O O O O O O O O O O O O O

Co-IP of HA-tagged tau with NSF from 293T cells. (E) Co-IP of NSF with haemagglutinin (HA)-tagged tau variants from cultured cells. HA-tagged tau variants and NSF were expressed in 293T cells followed by immunoprecipitation using HA antibody and immunoblot probed for NSf, tau and GAPDH. (n = 3 biological replicates).


( F ) Microscale thermophoretic ( MST ) measurement of direct tau and NSF interaction . MST binding curve of recombinant hexameric NT - 647 - NHS - labelled NSF ( 27 pM ) and recombinant tau ( 18 mM - 0 . 275 nM ) . Dissociation constant : KD = 146 ± 70 nM ( sigmoidal curve fit , R2 = 0 . 844 ) . ( n = 5 technical replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Microscale thermophoretic (MST) measurement of direct tau and NSF interaction. MST binding curve of recombinant hexameric NT-647-NHS-labelled NSF (27 pM) and recombinant tau (18 mM - 0.275 nM). Dissociation constant: KD = 146 ± 70 nM (sigmoidal curve fit, R2 = 0.844). (n = 5 technical replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( G ) Inhibition of NSF ATPase activity with increasing concentrations of tau . NSF activity ( 70 nM ) was assessed alone or in the presence of NSF - inhibiting peptide TAT - NSF or control TAT - Scramble peptides ( 140 nM ) ; or co - incubated with recombinant tau protein ( 8 nM , 16 nM , 24 nM ) , or heat - denatured tau ( dTau , 8 nM ) or albumin ( Alb , 16 nM ) . Data were fitted to Michaelis - Menten model to determine rate constants ( R2 = 0 . 904 - 0 . 994 for all conditions ; 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 O O O O O O O O O O O O O O O O O O O O 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) Inhibition of NSF ATPase activity with increasing concentrations of tau. NSF activity (70 nM) was assessed alone or in the presence of NSF-inhibiting peptide TAT-NSF or control TAT-Scramble peptides (140 nM); or co-incubated with recombinant tau protein (8 nM, 16 nM, 24 nM), or heat-denatured tau (dTau, 8 nM) or albumin (Alb, 16 nM). Data were fitted to Michaelis-Menten model to determine rate constants (R2 = 0.904 - 0.994 for all conditions; n = 3 technical replicates).


( H ) Rate constants derived from kinetic data in i . ( n = 3 technical replicates ) ATP hydrolysis is similar in NSF and NSF incubated with control peptide TAT - Scr , albumin , or denatured tau , yet lower with increasing concentrations of tau or after addition of NSF - inhibiting peptide TAT - NSF . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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

(H) Rate constants derived from kinetic data in i. (n = 3 technical replicates) ATP hydrolysis is similar in NSF and NSF incubated with control peptide TAT-Scr, albumin, or denatured tau, yet lower with increasing concentrations of tau or after addition of NSF-inhibiting peptide TAT-NSF. Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( A ) Impaired AMPA - type glutamate receptor surface expression in cultured tau - / - neurons . Cortical tau + / + or tau - / - neurons were surface biotinylated after incubation with non - competitive NMDAR antagonist MK - 801 ( 50 μM , 3 min ) and / or GABAAR antagonist bicuculline ( 30 μM , 10 min ) at 37 ° C . Surface GluA1 levels were detected by immunoblot after with streptavidin ( SA ) enrichment . Input was probed for total cellular GluA1 , NSF , tau and βIII - tubulin . ( n = 3 biological replicates ) Input and SA - enrichment from non - surface biotinylated neurons served as negative controls . ( B ) Reduced neuronal surface GluA1 in tau - / - neurons . Quantification of tau + / + and tau - / - surface GluA1 upon treatment and SA - enrichment shown in ( A ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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

(A) Impaired AMPA-type glutamate receptor surface expression in cultured tau-/- neurons. Cortical tau+/+ or tau-/- neurons were surface biotinylated after incubation with non-competitive NMDAR antagonist MK-801 (50 μM, 3 min) and/or GABAAR antagonist bicuculline (30 μM, 10 min) at 37°C. Surface GluA1 levels were detected by immunoblot after with streptavidin (SA) enrichment. Input was probed for total cellular GluA1, NSF, tau and βIII-tubulin. (n = 3 biological replicates) Input and SA-enrichment from non-surface biotinylated neurons served as negative controls. (B) Reduced neuronal surface GluA1 in tau-/- neurons. Quantification of tau+/+ and tau-/- surface GluA1 upon treatment and SA-enrichment shown in (A). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( C ) Cell surface GluA1 and total NSF immunostaining in primary mouse hippocampal neurons ( DIV15 ) . Tau + / + , tau - / - neurons and tau - / - expressing human tau ( tau - / - . AAVtau ) were treated with either vehicle ( Veh ; DMSO ) or MK - 801 ( 50 μM ) for 3 min . Representative confocal images of whole cell ( left ) and neurites ( right ) are shown ( n = 3 biological replicates ) . NSF , surface GluA1 and cell nuclei ( DAPI ) were visualized . Scale bar , 25 μm . ( D - E ) Quantification of neurite surface GluA1 and neurite NSF relative to tau + / + Veh control . Reduced surface GluA1 ( D ) and total NSF ( n = 10 - 12 neurites from 3 biological replicates ) ( E ) localized to neurites in tau - / - neurons treated with MK801 ( n = 21 - 23 neurites from 3 biological replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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

(C) Cell surface GluA1 and total NSF immunostaining in primary mouse hippocampal neurons (DIV15). Tau+/+, tau-/- neurons and tau-/- expressing human tau (tau-/-.AAVtau) were treated with either vehicle (Veh; DMSO) or MK-801 (50 μM) for 3 min. Representative confocal images of whole cell (left) and neurites (right) are shown (n = 3 biological replicates). NSF, surface GluA1 and cell nuclei (DAPI) were visualized. Scale bar, 25 μm. (D-E) Quantification of neurite surface GluA1 and neurite NSF relative to tau+/+ Veh control. Reduced surface GluA1 (D) and total NSF (n = 10-12 neurites from 3 biological replicates) (E) localized to neurites in tau-/- neurons treated with MK801 (n = 21-23 neurites from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( F ) Changes in secondary dendrite GluA2 expression in neurons ( DIV18 ) upon cLTP / cLTD induction . Tau - / - neurons were transfected with tau at DIV12 ( = tau - / - . tau ) or mock transfected ( tau - / - ) . Cells were stained for GluA2 , dendritic marker Map2 , and NSF at 2 h post - cLTP / cLTD induction or treatment with control artificial cerebrospinal fluid ( aCSF ) . Dendritic GluA2 cluster analysis was based on GluA2 - Map2 binary intensity map . Representative insets from n = 10 - 20 dendrites per condition from 2 mice per genotype . Scale bar , 3 μm . ( G ) Quantification of GluA2 mean intensity relative to Map2 area and normalised to aCSF controls . Secondary dendrite GluA2 increased in tau - expressing ( tau + / + and tau - / - . tau ) neurons upon cLTP induction . ( n = 10 - 19 dendrites from 3 biological replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Changes in secondary dendrite GluA2 expression in neurons (DIV18) upon cLTP/cLTD induction. Tau-/- neurons were transfected with tau at DIV12 (= tau-/-.tau) or mock transfected (tau-/-). Cells were stained for GluA2, dendritic marker Map2, and NSF at 2 h post-cLTP/cLTD induction or treatment with control artificial cerebrospinal fluid (aCSF). Dendritic GluA2 cluster analysis was based on GluA2-Map2 binary intensity map. Representative insets from n = 10-20 dendrites per condition from 2 mice per genotype. Scale bar, 3 μm. (G) Quantification of GluA2 mean intensity relative to Map2 area and normalised to aCSF controls. Secondary dendrite GluA2 increased in tau-expressing (tau+/+ and tau-/-.tau) neurons upon cLTP induction. (n = 10-19 dendrites from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( H ) Representative immunostaining for whole - cell NSF and GluA2 in tau + / + , tau - / - and tau - transfected tau - / - ( tau - / - . tau ) neurons at 2 h post - cLTP / cLTD induction or treatment with control artificial cerebrospinal fluid ( aCSF ) . ( n = 3 - 6 biological replicates per condition with 10 - 20 cells each ) . Scale bar , 10 μm . Arrows indicate areas of marked co - distribution . ( I ) Co - localisation analysis of NSF and GluA2 upon chemical LTP / LTD induction . Note significantly higher proportional co - distribution of NSF and GluA2 in tau - expressing ( tau + / + and tau - / - . tau ) cells upon cLTP induction . ( n = 3 - 6 cells from 3 biological replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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

(H) Representative immunostaining for whole-cell NSF and GluA2 in tau+/+, tau-/- and tau-transfected tau-/- (tau-/-.tau) neurons at 2 h post-cLTP/cLTD induction or treatment with control artificial cerebrospinal fluid (aCSF). (n = 3-6 biological replicates per condition with 10-20 cells each). Scale bar, 10 μm. Arrows indicate areas of marked co-distribution. (I) Co-localisation analysis of NSF and GluA2 upon chemical LTP/LTD induction. Note significantly higher proportional co-distribution of NSF and GluA2 in tau-expressing (tau+/+ and tau-/-.tau) cells upon cLTP induction. (n = 3-6 cells from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( B ) Examples of sagittal sections of tau - / - and AAV . syn - hTau - injected tau - / - ( tau - / - . AAVtau ) mice showing localized AAV - mediated expression of eGFP or tau and position of infusion cannulas ending in the hippocampus ( dashed line ) . GluA1 , immunolabelling for GluA1 AMPA receptor subunit . Scale bar , 50 μm .

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(B) Examples of sagittal sections of tau-/- and AAV.syn-hTau-injected tau-/- (tau-/-.AAVtau) mice showing localized AAV-mediated expression of eGFP or tau and position of infusion cannulas ending in the hippocampus (dashed line). GluA1, immunolabelling for GluA1 AMPA receptor subunit. Scale bar, 50 μm.


( C ) Cued fear conditioning memory in tau + / + , tau - / - and tau - / - . AAVtau mice upon bilateral infusion of TAT - NSF or TAT - Scr . Due to infusion of Tat - fusion peptides occurring under brief isoflurane anaesthesia , a general anaesthesia ( GA ) control is included . Note that infusion of TAT - NSF lowers memory performance to comparable levels in tau + / + , tau - / - and tau - / - . AAVtau mice . ( n = 2 - 5 biological replicates ) Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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(C) Cued fear conditioning memory in tau+/+, tau-/- and tau-/-.AAVtau mice upon bilateral infusion of TAT-NSF or TAT-Scr. Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included. Note that infusion of TAT-NSF lowers memory performance to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. (n = 2-5 biological replicates) Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( D ) Novel object preference ( fold preference over familiar object ) in tau + / + , tau - / - and tau - / - . AAVtau mice upon bilateral infusion of TAT - NSF or TAT - Scr ( n = 2 - 5 biological replicates ) . Due to infusion of Tat - fusion peptides occurring under brief isoflurane anaesthesia , a general anaesthesia ( GA ) control is included ( n = 2 for GA ) . Note that infusion of TAT - NSF lowers object discrimination to comparable levels in tau + / + , tau - / - and tau - / - . AAVtau mice . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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(D) Novel object preference (fold preference over familiar object) in tau+/+, tau-/- and tau-/-.AAVtau mice upon bilateral infusion of TAT-NSF or TAT-Scr (n = 2-5 biological replicates). Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included (n = 2 for GA). Note that infusion of TAT-NSF lowers object discrimination to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( E ) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates ( tau5 ) from cortical lysates of indicated genotypes , including from tau - transgenic mice ( ALZ17 , Tau58 / 2 ) ( n = 5 - 8 biological replicates ) . IgG , control immunoprecipitate with non - specific IgG from tau + / + lysate . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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(E) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau5) from cortical lysates of indicated genotypes, including from tau-transgenic mice (ALZ17, Tau58/2) (n = 5-8 biological replicates). IgG, control immunoprecipitate with non-specific IgG from tau+/+ lysate. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( F ) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates ( tau5 ) from brain lysates of healthy controls ( HC ) , mild cognitive impairment ( MCI ) , and Alzheimer ' s disease ( AD ) ( n = 3 - 4 biological replicates ) . IgG , control immunoprecipitate . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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(F) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau5) from brain lysates of healthy controls (HC), mild cognitive impairment (MCI), and Alzheimer's disease (AD) (n = 3-4 biological replicates). IgG, control immunoprecipitate. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( D ) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp ( without ATG ) to the sequence from 5 ' cap of pri - miR - 31 to ATG site or which mutated to ATT of the sORF . Cells were selected by G418 for 2 days . Analysis of the GFP expression by confocal microscopy or flow cytometry , scale bar 100 μm .

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(D) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp (without ATG) to the sequence from 5' cap of pri-miR-31 to ATG site or which mutated to ATT of the sORF. Cells were selected by G418 for 2 days. Analysis of the GFP expression by confocal microscopy or flow cytometry, scale bar 100 μm.


( E ) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP - N1 - miPEP31 or pEGFP - N1 , the red arrow indicate predicted size of miPEP31 - GFP band .

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(E) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP-N1-miPEP31 or pEGFP-N1, the red arrow indicate predicted size of miPEP31-GFP band.


( F ) Flow - fish to detect the localization of pri - miR - 31 . The location of pri - miR - 31 was analyzed by confocal microscopy . Scale bar 5 μm .

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(F) Flow-fish to detect the localization of pri-miR-31. The location of pri-miR-31 was analyzed by confocal microscopy. Scale bar 5 μm.


( G ) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31 . Values are expressed as fold changes relative to controls and normalized to GAPDH , the red arrow indicate predicted size of miPEP31 .

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(G) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31. Values are expressed as fold changes relative to controls and normalized to GAPDH, the red arrow indicate predicted size of miPEP31.


( H ) Immunoblot analysis of spleen , lymph node and thymus from WT or miR - 31 - / - mice with miPEP31 antibody , the red arrow indicate predicted size of miPEP31 .

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(H) Immunoblot analysis of spleen, lymph node and thymus from WT or miR-31-/- mice with miPEP31 antibody, the red arrow indicate predicted size of miPEP31.


( A ) CD4 + T cells were activated with anti - CD3 / 28 and treated with different concentration of FAM - miPEP31 for 24 hours , the fluorescein was detected by Flow Cytometry .

(A) CD4+ T cells were activated with anti-CD3/28 and treated with different concentration of FAM-miPEP31 for 24 hours, the fluorescein was detected by Flow Cytometry.


( B ) Confocal Microscopy was used to analyze the fluorescence of FAM - miPEP31 in anti - CD3 / 28 activated CD4 + T cells treated with 10 μM FAM - miPEP31 . Scale bar 5 μm .

(B) Confocal Microscopy was used to analyze the fluorescence of FAM-miPEP31 in anti-CD3/28 activated CD4+ T cells treated with 10 μM FAM-miPEP31. Scale bar 5 μm.


Anti - CD3 / 28 activated CD4 + T cells were cultured with 10 μM miPEP31 for 24 hours . ( C ) Western Blotting analysis of Cytochrome C and Histone H2A in total lysates and nuclear lysates .

Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (C) Western Blotting analysis of Cytochrome C and Histone H2A in total lysates and nuclear lysates.


Anti - CD3 / 28 activated CD4 + T cells were cultured with 10 μM miPEP31 for 24 hours . ( D ) ELISA to detect the concentration of miPEP31 in protein of total cell and nuclear extracts in CD4 + T and Treg cells treated with or without 10 μM miPEP31 . Data in ( D ) are presented as mean ± SEM of four biological replicates . ns , not significant ( CD4 + T versus Treg ) , unpaired two - tailed Student ' s t - test . .

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Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (D) ELISA to detect the concentration of miPEP31 in protein of total cell and nuclear extracts in CD4+ T and Treg cells treated with or without 10 μM miPEP31. Data in (D) are presented as mean ± SEM of four biological replicates. ns, not significant (CD4+ T versus Treg), unpaired two-tailed Student's t-test..


( E ) Naïve CD4 + T cells were isolated from WT C57BL / 6 mice , and activated by anti - CD3 / 28 for 48 hours . Subsequently , FAM - labeled miPEP31 was added . Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4 + T cells . Scale bar 7 μm .

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(E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, FAM-labeled miPEP31 was added. Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4+ T cells. Scale bar 7 μm.


( F , G ) C57BL / 6 mice were injected intravenously with 100 μg FAM - labeled miPEP31 . Splenocytes were isolated and stained for CD4 and DAPI . Imaging Flow Cytometry analysis of miPEP31 in CD4 + T cells ( F ) and non - CD4 splenocytes ( G ) , scale bar 10 μm .

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(F, G) C57BL/6 mice were injected intravenously with 100 μg FAM-labeled miPEP31. Splenocytes were isolated and stained for CD4 and DAPI. Imaging Flow Cytometry analysis of miPEP31 in CD4+ T cells (F) and non-CD4 splenocytes (G) , scale bar 10 μm.


( D , E ) Naïve CD4 + T cells were isolated from WT C57BL / 6 mice , and activated by anti - CD3 / 28 for 48 hours . Subsequently , 10 μM FAM - labeled miPEP31 was added for 20 min at 4 ℃ or 37 ℃ . Confocal Microscopy analysis of the fluorescence . Scale bar 10 μm . The localization of miPEP31 with DAPI was shown in ( E ) . Data are representative nuclear co - localization of miPEP31 in four representative images from four biological experiments . ( mean ± SEM ) . * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t - test .

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(D, E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, 10 μM FAM-labeled miPEP31 was added for 20 min at 4℃ or 37℃. Confocal Microscopy analysis of the fluorescence. Scale bar 10 μm. The localization of miPEP31 with DAPI was shown in (E). Data are representative nuclear co-localization of miPEP31 in four representative images from four biological experiments. (mean ± SEM). ****p < 0.0001, unpaired two-tailed Student's t-test.


scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization . ( D , E ) H & E and Fast blue staining of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization . Histopathology scores of inflammation and demyelination were shown in ( E ) . Scale bars , 70 μm . Data in ( C , E ) are presented as mean ± SEM of three independent experiments with nine to ten mice per group in each . * * * *

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scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization. (D, E) H&E and Fast blue staining of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization. Histopathology scores of inflammation and demyelination were shown in (E). Scale bars, 70 μm. Data in (C, E) are presented as mean ± SEM of three independent experiments with nine to ten mice per group in each. ****


Purified NK cells were activated with IL - 2 or IL - 15 in the presence or absence of PGE2 ( 1µM ) and tested for ( A ) proliferation by flow cytometry for Ki - 67 staining after four days of culture ( n = 6 , biological replicates ) ,

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (A) proliferation by flow cytometry for Ki-67 staining after four days of culture (n=6, biological replicates),


Purified NK cells were activated with IL - 2 or IL - 15 in the presence or absence of PGE2 ( 1µM ) and tested for , ( B ) ability to kill K562 cells after two days of culture in a 4 - hour chromium - assay at an E : T ratio of 5 : 1 ( n = 4 , biological replicates ) .

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for , (B) ability to kill K562 cells after two days of culture in a 4-hour chromium-assay at an E:T ratio of 5:1 (n=4, biological replicates).


Purified NK cells were activated with IL - 2 or IL - 15 in the presence or absence of PGE2 ( 1µM ) and tested for ( C ) Viability by flow cytometry using fixable live / dead staining after two days of culture ( n = 7 , biological replicates ) .

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (C) Viability by flow cytometry using fixable live/dead staining after two days of culture (n=7, biological replicates).


D ) Intracellular cAMP detected in two - day IL - 2 or IL - 15 activated NK cells before and 5 minutes after the addition of PGE2 ( 1 µM ) ( n = 3 , biological replicates )

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D) Intracellular cAMP detected in two-day IL-2 or IL-15 activated NK cells before and 5 minutes after the addition of PGE2 (1 µM) (n=3, biological replicates)


E ) mRNA levels of selected PGE2 response elements as measured by qPCR in NK cells ( n = 3 - 4 , biological replicates ) .

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E) mRNA levels of selected PGE2 response elements as measured by qPCR in NK cells (n=3-4, biological replicates).


F ) Fold change comparing flow cytometry mean fluorescence intensity ( MFI ) values of PDE4A expression between IL - 2 and IL - 15 activated NK cells after two days activation with cytokines ( n = 5 , biological replicates ) .

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F) Fold change comparing flow cytometry mean fluorescence intensity (MFI) values of PDE4A expression between IL-2 and IL-15 activated NK cells after two days activation with cytokines (n=5, biological replicates).


G ) Representative images showing PDE4A expression in NK cells activated by IL - 2 or IL - 15 for two days . Scale bar : 10 μm .

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G) Representative images showing PDE4A expression in NK cells activated by IL-2 or IL-15 for two days. Scale bar: 10 μm.


A - B ) NK cells were activated with IL - 2 or IL - 15 for two days in the presence or absence of PGE2 ( 1µM ) and analyzed for A ) the levels of phosphorylated S6 ( pS235 / pS236 ) , STAT3 ( S727 ) , STAT5 ( Y694 ) , Akt ( S473 ) ( n = 4 , biological replicates ) and B ) 16 categories of phosphorylation array showed . Red box indicates category 1 ( pS6 + , pSTAT3 + , pSTAT5 + , pAKT + ) .

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A-B) NK cells were activated with IL-2 or IL-15 for two days in the presence or absence of PGE2 (1µM) and analyzed for A) the levels of phosphorylated S6 (pS235/pS236), STAT3 (S727), STAT5 (Y694), Akt (S473) (n=4, biological replicates) and B)16 categories of phosphorylation array showed. Red box indicates category 1 (pS6+, pSTAT3+, pSTAT5+, pAKT+).


C ) Relative changes in frequency of NK cells expressing pS6 in the presence and absence of PGE2 ( n = 4 , biological replicates ) .

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C) Relative changes in frequency of NK cells expressing pS6 in the presence and absence of PGE2 (n=4, biological replicates).


D ) Flow cytometry analysis of PDE4A in the presence or absence of Torin - 1 ( 1µM ) ( left , n = 7 , biological replicates ) with representative histograms ( right ) .

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D) Flow cytometry analysis of PDE4A in the presence or absence of Torin-1 (1µM) (left, n=7, biological replicates) with representative histograms (right).


E ) NK cell - mediated cytotoxicity of K562 cells by 4 - hour chromium - release assay of two - day cytokine activated NK cells in the presence or absence of Torin - 1 ( 1µM ) and PGE2 ( 1µM ) ( n = 6 , biological replicates ) . E : T ratio = 5 : 1 .

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E) NK cell-mediated cytotoxicity of K562 cells by 4-hour chromium-release assay of two-day cytokine activated NK cells in the presence or absence of Torin-1 (1µM) and PGE2 (1µM) (n=6, biological replicates). E:T ratio=5:1.


F , G ) ( F ) Flow cytometry analysis of pS6 and ( G ) 4 - hour chromium - release assay of PGE2 - treated NK cells against K562 measured after 48 hours treatment with Roflumilast ( PDE4i , 1µM ) ( n = 3 , biological replicates ) E : T ratio = 5 : 1 .

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F, G) (F) Flow cytometry analysis of pS6 and (G) 4-hour chromium-release assay of PGE2-treated NK cells against K562 measured after 48 hours treatment with Roflumilast (PDE4i, 1µM) (n=3, biological replicates) E:T ratio=5:1.


B ) Volcano plot showing genes compared after two - days cytokine activated NK cells . Genes with significant changes ( p value < 0 . 05 ) and > 1 . 5 log2 fold change between IL - 2 and IL - 15 activated NK cells are labeled in red . Genes belonging to cell surface ( GO : 0009986 ) are annotated with gene name .

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B) Volcano plot showing genes compared after two-days cytokine activated NK cells. Genes with significant changes (p value<0.05) and >1.5 log2 fold change between IL-2 and IL-15 activated NK cells are labeled in red. Genes belonging to cell surface (GO:0009986) are annotated with gene name.


C ) Quantification of CD25 + CD54 + population changes after two days treatment ( n = 4 , biological replicates ) .

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C) Quantification of CD25+ CD54+ population changes after two days treatment (n=4, biological replicates).


D ) Representative flow cytometry graphs for CD25 and CD54 staining in IL - 2 and IL - 15 NK cells in the presence or absence of PGE2 .

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D) Representative flow cytometry graphs for CD25 and CD54 staining in IL-2 and IL-15 NK cells in the presence or absence of PGE2.


E ) Quantification of the number of cell clusters . NK cell clusters were defined as cell aggregates occupying an area at least 2000 μm2 and an eccentricity of < 0 . 8 ) . ( n = 4 , biological replicates ) .

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E) Quantification of the number of cell clusters. NK cell clusters were defined as cell aggregates occupying an area at least 2000 μm2 and an eccentricity of <0.8). (n=4, biological replicates).


F ) Representative phase contrast image of IL - 2 and IL - 15 activated NK cells in the presence or absence of PGE2 . Scale bar 50 µm .

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F) Representative phase contrast image of IL-2 and IL-15 activated NK cells in the presence or absence of PGE2. Scale bar 50 µm.


G ) Chromium - release cytotoxicity assay by two - day cytokine activated NK cells in the presence or absence of PGE2 ( 1µM ) and anti - CD54 ( 20 µg / ml ) ( n = 4 , biological replicates ) , E : T ratio = 5 : 1 .

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G) Chromium-release cytotoxicity assay by two-day cytokine activated NK cells in the presence or absence of PGE2 (1µM) and anti-CD54 (20 µg/ml) (n=4, biological replicates), E:T ratio=5:1.


A ) NK cells were stimulated with either IL - 2 or IL - 15 in the presence or absence of PGE2 for two days and thereafter purified as CD25 + / CD54 + and CD25 - / CD54 - and tested in a 51Cr - release assay against K562 . ( E : T = 5 : 1 , n = 4 , biological replicates ) . Stars indicate significant p - values comparing CD25 + / CD54 + vs . CD25 - / CD54 - NK cells for each condition .

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A) NK cells were stimulated with either IL-2 or IL-15 in the presence or absence of PGE2 for two days and thereafter purified as CD25+/CD54+ and CD25-/CD54- and tested in a 51Cr-release assay against K562. (E: T=5:1, n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54- NK cells for each condition.


B - F ) NK cells were stimulated with IL - 15 and purified as CD25 + / CD54 + and CD25 - / CD54 - and co - cultures with K562 cells ( 5 : 1 ratio ) for six hours and analyzed for the expression of B ) CD107a , C ) IFNγ , D ) Perforin , E ) TRAIL and F ) Granzyme B were by flow cytometry ( n = 4 , biological replicates ) .

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B-F) NK cells were stimulated with IL-15 and purified as CD25+/CD54+ and CD25-/CD54- and co-cultures with K562 cells (5:1 ratio) for six hours and analyzed for the expression of B) CD107a, C) IFNγ, D) Perforin, E) TRAIL and F) Granzyme B were by flow cytometry (n=4, biological replicates).


G ) Flow cytometry analysis of levels ( Left = % frequency , right = fold change of mean fluorescence intensity ) of phosphorylated S6 ( S235 / 236 ) in CD25 + / CD54 + and CD25 - / CD54 - IL - 15 activated NK cells ( n = 4 , biological replicates ) . H ) Flow cytometry analysis of PDE4A expression in CD25 + / CD54 + and CD25 - / CD54 - IL - 15 activated NK cells ( n = 4 , biological replicates ) .

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G) Flow cytometry analysis of levels (Left=% frequency, right=fold change of mean fluorescence intensity) of phosphorylated S6 (S235/236) in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates). H) Flow cytometry analysis of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates).


I ) Representative image of PDE4A expression in CD25 + / CD54 + and CD25 - / CD54 - IL - 15 activated NK cells . Scale bar : 10 µm .

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I) Representative image of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells. Scale bar: 10 µm.


J ) NK cells were first stimulated with IL - 15 for two days in the absence of PGE2 , then purified as CD25 + / CD54 + or CD25 - / CD54 - NK cells and tested in a 51Cr - release cytotoxicity assay against K562 cells at an E : T ratio of 5 : 1 in the presence ( filled bars ) or absence ( open bars ) of PGE2 ( 1 µM ) ( n = 3 , biological replicates ) .

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J) NK cells were first stimulated with IL-15 for two days in the absence of PGE2, then purified as CD25+/CD54+ or CD25-/CD54- NK cells and tested in a 51Cr-release cytotoxicity assay against K562 cells at an E:T ratio of 5:1 in the presence (filled bars) or absence (open bars) of PGE2 (1 µM) (n=3, biological replicates).


K ) Mitochondria mass from purified CD25 + / CD54 + and CD25 - / CD54 - NK cells measured by flow cytometry ( n = 5 , biological replicates ) .

K) Mitochondria mass from purified CD25+/CD54+ and CD25-/CD54- NK cells measured by flow cytometry (n=5, biological replicates).


L ) Representative image of Mitotracker Red FM in CD25 + / CD54 + NK cells and CD25 - / CD54 - NK cells . Scale bar : 20 μm .

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L) Representative image of Mitotracker Red FM in CD25+/CD54+ NK cells and CD25-/CD54- NK cells. Scale bar: 20 μm.


A ) Expression of PTGES in normal and tumor tissue .

A) Expression of PTGES in normal and tumor tissue.


B ) Kaplan - Meier analysis of overall ( left ) and progression - free ( right ) survival in patients with lung adenocarcinoma with high PTGES ( top 10 % ) and low PTGES ( bottom 90 % ) expression .

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B) Kaplan-Meier analysis of overall (left) and progression-free (right) survival in patients with lung adenocarcinoma with high PTGES (top 10%) and low PTGES (bottom 90%) expression.


C ) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma .

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C) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma.


D ) IL15 gene expression analysis in high and low NK gene signature ( NK GS ) .

D) IL15 gene expression analysis in high and low NK gene signature (NK GS).


E ) Overall survival in patients with stage I ( n = 269 ) lung adenocarcinoma with high ( top 50 % ) and low ( bottom 50 % ) NK cell gene expression signature expression .

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E) Overall survival in patients with stage I (n=269) lung adenocarcinoma with high (top 50%) and low (bottom 50%) NK cell gene expression signature expression.


F ) Overall survival in patients with high ( top 50 % ) PTGES expression in relation to high ( top 50 % ) and low ( bottom 50 % ) NK cell gene expression signature .

F) Overall survival in patients with high (top 50%) PTGES expression in relation to high (top 50%) and low (bottom 50%) NK cell gene expression signature.


A ) Real time image - based killing of A549 cells by NK cells isolated from two individual healthy donors at an E : T ratio of 10 : 1 .

A) Real time image-based killing of A549 cells by NK cells isolated from two individual healthy donors at an E:T ratio of 10:1.


B ) NK cells were stimulated with IL - 2 or IL - 15 in the presence or absence of PGE2 and then purified as CD25 + / CD54 + or CD25 - / CD54 - cells and tested for cytotoxicity against A549 tumor cell in a 51Cr - release assay . E : T ratio = 5 : 1 ( n = 4 , biological replicates ) . Stars indicate significant p - values comparing CD25 + / CD54 + vs . CD25 - / CD54 - .

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B) NK cells were stimulated with IL-2 or IL-15 in the presence or absence of PGE2 and then purified as CD25+/CD54+ or CD25-/CD54- cells and tested for cytotoxicity against A549 tumor cell in a 51Cr-release assay. E:T ratio = 5:1 (n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54-.


C ) Representative image of NK cell infiltration ( red ) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells , scale bar : 400 μm .

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C) Representative image of NK cell infiltration (red) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells, scale bar: 400 μm.


D , E ) Incucyte quantification of D ) total and E ) tumor - infiltrating CD25 + / CD54 + and CD25 - / CD54 - NK cells . RCU , Red Calibrated Unit , ( n = 4 , biological replicates ) .

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D, E) Incucyte quantification of D) total and E) tumor-infiltrating CD25+/CD54+ and CD25-/CD54- NK cells. RCU, Red Calibrated Unit, (n=4, biological replicates).


F , G ) Flow cytometry quantification of ( F ) tumor - infiltrating and ( G ) non - infiltrating NK cells . Results are depicted as total NK cells among CD45 positive live cells . ( n = 4 , biological replicates ) .

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F, G) Flow cytometry quantification of (F) tumor-infiltrating and (G) non-infiltrating NK cells. Results are depicted as total NK cells among CD45 positive live cells. (n=4, biological replicates).


H ) 48 hours dpi zebrafish injected with A549 , A549 + CD25 + / CD54 + , or A549 + CD25 - / CD54 - NK cells after 24 hours . Fluorescent image was taken by Leica epi - fluorescent stereomicroscope . Scale bar : 500 µm . The tumor injection area ( perivitelline space ) is depicted with a dashed line .

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H) 48 hours dpi zebrafish injected with A549, A549+ CD25+/CD54+, or A549+ CD25-/CD54- NK cells after 24 hours. Fluorescent image was taken by Leica epi-fluorescent stereomicroscope. Scale bar: 500 µm. The tumor injection area (perivitelline space) is depicted with a dashed line.


I ) 3D view of tumor injection area 24 hours posts tumor inoculation ( left ) . Scale bar = 50 µm . The tumor injection area ( perivitelline space ) is depicted with a dashed line . Magnified view for tumor area ( right ) . The A549 - td tumor cells are annotated with a purple translucent mask and NK cells are color - coded based on the distance to the tumor area ( near - far : blue - red ) . Scale bar = 10 µm .

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I) 3D view of tumor injection area 24 hours posts tumor inoculation (left). Scale bar=50 µm. The tumor injection area (perivitelline space) is depicted with a dashed line. Magnified view for tumor area (right). The A549-td tumor cells are annotated with a purple translucent mask and NK cells are color-coded based on the distance to the tumor area (near-far: blue-red). Scale bar=10 µm.


J ) The distance between NK cells and tumor area in CD25 - / CD54 - and CD25 + / CD54 + NK cell treated fish ( n = 5 , biological replicates ) . K ) Frequency of tumor infiltrating NK cells in in CD25 - / CD54 - and CD25 + / CD54 + NK cell treated fish ( n = 5 , biological replicates ) .

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J) The distance between NK cells and tumor area in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates). K) Frequency of tumor infiltrating NK cells in in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates).


L , M ) Quantification of ( L ) tumor cell and ( M ) NK cell counts 24 hours after injection of NK cells by Imaris ( n = 5 , biological replicates ) .

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L, M) Quantification of (L) tumor cell and (M) NK cell counts 24 hours after injection of NK cells by Imaris (n=5, biological replicates).


N ) Frequency of tissue - infiltrating total NK cells ( CD56 + / CD3 - ) ( left ) and CD54 positive NK cells ( right ) among CD45 positive live cells ( n = 10 , biological replicates ) .

N) Frequency of tissue-infiltrating total NK cells (CD56+/CD3-) (left) and CD54 positive NK cells (right) among CD45 positive live cells (n=10, biological replicates).


B . Instability of BU - 1 expression in timeless cells . FACS plots of wild type and timeless ( clone 1 ) DT40 cells stained with anti - Bu - 1 conjugated with phycoerythrin . Each line represents the Bu - 1 expression profile of an individual clonal population . Unstained controls are shown in blue .

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B. Instability of BU-1 expression in timeless cells. FACS plots of wild type and timeless (clone 1) DT40 cells stained with anti-Bu-1 conjugated with phycoerythrin. Each line represents the Bu-1 expression profile of an individual clonal population. Unstained controls are shown in blue.


C . Fluctuation analysis for Bu - 1 loss in wild - type DT40 cells and two independent timeless clones generated by CRISPR - Cas9 targeting ( clones 1 and 2 ; , timeless ( clone 1 ) complemented by expression of human Timeless cDNA and a timeless mutant on a background in which the endogenous + 3 . 5 G4 has been deleted ( ΔG4 )

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C. Fluctuation analysis for Bu-1 loss in wild-type DT40 cells and two independent timeless clones generated by CRISPR-Cas9 targeting (clones 1 and 2; , timeless (clone 1) complemented by expression of human Timeless cDNA and a timeless mutant on a background in which the endogenous +3.5 G4 has been deleted (ΔG4)


D . Fluctuation analysis for Bu - 1 loss in DT40 wild - type and tipin cells .

D. Fluctuation analysis for Bu-1 loss in DT40 wild-type and tipin cells.


D . The DNA - binding affinity of DBD was measured by fluorescence anisotropy , titrating the DBD protein against Cy3 3 ' - labelled ssDNA , dsDNA and G4 DNA The top panel shows binding curves for ss - and dsDNA , the bottom panel shows the binding curve for the G4 DNA substrate .

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D. The DNA-binding affinity of DBD was measured by fluorescence anisotropy, titrating the DBD protein against Cy3 3'-labelled ssDNA, dsDNA and G4 DNA The top panel shows binding curves for ss- and dsDNA, the bottom panel shows the binding curve for the G4 DNA substrate.


Fluorescence anisotropy was used to measure the binding affinity of Timeless - Tipin for the indicated DNA sequences . A . ssG4 : G4 flanked by single - stranded DNA ; ssHP : hairpin flanked by single - stranded DNA ; ss : single - stranded DNA

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Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. A. ssG4: G4 flanked by single-stranded DNA; ssHP: hairpin flanked by single-stranded DNA; ss: single-stranded DNA


Fluorescence anisotropy was used to measure the binding affinity of Timeless - Tipin for the indicated DNA sequences . B . Binding affinity of Timeless - Tipin for a range of G4 DNA sequences Single - stranded ( ss20 : 5 ′ - 6FAM - ATAAGAGTGGTTAGAGTGTA ) and double - stranded ( ds20 : ss20 annealed to complementary sequence ) DNA were also tested as controls .

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Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. B. Binding affinity of Timeless-Tipin for a range of G4 DNA sequences Single-stranded (ss20: 5′-6FAM-ATAAGAGTGGTTAGAGTGTA) and double-stranded (ds20: ss20 annealed to complementary sequence) DNA were also tested as controls.


Fluctuation analysis for the generation of Bu - 1 loss variants . Top to bottom : wild type , timeless ( clone 1 ) , a timeless mutant ( timeless ∆ C ) generated by CRISPR - Cas9 targeting exon 16 which truncates the protein removing the CTD containing both the DBD and the PARP binding domains . Then , complementation of timeless # 1 with human Timeless ( hTim ) , hTim ∆ 816 - 1208 ( lacking both the DBD and PBD ) , hTim ∆ 816 - 965 ( lacking the DBD ) and hTim [ 1 : 1000 ] , lacking the PBD . At least two independent fluctuation analyses were performed with 24 - 36 individual clones each cell line per repeat .

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Fluctuation analysis for the generation of Bu-1 loss variants. Top to bottom: wild type, timeless (clone 1), a timeless mutant (timeless ∆C) generated by CRISPR-Cas9 targeting exon 16 which truncates the protein removing the CTD containing both the DBD and the PARP binding domains. Then, complementation of timeless#1 with human Timeless (hTim), hTim∆816-1208 (lacking both the DBD and PBD), hTim∆816-965 (lacking the DBD) and hTim[1:1000], lacking the PBD. At least two independent fluctuation analyses were performed with 24-36 individual clones each cell line per repeat.


A . Enhanced recruitment of DDX11 to chromatin associated PCNA following exposure to 4 µM PDS for 24 hours . PCNA was precipitated from cross - linked chromatin and the immunoprecipitate blotted for FLAG - DDX11 .

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A. Enhanced recruitment of DDX11 to chromatin associated PCNA following exposure to 4 µM PDS for 24 hours. PCNA was precipitated from cross-linked chromatin and the immunoprecipitate blotted for FLAG-DDX11.


B . Fluctuation analysis for Bu - 1a loss in wild - type DT40 cells , two independent ddx11 clones generated by CRISPR - Cas9 targeting ( clones 1 and 2 ) and one ddx11 clone generated by conventional homologous recombination gene targeting ( clone 3 ) . Each symbol represents the percentage of cells in an individual clone expanded for 2 - 3 weeks that have lost Bu - 1ahigh expression .

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B. Fluctuation analysis for Bu-1a loss in wild-type DT40 cells, two independent ddx11 clones generated by CRISPR-Cas9 targeting (clones 1 and 2) and one ddx11 clone generated by conventional homologous recombination gene targeting (clone 3). Each symbol represents the percentage of cells in an individual clone expanded for 2-3 weeks that have lost Bu-1ahigh expression.


C . Fluctuation analysis for Bu - 1a loss variant generation in wild - type cells , ddx11 ( clone 1 ) cells , ddx11 ( clone 1 ) complemented by expression of chicken DDX11 WT cDNA , ddx11 ( clone 1 ) complemented by expression of helicase - dead form of chicken DDX11 ( K87A ) cDNA , and a ddx11 clone generated in cells in which the endogenous + 3 . 5 G4 has been deleted ( ΔG4 ) .

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C. Fluctuation analysis for Bu-1a loss variant generation in wild-type cells, ddx11 (clone 1) cells, ddx11 (clone 1) complemented by expression of chicken DDX11 WT cDNA, ddx11 (clone 1) complemented by expression of helicase-dead form of chicken DDX11 (K87A) cDNA, and a ddx11 clone generated in cells in which the endogenous +3.5 G4 has been deleted (ΔG4).


D . Fluctuation analysis for Bu - 1 loss in two independent timeless / ddx11 double - mutant clones ( # 1 and # 2 ) , ddx11 expressing DDX11KAK and fancj and fancj / ddx11 double - mutants . Fluctuation analyses for wild type , timeless # 1 and ddx11 # 1 are shown for comparison .

D. Fluctuation analysis for Bu-1 loss in two independent timeless/ddx11 double-mutant clones (#1 and #2), ddx11 expressing DDX11KAK and fancj and fancj/ddx11 double-mutants. Fluctuation analyses for wild type, timeless #1 and ddx11 #1 are shown for comparison.


A . Growth curves for DT40 wild type , ddx11 , timeless and ddx11 / timeless cells , with and without 4 μM pyridostatin ( PDS ) . Cells were seeded at 5 x104 cells / ml on day 0 and the viable cells were counted each 24 h for 4 days . Bars represent SD of two independent experiments performed in duplicate . Doubling times ( DMSO ) : WT 13 hours , timeless 18 hours , ddx11 16 hours , ddx11 / timeless 24 hours . Doubling times ( PDS ) : WT 13 . 6 hours , timeless 27 hours , ddx11 25 . 7 hours , ddx11 / timeless 47 . 5 hours .

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A. Growth curves for DT40 wild type, ddx11, timeless and ddx11/timeless cells, with and without 4 μM pyridostatin (PDS). Cells were seeded at 5 x104 cells/ml on day 0 and the viable cells were counted each 24 h for 4 days. Bars represent SD of two independent experiments performed in duplicate. Doubling times (DMSO): WT 13 hours, timeless 18 hours, ddx11 16 hours, ddx11/timeless 24 hours. Doubling times (PDS): WT 13.6 hours, timeless 27 hours, ddx11 25.7 hours, ddx11/timeless 47.5 hours.


B . DDR signalling detected by phosphorylation of histone H2AX ( γ - H2AX ) by flow cytometry in untreated cells or cells exposed to 4 μM PDS for 3 days . Pale histogram , untreated ; dark histogram , treated ; black dotted line , positive control cells treated with 0 . 1 µM cisplatin , also for 3 days . C . Quantification of γ - H2AX in DT40 wild type , ddx11 , timeless and ddx11 / timeless cells treated with 4 μM PDS for 3 days . The central band represents the median , the box the 25th - 75th centile and whiskers the minimum to maximum range of three independent experiments performed in duplicate .

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B. DDR signalling detected by phosphorylation of histone H2AX (γ-H2AX) by flow cytometry in untreated cells or cells exposed to 4 μM PDS for 3 days. Pale histogram, untreated; dark histogram, treated; black dotted line, positive control cells treated with 0.1 µM cisplatin, also for 3 days. C. Quantification of γ-H2AX in DT40 wild type, ddx11, timeless and ddx11/timeless cells treated with 4 μM PDS for 3 days. The central band represents the median, the box the 25th - 75th centile and whiskers the minimum to maximum range of three independent experiments performed in duplicate.


A . Correlation of magnitude and direction of change of genes dysregulated ( relative to wild type ) in timeless vs . ddx11 DT40 cells . rs ( Spearman rho ) is shown for each correlation .

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A. Correlation of magnitude and direction of change of genes dysregulated (relative to wild type) in timeless vs. ddx11 DT40 cells. rs (Spearman rho) is shown for each correlation.


B . Genes dysregulated in timeless , ddx11 and fancj mutants have a higher density of G4s around their TSS . Cumulative fraction of the genes dysregulated in timeless ( red ) , ddx11 ( blue ) and fancj ( green ) containing n ( x - axis ) G4 motifs within 1 . 5kb of the TSS compared with all genes ( black ) .

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B. Genes dysregulated in timeless, ddx11 and fancj mutants have a higher density of G4s around their TSS. Cumulative fraction of the genes dysregulated in timeless (red), ddx11 (blue) and fancj (green) containing n (x-axis) G4 motifs within 1.5kb of the TSS compared with all genes (black).


C . Metagene analysis showing G4 frequency around the TSS of genes dysregulated ( up or down ) in timeless ( left panel ) , ddx11 ( centre panel ) and fancj ( right panel ) compared with all genes ( black line ) . G4 frequency is calculated separately for coding ( above the x - axis ) and template strands ( below the x - axis ) .

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C. Metagene analysis showing G4 frequency around the TSS of genes dysregulated (up or down) in timeless (left panel), ddx11 (centre panel) and fancj (right panel) compared with all genes (black line). G4 frequency is calculated separately for coding (above the x-axis) and template strands (below the x-axis).


( A - E ) GFP - pho8Δ60 - expressing wild - type ( WT ) , atg5Δ , atg7Δ , and atg5Δpep4Δ cells were starved ( A ) or treated with AmphoB ( B - E ) and subjected to western blotting . Cells were treated with 2 . 5 µg / ml AmphoB for the indicated time ( B ) or with the indicated doses for 24 hr ( C - E ) . Generation of free GFP was a marker of proteolysis .

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(A-E) GFP-pho8Δ60-expressing wild-type (WT), atg5Δ, atg7Δ, and atg5Δpep4Δ cells were starved (A) or treated with AmphoB (B-E) and subjected to western blotting. Cells were treated with 2.5 µg/ml AmphoB for the indicated time (B) or with the indicated doses for 24 hr (C-E). Generation of free GFP was a marker of proteolysis.


( F ) GFP - pho8Δ60 - and Pep4 - mRFP - expressing atg5Δ cells were cultured with ( AmphoB ) or without ( NT ) AmphoB ( 2 . 5 µg / ml , 24 hr ) , and localization of GFP - pho8Δ60 and Pep4 - mRFP was observed by confocal microscopy . Arrowheads indicate the cells in which GFP - pho8Δ60 was delivered to the vacuoles . Scale bars = 2 µm .

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(F) GFP-pho8Δ60- and Pep4-mRFP-expressing atg5Δ cells were cultured with (AmphoB) or without (NT) AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-pho8Δ60 and Pep4-mRFP was observed by confocal microscopy. Arrowheads indicate the cells in which GFP-pho8Δ60 was delivered to the vacuoles. Scale bars = 2 µm.


( G , H ) Accumulation of autophagic body ( AB ) - like structures by AmphoB in atg5Δpep4Δ cells . pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . Scale bars = 2 µm . Cells containing AB - like structures were counted under phase - contrast microscopy . Representative images ( G ) and the percentage of cells with AB - like structures ( H ) are shown . * p < 0 . 01 vs the value of pep4Δ cells . ( I - K ) Induction of AB - like structures in atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . Representative images of quick - frozen replicas ( I ) and thin section of the frozen - substituted material ( J ) are shown .

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

(G, H) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I-K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown.


( I ) AB - like structures accumulated in the vacuole . Scale bar = 0 . 5 µm . ( J ) AB - like structures containing lipid particles and mitochondria accumulated in the cross - section of the vacuole . An autophagosome ( AP ) - like double - membrane compartment containing a ribosome and Golgigranule is present in the cytosol . Scale bar = 0 . 5 µm . A magnified photo is available in Appendix Fig . S4B . ( K ) The cells containing AB - like structures were counted under electron microscopy [ no treatment ( NT ) : n = 20 , AmphoB : n = 192 ] . * p < 0 . 01 vs the value of NT .

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

(I) AB-like structures accumulated in the vacuole. Scale bar = 0.5 µm. (J) AB-like structures containing lipid particles and mitochondria accumulated in the cross-section of the vacuole. An autophagosome (AP)-like double-membrane compartment containing a ribosome and Golgigranule is present in the cytosol. Scale bar = 0.5 µm. A magnified photo is available in Appendix Fig. S4B. (K) The cells containing AB-like structures were counted under electron microscopy [no treatment (NT): n = 20, AmphoB: n = 192]. * p < 0.01 vs the value of NT.


( A - D ) Generation of Golgi stacks by AmphoB . Stacked Golgicisternae are observed in quick - frozen replicas ( A - C ) and substituted thin section ( D ) of atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml ) for the indicated time . ( A ) Scale bar = 1 µm . ( B ) The magnified image shows the area indicated by the square in ( A ) . Scale bar = 0 . 5 µm . ( C , D ) A representative Golgi stack with four cisternae is shown . Scale bar = 0 . 5 µm .

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 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

(A-D) Generation of Golgi stacks by AmphoB. Stacked Golgicisternae are observed in quick-frozen replicas (A-C) and substituted thin section (D) of atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml) for the indicated time. (A) Scale bar = 1 µm. (B) The magnified image shows the area indicated by the square in (A). Scale bar = 0.5 µm. (C, D) A representative Golgi stack with four cisternae is shown. Scale bar = 0.5 µm.


( E - G ) Generation of AP - like double - membrane compartment from the stacked Golgi . A quick - frozen replica ( E ) and a substituted thin section ( F , G ) are shown . Scale bars = 0 . 5 µm . The distal stacks are extended , curved , and form AP - like structure ( E , F ) . Mitochondria are encircled by the Golgi membrane ( F ) . ( G ) The AP - like structure was separated away from Golgi stacks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Generation of AP-like double-membrane compartment from the stacked Golgi. A quick-frozen replica (E) and a substituted thin section (F, G) are shown. Scale bars = 0.5 µm. The distal stacks are extended, curved, and form AP-like structure (E, F). Mitochondria are encircled by the Golgi membrane (F). (G) The AP-like structure was separated away from Golgi stacks.


( H , I ) The scene that AP - like structure was fused with vacuole . A magnified image of ( H ) is shown in ( I ) . In atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) , outer membrane of AP - like structure ( red arrows ) was fused with vacuolar membrane ( blue arrows ) . Inner membranes of AP - like structure ( yellow arrowheads ) were entered into the vacuoles to become AB - like structure . Green arrows indicate the membrane fusion site . Scale bars = 0 . 5 µm ( H ) and 0 . 2 µm ( I ) .

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

(H, I) The scene that AP-like structure was fused with vacuole. A magnified image of (H) is shown in (I). In atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr), outer membrane of AP-like structure (red arrows) was fused with vacuolar membrane (blue arrows). Inner membranes of AP-like structure (yellow arrowheads) were entered into the vacuoles to become AB-like structure. Green arrows indicate the membrane fusion site. Scale bars = 0.5 µm (H) and 0.2 µm (I).


( J ) The number of elongated single cisternae ( green bars ) , Golgi stacks ( red bars ) , and AB - like structures ( blue bars ) was counted over time using electron microscopy images . Error bars indicate s . e . m . ( 3 hr : n = 21 , 6 hr : n = 20 , 12 hr : n = 193 , 24 hr : n = 192 ) .

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

(J) The number of elongated single cisternae (green bars), Golgi stacks (red bars), and AB-like structures (blue bars) was counted over time using electron microscopy images. Error bars indicate s.e.m. (3 hr: n = 21, 6 hr: n = 20, 12 hr: n = 193, 24 hr: n = 192).


( K ) Translocation of the trans - Golgi protein to the vacuolar membrane induced by AmphoB treatment . GFP - Sft2 - expressing atg5Δ cells ( trans - Golgi marker protein ) were incubated with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) , and localization of GFP - Sft2 was assessed by confocal microscopy . Arrowhead indicates the GFP signal on the vacuolar membrane . Scale bars = 2 µm .

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 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

(K) Translocation of the trans-Golgi protein to the vacuolar membrane induced by AmphoB treatment. GFP-Sft2-expressing atg5Δ cells (trans-Golgi marker protein) were incubated with or without AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-Sft2 was assessed by confocal microscopy. Arrowhead indicates the GFP signal on the vacuolar membrane. Scale bars = 2 µm.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( L , M ) Representative electron microscopy images of grh1Δatg5Δpep4Δ ( L ) and gvp36Δatg5Δpep4Δ cells ( M ) treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . AP - like and AB - like structures were absent . ( L ) A unilamellar Golgi cisterna was observed in the cytoplasm ( dotted square ) . Scale bar = 1 µm . ( M ) Formation of a Golgi stack , but not Golgi curvature , was observed ( dotted square ) . Bar = 0 . 5 µm .

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

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (L, M) Representative electron microscopy images of grh1Δatg5Δpep4Δ (L) and gvp36Δatg5Δpep4Δ cells (M) treated with AmphoB (2.5 µg/ml, 24 hr). AP-like and AB-like structures were absent. (L) A unilamellar Golgi cisterna was observed in the cytoplasm (dotted square). Scale bar = 1 µm. (M) Formation of a Golgi stack, but not Golgi curvature, was observed (dotted square). Bar = 0.5 µm.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( N ) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB - like structures was counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

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

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (N) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB-like structures was counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( O ) GFP - pho8Δ60 - expressing atg5Δ , grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) and subjected to western blotting for GFP . An unidentified band at 32kD ( asterisk ) is non - specific band because it was present in Pep4 - lacking grh1Δatg5Δ cells .

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

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (O) GFP-pho8Δ60-expressing atg5Δ, grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP. An unidentified band at 32kD (asterisk) is non-specific band because it was present in Pep4-lacking grh1Δatg5Δ cells.


( A ) Localization of cellular PI ( 4 ) P . GFP - 2xPHOsh2 - expressing atg5Δ , sac1 - 23 / atg5Δ , and pik1 - 83 / atg5Δ cells were incubated with or without AmphoB ( 2 . 5 µg / ml ) for 6 hr at 37 ° C , and their localization was observed by confocal microscopy . Scale bars = 2 µm .

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

(A) Localization of cellular PI(4)P. GFP-2xPHOsh2-expressing atg5Δ, sac1-23/atg5Δ, and pik1-83/atg5Δ cells were incubated with or without AmphoB (2.5 µg/ml) for 6 hr at 37 °C, and their localization was observed by confocal microscopy. Scale bars = 2 µm.


( B ) The same experiments were performed using GFP - GOLPH3 ( a GolgiPI ( 4 ) P - monitoring protein ) instead of GFP - 2xPHOsh2 . Scale bars = 2 µm .

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

(B) The same experiments were performed using GFP-GOLPH3 (a GolgiPI(4)P-monitoring protein) instead of GFP-2xPHOsh2. Scale bars = 2 µm.


( C , D ) Reduction of AmphoB - induced GOMED by the deletion of Golgi PI ( 4 ) phosphatases . ( C ) Indicated cells expressing GFP - pho8Δ60 were cultured with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) and subjected to western blotting for GFP .

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

(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (C) Indicated cells expressing GFP-pho8Δ60 were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP.


( C , D ) Reduction of AmphoB - induced GOMED by the deletion of Golgi PI ( 4 ) phosphatases . ( D ) The number of AP - like and AB - like structures was counted using EM images ( Fig . EV2B , C ) . ( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase .

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

(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (D) The number of AP-like and AB-like structures was counted using EM images (Fig. EV2B, C). (E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( E ) Indicated cells were incubated at 37 ° C ( temperature shift ) for 6 hr , and cells containing AB - like structures were counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (E) Indicated cells were incubated at 37 °C (temperature shift) for 6 hr, and cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( F ) Representative electron microscopy image of pik1 - 83 / atg5Δpep4Δ cells at 3 hr after temperature shift . AP - like structure ( arrow ) and AB - like structures were generated . Scale bar = 0 . 2 µm . ( G ) The number of AP - like and AB - like structures was counted using EM images . Error bars indicate s . e . m . ( atg5Δpep4Δ cells at 37 ° C : n = 54 and pik1 - 83 / atg5Δpep4Δ cells at 37 ° C : n = 63 ) . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (F) Representative electron microscopy image of pik1-83/atg5Δpep4Δ cells at 3 hr after temperature shift. AP-like structure (arrow) and AB-like structures were generated. Scale bar = 0.2 µm. (G) The number of AP-like and AB-like structures was counted using EM images. Error bars indicate s.e.m. (atg5Δpep4Δ cells at 37 °C: n = 54 and pik1-83/atg5Δpep4Δ cells at 37 °C: n = 63). *p < 0.01 vs the value of atg5Δpep4Δ cells.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( H ) Sec7 - HA - expressing pik1 - 83 / atg5Δ cells were incubated at 37 ° C ( temperature shift ) for 3 hr , and freeze replica immunolabelling was performed as described in Methods . Sec7 - HA - positive signals ( labelled with 15 nm gold particle ) were observed on the surface membrane of AP - like structure and Golgi membrane . Scale bar = 0 . 1 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (H) Sec7-HA-expressing pik1-83/atg5Δ cells were incubated at 37 °C (temperature shift) for 3 hr, and freeze replica immunolabelling was performed as described in Methods. Sec7-HA-positive signals (labelled with 15 nm gold particle) were observed on the surface membrane of AP-like structure and Golgi membrane. Scale bar = 0.1 µm.


( I − L ) Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( I ) Indicated cells were incubated at 37 ° C ( temperature switch ) for 3 hr , and the cells containing AB - like structures were counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

(I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (I) Indicated cells were incubated at 37 °C (temperature switch) for 3 hr, and the cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( I − L ) Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( J ) Indicated cells were also observed using EM . Arrow indicates AP - like structure . Scale bar = 0 . 5 µm .

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

(I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (J) Indicated cells were also observed using EM. Arrow indicates AP-like structure. Scale bar = 0.5 µm.


Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( K , L ) atg5Δpep4Δ cells were treated with ( CBM ) or without ( NT ) CBM ( 1 mM , 3 hr ) , and cells containing AB - like structures were counted by phase - contrast microscopy ( K ) . * p < 0 . 01 vs the value of NT .

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

Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr), and cells containing AB-like structures were counted by phase-contrast microscopy (K). * p < 0.01 vs the value of NT.


Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( K , L ) atg5Δpep4Δ cells were treated with ( CBM ) or without ( NT ) CBM ( 1 mM , 3 hr ) . Cells were also observed using EM ( L ) . AB - like structures were observed . Scale bar = 0 . 5 µm .

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

Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr). Cells were also observed using EM (L). AB-like structures were observed. Scale bar = 0.5 µm.


( M − O ) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED . Indicated cells expressing Hsp150 - HA ( 3 × ) were treated as follows : NT , no treatment ; starve , starvation for 6 hr ; AmphoB , 2 . 5 µg / ml for 24 hr ( M ) ; CBM , 1 mM for 24 hr ( N ) ; temperature shift , from 25 ° C to 37 ° C for 3 hr ( O ) . The cells were then lysed and examined for Hsp150 - HA ( 3 × ) expression by western blotting . Accumulation of O - glycosylated Hsp150 - HA ( 3 × ) indicated the trafficking failure from the Golgi to the PM .

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

(M−O) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED. Indicated cells expressing Hsp150-HA(3×) were treated as follows: NT, no treatment; starve, starvation for 6 hr; AmphoB, 2.5 µg/ml for 24 hr (M); CBM, 1 mM for 24 hr (N); temperature shift, from 25 °C to 37 °C for 3 hr (O). The cells were then lysed and examined for Hsp150-HA(3×) expression by western blotting. Accumulation of O-glycosylated Hsp150-HA(3×) indicated the trafficking failure from the Golgi to the PM.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( A , B ) EM analysis revealed the formation of AP - like and AL - like structures . Atg5 KO MEFs were treated with CBM ( 2 mM ) for 3 hr . A representative low - magnification image ( A ) and high - magnification images ( B ) are shown . Scale bar = 1 µm ( A ) and 0 . 2 µm ( B ) . The arrowheads and arrows indicate AL - like and AP - like structures , respectively .

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 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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (A, B) EM analysis revealed the formation of AP-like and AL-like structures. Atg5 KO MEFs were treated with CBM (2 mM) for 3 hr. A representative low-magnification image (A) and high-magnification images (B) are shown. Scale bar = 1 µm (A) and 0.2 µm (B). The arrowheads and arrows indicate AL-like and AP-like structures, respectively.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( C ) CLEM analysis of mCherry - syntaxin 6 - expressing Atg5 KO MEFs . Cells were treated with CBM ( 2 mM ) for 24 hr and observed using fluorescence and electron microscopy . mCherry - syntaxin 6puncta merged with the AL - like structures . Scale bar = 5 µm . " N " indicates nucleus . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (C) CLEM analysis of mCherry-syntaxin 6-expressing Atg5 KO MEFs. Cells were treated with CBM (2 mM) for 24 hr and observed using fluorescence and electron microscopy. mCherry-syntaxin 6puncta merged with the AL-like structures. Scale bar = 5 µm. "N" indicates nucleus. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.