D ) Q - PCR of Wnt YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation . Note inhibition of Axin2 , Sox9 , YAP , Ctgf , Cyr61 , TEAD family expression upon treatment with Porcupine inhibitor LGK974 . mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation ( 1dpi ) becomes more pronounced at 3 days after irradiation ( 3dpi ) . n = 4 biological replicates per condition , statistical was performed with Student ' s 2 - t - , error bars = 1 SD , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

D) Q-PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation. Note the inhibition of Axin2, Sox9, YAP, Ctgf, Cyr61, and TEAD family expression upon treatment with Porcupine inhibitor LGK974. The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation (1dpi) becomes more pronounced at 3 days after irradiation (3dpi). n = 4 biological replicates per condition, statistical analysis was performed with Student's 2-tailed t-test, error bars = 1 SD, * p<0.05, ** p<0.01, ***p<0.001.


A . Volcano plots of genes that are differentially expressed in infected relative to mock - infected at 12 hpi 24 hpi in ileum organoids . statistical significance ( - log10 of adjusted p - value ) is shown as a function of log2 fold change . MAST were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in panels are gene names of selected differentially expressed genes between compared two populations .

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A. Volcano plots of genes that are differentially expressed in infected cells relative to mock-infected cells at 12 hpi and 24 hpi in ileum organoids. The statistical significance (-log10 of the adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two populations.


F . Multiplex in situ hybridization of ACE2 SARS - CoV - 2 of mock - infected infected 2D ileum organoids at 12 hpi 24 hpi . arrows point at SARS - CoV - 2 - infected . A representative image is shown .

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F. Multiplex in situ RNA hybridization of ACE2 and SARS-CoV-2 of mock-infected and infected 2D ileum organoids at 12 hpi and 24 hpi. White arrows point at SARS-CoV-2-infected cells. A representative image is shown.


Volcano plots displaying genes that are differentially expressed in immature enterocytes 2 upon SARS - CoV - 2 infection of colon organoids . infected vs . mock infected statistical significance ( - log10 adjusted p - value ) is shown as a function of log2 fold change . MAST were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in blue in panels are gene names of selected differentially expressed genes between compared two populations . Labeled dots in red in panels are gene corresponding to interferon if detected .

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

Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. infected vs. mock infected cells The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected.


A . Human ileum - derived organoids were seeded in 2D on iBIDi chamber slides . 12 24 hpi were fixed amount of SARS - CoV - 2 infected ( red ) induction of ISG15 ( ) was analyzed by single molecule FISH . Nuclei were visualized with DAPI ( blue ) . arrows indicate SARS - CoV - 2 positive . N = 3 biological samples . Representative image is shown . Scale bar = 100 um .

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A. Human ileum-derived organoids were seeded in 2D on iBIDi chamber slides. 12 and 24 hpi cells were fixed and the amount of SARS-CoV-2 infected cells (red) and the induction of ISG15 (white) was analyzed by single molecule RNA FISH. Nuclei were visualized with DAPI (blue). White arrows indicate SARS-CoV-2 positive cells. N=3 biological samples. Representative image is shown. Scale bar=100 um.


D . T84 IRF3 knock - out were infected with SARS - CoV - 2 or human astrovirus 1 at an MOI = 3 . 24 hpi , were incubated in presence or absence of 2000 IU / mL of IFNβ1 . 12 h post - treatment was harvested induction of ISG15 was analyzed by q - RT - PCR normalized to housekeeping gene TBP . N = 3 biological replicates . Error bar indicates standard deviation . Statistics were determined by unpaired t - .

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D. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment RNA was harvested and the induction of ISG15 was analyzed by q-RT-PCR and normalized to the housekeeping gene TBP. N=3 biological replicates. Error bar indicates standard deviation. Statistics were determined by unpaired t-test.


E . T84 IRF3 knock - out were infected with SARS - CoV - 2 or human astrovirus 1 at an MOI = 3 . 24 hpi , were fixed stained for either SARS - CoV - 2 N or for human astrovirus 1 ( HAstV1 ) capsid . N = 3 biological replicates . Representative image is shown . Scale bar = 100 um .

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E. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were fixed and stained for either the SARS-CoV-2 N protein or for human astrovirus 1 (HAstV1) capsid protein. N=3 biological replicates. Representative image is shown. Scale bar=100 um.


G . T84 MX - 1 mcherry were infected with SARS - CoV - 2 at an MOI = 3 or MOI = 1 . 24 hpi were incubated in presence or absence of 2000 IU / mL of IFNβ1 . 12 h post - treatment were fixed stained for SARS - CoV - 2 N . number of MX - 1 positive , SARS - CoV - 2 positive double positive was quantified . N = 3 biological replicates were performed . Error bar indicates standard deviation .

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G. T84 MX-1 mcherry were infected with SARS-CoV-2 at an MOI=3 or MOI=1. 24 hpi cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment cells were fixed and stained for SARS-CoV-2 N protein. The number of MX-1 positive, SARS-CoV-2 positive and double positive cells was quantified. N=3 biological replicates were performed. Error bar indicates standard deviation.


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

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(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 BioID2 - tau . Controls : lysates from ( 1 ) BioID2 - or ( 2 ) BioID2 - tau - expressing ( 3 ) non - transduced mouse brain . Loading , glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) . * , unspecific band . Relative confirmed similar expression of BioID2 BioID2 - tau . Means ± S . E . M . ns , not significant ( Student ' s t - )

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(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 ) for nuclei ( DAPI ) ( n = 3 - 5 biological replicates ) . Scale bar , 50 μm .

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

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(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 NSF from mouse brain . ( B ) Endogenous 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 α - actin . Endogenous NSF was co - immunoprecipitated with transgenic endogenous tau from ALZ17tg or ALZ17non - tg but not from tau - / - with . GAPDH , input loading control ( n = 3 biological replicates ) . Recombinant tau NSF served as controls . ( C ) Densitometric 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 .

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

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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 NSF interaction . MST binding curve of recombinant hexameric NT - 647 - NHS - labelled NSF ( 27 pM ) 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 .

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(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 presence of NSF - inhibiting peptide TAT - NSF or control TAT - Scramble peptides ( 140 nM ) ; or co - incubated with recombinant tau ( 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 conditions ; n = 3 technical replicates ) .

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(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 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 .

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(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 ) / or GABAAR antagonist bicuculline ( 30 μM , 10 min ) at 37 ° C . Surface GluA1 were detected by immunoblot after with streptavidin ( SA ) enrichment . Input was probed for total GluA1 , NSF , tau βIII - tubulin . ( n = 3 biological replicates ) Input SA - enrichment from non - surface biotinylated neurons served as negative controls . ( B ) Reduced neuronal surface GluA1 in tau - / - neurons . of tau + / + tau - / - surface GluA1 upon treatment 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 .

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

(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 ) surface GluA1 total NSF immunostaining in mouse hippocampal neurons ( DIV15 ) . Tau + / + , tau - / - neurons tau - / - expressing human tau ( tau - / - . AAVtau ) were treated with either vehicle ( Veh ; DMSO ) or MK - 801 ( 50 μM ) for 3 min . Representative confocal of whole ( left ) neurites ( right ) are shown ( n = 3 biological replicates ) . NSF , surface GluA1 nuclei ( DAPI ) were visualized . Scale bar , 25 μm . ( D - E ) of neurite surface GluA1 neurite NSF relative to tau + / + Veh control . Reduced surface GluA1 ( D ) 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 .

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

(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 - / - ) . were stained for GluA2 , dendritic marker Map2 , NSF at 2 h post - cLTP / cLTD induction or treatment with control artificial cerebrospinal fluid ( aCSF ) . Dendritic GluA2 cluster was on GluA2 - Map2 binary map . Representative insets from n = 10 - 20 dendrites per condition from 2 mice per genotype . Scale bar , 3 μm . ( G ) of GluA2 mean relative to Map2 normalised to aCSF controls . Secondary dendrite GluA2 increased in tau - expressing ( tau + / + 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 .

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(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 - NSF GluA2 in tau + / + , tau - / - 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 each ) . Scale bar , 10 μm . Arrows indicate areas of marked co - distribution . ( I ) Co - localisation of NSF GluA2 upon chemical LTP / LTD induction . Note significantly higher proportional co - distribution of NSF GluA2 in tau - expressing ( tau + / + tau - / - . tau ) upon cLTP induction . ( n = 3 - 6 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - / - AAV . syn - hTau - injected tau - / - ( tau - / - . AAVtau ) mice showing localized AAV - mediated expression of eGFP or tau position of infusion cannulas ending in hippocampus ( dashed ) . 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 - / - 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 in tau + / + , tau - / - 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 .

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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 - / - 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 in tau + / + , tau - / - 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 .

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

(D) 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 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 .

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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 of recombinant NSF upon incubation with tau coimmunoprecipitates ( tau5 ) from brain lysates of healthy controls ( HC ) , mild cognitive impairment ( MCI ) , 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 .

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


( B ) Electrophoretic mobility shift ( EMSA ) with increasing ( 0 , 15nM , 30nM , 60nM , 1 . 25μM 2 . 5μM ) of GST - FOXD3 fixed ( 50nM ) of 5 ' - Cy5 labelled dsDNA oligonucleotides ( 35 bp each ) from Sox15 promoter ( positive control ) , MSR , MERVL - LTR , MERVL - int , L1MdA Hprt promoter ( negative control ) . experiment was repeated 3 times . ( D ) ChIP - qPCR enrichment of FOXD3 in mESCs using primers specific for MSR , MERVL - LTR , MERVL - int ( including excluding FOXD3 cognate motif ) , L1MdA promoter sequences . Sox15 promoter Actb promoter primers are used as positive negative control respectively . Data is represented as of input average of 3 biological replicates is plotted . Error bar indicates standard error of mean ( ) . Asterisks indicate statistically significant differences compared to no antibody control ( * : p < 0 . 05 , paired t - ) .

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(B) Electrophoretic mobility shift assay (EMSA) with increasing concentration (0, 15nM, 30nM, 60nM, 1.25μM and 2.5μM) of GST-FOXD3 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) from Sox15 promoter (positive control), MSR, MERVL-LTR, MERVL-int, L1MdA and Hprt promoter (negative control). The experiment was repeated 3 times. (D) ChIP-qPCR enrichment of FOXD3 in mESCs using primers specific for MSR, MERVL-LTR, MERVL-int (including and excluding the FOXD3 cognate motif), L1MdA promoter sequences. Sox15 promoter and Actb promoter primers are used as positive and negative control respectively. Data is represented as percentage of input and the average of 3 biological replicates is plotted. Error bar indicates standard error of the mean (SEM). Asterisks indicate statistically significant differences compared to no antibody control levels (*: p< 0.05, paired t-test).


( B ) Validation of select upregulated repeats in Foxd3 KO by RT - qPCR . data is plotted as average fold change relative to control , after normalization to Gapdh . Error bars indicate ( n = 3 biological replicates ) . Asterisks indicate statistically significant differences when compared to control ( * * : p < 0 . 005 , * : p < 0 . 05 , paired t - ) .

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(B) Validation of select upregulated repeats in Foxd3 KO cells by RT-qPCR. The data is plotted as average fold change relative to control, after normalization to Gapdh. Error bars indicate SEM (n=3 biological replicates). Asterisks indicate statistically significant differences when compared to control (**: p<0.005, *: p<0.05, paired t-test).


( C ) Immunofluorescence of MERVL - gag in control Foxd3 KO . Nuclei are labelled with DAPI . of counted ( ~ 150 per experiment ) exhibiting immunofluorescence signal is indicated . Scale bar represents 10 μm , n = 3 biological replicates .

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(C) Immunofluorescence analysis of the MERVL-gag protein in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting immunofluorescence signal is indicated. Scale bar represents 10 μm, n= 3 biological replicates.


( E ) Immunofluorescence of 2CLC markers ZSCAN4 DPPA3 in control Foxd3 KO . Nuclei are labelled with DAPI . of counted ( ~ 150 per experiment ) exhibiting immunofluorescence signal is indicated . Scale bar represents 10 μm , n = 3 biological replicates .

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(E) Immunofluorescence analysis of 2CLC markers ZSCAN4 and DPPA3 in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting the immunofluorescence signal is indicated. Scale bar represents 10 μm, n=3 biological replicates.


( A ) Electrophoretic mobility shift ( EMSA ) with increasing concentrations ( 0 , 30nM , 60nM , 1 . 25μM , 2 . 5μM 5μM ) of recombinant GST - FOXD3 , binding mutants GST - FOXD3 M1 GST - FOXD3 M2 fixed ( 50nM ) of 5 ' - Cy5 labelled dsDNA oligonucleotides ( 35 bp each ) representing MERVL MSR . experiment was repeated 3 times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electrophoretic mobility shift assay (EMSA) with increasing concentrations (0, 30nM, 60nM, 1.25μM, 2.5μM and 5μM) of recombinant GST-FOXD3, DNA binding mutants GST-FOXD3 M1 and GST- FOXD3 M2 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) representing MERVL and MSR. The experiment was repeated 3 times.


( B ) RT - qPCR of MERVL , MSR L1MdA in Foxd3 cKO , Foxd3 cKO over expressing full FOXD3 or FOXD3 M1 without with 4 - OHT . Data is represented as fold change relative to respective untreated after normalizing to Gapdh . average data for 3 biological replicates is plotted , error bars represent standard error mean ( ) . Asterisks indicate statistically significant differences . ( * * : p < 0 . 005 , * : p < 0 . 05 paired t - ) .

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(B) RT-qPCR analysis of MERVL, MSR and L1MdA in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).


( D ) RT - qPCR of , 2C genes Zscan4c Dppa3 in Foxd3 cKO , Foxd3 cKO over expressing full FOXD3 or FOXD3 M1 without with 4 - OHT . Data is represented as fold change relative to respective untreated after normalizing to Gapdh . average data for 3 biological replicates is plotted , error bars represent standard error mean ( ) . Asterisks indicate statistically significant differences . ( * * : p < 0 . 005 , * : p < 0 . 05 paired t - ) .

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(D) RT-qPCR analysis of, 2C genes Zscan4c and Dppa3 in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).


( A ) GFP immunoprecipitation using GFP - SUV39H1 GFP - SUV39H2 expressing Suv39h dn mESCs . top panel represents immunoblot using GFP antibody bottom panel represents immunoblot using FOXD3 antibody . experiment was repeated 3 times using biological replicates .

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(A) GFP immunoprecipitation analysis using GFP-SUV39H1 and GFP-SUV39H2 expressing Suv39h dn mESCs. The top panel represents immunoblot using GFP antibody and the bottom panel represents immunoblot using FOXD3 antibody. The experiment was repeated 3 times using biological replicates.


( D ) ChIP - qPCR of endogenous SUV39H1 enrichment over MERVL , MSR L1MdA in control Foxd3 KO . Actb promoter is used as negative control . Data is represented as average input from 3 biological replicates . Error bar indicates ( * : p < 0 . 05 paired t - ) .

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(D) ChIP-qPCR analysis of endogenous SUV39H1 enrichment over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data is represented as average percentage input from 3 biological replicates. Error bar indicates SEM (*: p<0.05 paired t-test).


( E ) ChIP - qPCR revealing H3K9me3 enrichment over MERVL , MSR L1MdA in control Foxd3 KO . Actb promoter is used as negative control . Data is represented as average input from 3 biological replicates . Error bar indicates ( * * : p < . 005 , * : p < 0 . 05 paired t - ) .

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(E) ChIP-qPCR analysis revealing H3K9me3 enrichment over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data is represented as average percentage input from 3 biological replicates. Error bar indicates SEM (**: p<.005, *: p<0.05 paired t-test).


( G ) Immunofluorescence of control Foxd3i GFP - SUV39H1 expressing Suv39h dn mESCs stained with DAPI , FOXD3 H3K9me3 . of counted ( ~ 150 per experiment ) exhibiting signal is indicated . Scale bar = 2μm . n = 3 biological replicates .

(G) Immunofluorescence analysis of control and Foxd3i GFP-SUV39H1 expressing Suv39h dn mESCs stained with DAPI, FOXD3 and H3K9me3. The percentage of counted cells (~150 cells per experiment) exhibiting fluorescence signal is indicated. Scale bar=2μm. n= 3 biological replicates.


( C ) Equal amounts of WT indicated single or double mutants in serial dilutions were incubated on agar plates at indicated temperatures . synthetic growth defects of vps4 ∆ tul1 ∆ or vps4 ∆ ubx3 ∆ double mutants at 26 ° C 37 ° C were complemented with plasmids encoding VPS4 or TUL1 or UBX3 . dashed boxes indicate non - complemented double mutants .

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(C) Equal amounts of WT cells and indicated single or double mutants in serial dilutions were incubated on agar plates at the indicated temperatures. The synthetic growth defects of vps4∆ tul1∆ or vps4∆ ubx3∆ double mutants at 26°C and 37°C were complemented with plasmids encoding VPS4 or TUL1 or UBX3. The dashed boxes indicate the non-complemented double mutants.


( A ) Vacuolar proteolysis - deficient pep4 ∆ tul1 ∆ pep4 ∆ were labeled to saturation with heavy 13C6 , 15C2 - L - lysine then chased for 3 hours in presence of L - lysine , following quantitative proteome by LC - MS ( two biological replicates ) . To analyze Dsc - dependent turnover , of H / L ratios of tul1 ∆ pep4 ∆ over pep4 ∆ of 2511 quantified in both strains was plotted , with a of H / L ratios > 2 were selected . Red squares : regulated with at least one predicted transmembrane ( TM ) domain ; black triangles : regulated without TM domains .

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(A) Vacuolar proteolysis-deficient pep4∆ and tul1∆ pep4∆ cells were labeled to saturation with heavy 13C6,15C2-L-lysine and then chased for 3 hours in presence of light L-lysine, following quantitative proteome analysis by LC-MS (two biological replicates). To analyze Dsc complex-dependent protein turnover, the ratio of H/L ratios of tul1∆ pep4∆ over pep4∆ cells of 2511 proteins quantified in both strains was plotted, and proteins with a ratio of H/L ratios >2 were selected. Red squares: regulated proteins with at least one predicted transmembrane (TM) domain; black triangles: regulated proteins without TM domains.


SDS - PAGE Western blot with indicated antibodies of total yeast lysates from WT indicated

SDS-PAGE and Western blot analysis with the indicated antibodies of total yeast lysates from WT cells and the indicated mutants


SDS - PAGE Western blot with indicated antibodies ( E ) of input elution ( with FLAG peptide ) from denaturing FLAG - Orm2 immunoprecipitations ( IP ) from WT indicated mutants . Control expressed untagged

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

SDS-PAGE and Western blot analysis with the indicated antibodies (E) of input and elution (with FLAG peptide) from denaturing FLAG-Orm2 immunoprecipitations (IP) from WT cells and the indicated mutants. Control cells expressed untagged Orm2


SDS - PAGE Western blot with indicated antibodies ( F ) of input elution ( with FLAG peptide ) from native FLAG - Orm2 co - immunoprecipitations from WT indicated mutants . control expresses untagged Orm2 .

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SDS-PAGE and Western blot analysis with the indicated antibodies (F) of input and elution (with FLAG peptide) from native FLAG-Orm2 co-immunoprecipitations from WT cells and the indicated mutants. The control strain expresses untagged Orm2.


( A ) Epifluorescence phase contrast microscopy of living WT yeast indicated mutants expressing GFP - Orm2 or GFP - Orm2 - K25 , 33R ( green ) with dsRed - HDEL ( red ) . Arrowheads point to GFP - Orm2 accumulations outside endoplasmic reticulum ( ER ) . Data information : Scale bars = 5 µm .

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(A) Epifluorescence and phase contrast microscopy of living WT yeast cells and the indicated mutants expressing GFP-Orm2 or GFP-Orm2-K25,33R (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER). Data information: Scale bars = 5 µm.


( B ) Epifluorescence phase contrast microscopy of tul1 ∆ mutants expressing GFP - Orm2 ( green ) with Vps4 - mCherry ( red , endosomes ) or with Sec7 - mCherry ( red , Golgi ) or Vps4 - eGFP ( green ) with Sec7 - mCherry . Data information : Scale bars = 5 µm .

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(B) Epifluorescence and phase contrast microscopy of tul1∆ mutants expressing GFP-Orm2 (green) with Vps4-mCherry (red, endosomes) or with Sec7-mCherry (red, Golgi) or Vps4-eGFP (green) with Sec7-mCherry. Data information: Scale bars = 5 µm.


( C ) Left panel : Epifluorescence phase contrast microscopy of indicated mutants expressing GFP - Orm2 ( green ) with mCherry - Cps1 ( red , MVB cargo ) . E indicates class E compartments ; Right panel : Equal amounts of indicated mutants in serial dilutions were incubated on agar plates at indicated temperatures . Data information : Scale bars = 5 µm .

(C) Left panel: Epifluorescence and phase contrast microscopy of the indicated mutants expressing GFP-Orm2 (green) with mCherry-Cps1 (red, MVB cargo). E indicates class E compartments; Right panel: Equal amounts of the indicated mutants in serial dilutions were incubated on agar plates at the indicated temperatures. Data information: Scale bars = 5 µm.


WT indicated mutants were left untreated ( 0 min ) or were treated with cycloheximide ( CHX ) to block synthesis for 90 min 180 min at 26 ° C or at non - permissive ( 37 ° C ) where indicated . Total lysates were analyzed by SDS - PAGE Western blotting with indicated antibodies . ( A ) Graphs display FLAG - Orm2 determined by densitometric of Western blots from lysates of WT indicated mutants 0 , 90 180 min after addition of CHX . Each experiment was repeated at least three times , FLAG - Orm2 were normalized to Pgk1 loading controls , each time - point was related to t = 0 min ( set to 1 ) . Data are presented as mean ± standard deviation .

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WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (A) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots from cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.


WT indicated mutants were left untreated ( 0 min ) or were treated with cycloheximide ( CHX ) to block synthesis for 90 min 180 min at 26 ° C or at non - permissive ( 37 ° C ) where indicated . Total lysates were analyzed by SDS - PAGE Western blotting with indicated antibodies . Each experiment was repeated at least three times , FLAG - Orm2 were normalized to Pgk1 loading controls , each time - point was related to t = 0 min ( set to 1 ) .

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

WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1).


WT indicated mutants were left untreated ( 0 min ) or were treated with cycloheximide ( CHX ) to block synthesis for 90 min 180 min at 26 ° C or at non - permissive ( 37 ° C ) where indicated . Total lysates were analyzed by SDS - PAGE Western blotting with indicated antibodies . ( C ) SEC13 WT sec13 - 4 were preincubated for 30 minutes at 37 ° C before CHX was added .

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

WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (C) SEC13 WT and sec13-4 cells were preincubated for 30 minutes at 37°C before CHX was added.


( D ) Epifluorescence phase contrast microscopy of living WT cdc48 - 3 mutants expressing GFP - Orm2 incubated for 5h at non - permissive ( 37 ° C ) . Data information : Scale bars 5µm .

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

(D) Epifluorescence and phase contrast microscopy of living WT cells and cdc48-3 mutants expressing GFP-Orm2 incubated for 5h at the non-permissive temperature (37°C). Data information: Scale bars 5µm.


( E ) Epifluorescence phase contrast microscopy of living tul1 ∆ tul1 ∆ sec13 - 4 mutants expressing GFP - Orm2 ( green ) dsRed - HDEL ( red ) incubated at 26 ° C or shifted for 90 minutes to non - permissive ( 37 ° C ) . Data information : Scale bars 5µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Epifluorescence and phase contrast microscopy of living tul1∆ and tul1∆ sec13-4 mutants expressing GFP-Orm2 (green) and dsRed-HDEL (red) incubated at 26°C or shifted for 90 minutes to the non-permissive temperature (37°C). Data information: Scale bars 5µm.


( A ) WT ( pdr5 ∆ ) expressing FLAG - Orm2 Sec61 - GFP ( integral ER ) treated with MG - 132 were fractionated into insoluble ( P100 ) soluble ( S100 ) fractions analyzed by SDS - PAGE Western blot with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O 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) WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP (integral ER membrane protein) treated with MG-132 were fractionated into insoluble membrane (P100) and soluble cytoplasmic (S100) fractions and analyzed by SDS-PAGE and Western blot with the indicated antibodies.


WT , tul1 ∆ mutants ( in a pdr5 ∆ background ) were left untreated or treated with proteasome inhibitor ( MG - 132 ) as indicated subsequently subjected to subcellular fractionation S100 P100 fractions were subjected to denaturing FLAG - Orm2 immunoprecipitations ( IP ) . Input eluted fractions ( with FLAG peptide ) were analyzed by SDS - PAGE Western blotting with indicated antibodies .

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

WT cells, tul1∆ mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.


WT cdc48 - 3 mutants ( in a pdr5 ∆ background ) were left untreated or treated with proteasome inhibitor ( MG - 132 ) as indicated subsequently subjected to subcellular fractionation S100 P100 fractions were subjected to denaturing FLAG - Orm2 immunoprecipitations ( IP ) . Input eluted fractions ( with FLAG peptide ) were analyzed by SDS - PAGE Western blotting with indicated antibodies .

WT cells and cdc48-3 mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.


( D ) P100 fractions from MG - 132 - treated WT ( pdr5 ∆ ) expressing FLAG - Orm2 Sec61 - GFP were extracted with Na2CO3 solution at indicated pH subsequently fractionated by 100 . 000 x g centrifugation into soluble ( S ) insoluble ( M ) fractions , subjected to denaturing FLAG - immunoprecipitations analyzed by Western blotting with indicated antibodies . asterisk indicates reactivity of secondary antibody with IgG heavy chains .

O O 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

(D) P100 fractions from MG-132-treated WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP were extracted with Na2CO3 solution at the indicated pH and subsequently fractionated by 100.000 x g centrifugation into soluble (S) and insoluble membrane (M) fractions, subjected to denaturing FLAG-immunoprecipitations and analyzed by Western blotting with the indicated antibodies. The asterisk indicates reactivity of the secondary antibody with IgG heavy chains.


( A ) of two major ceramide ( Cer ) species in lipid extracts from WT ( black ) , tul1Δ ( gray ) or Orm2 - K25 , 33R mutants ( grey ) were measured using LC - MS quantified using spiked non - yeast ceramides as internal standard . Data are normalized to Cer44 : 0 ; 4 of WT ( set to 1 ) presented as mean ± standard deviation from three independent experiments . Pairwise statistical significance was assessed by Student ' s t - . p values are given above respective bars .

O O O O O O B-MEASURED_VAR 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 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

(A) The levels of two major ceramide (Cer) species in lipid extracts from WT (black), tul1Δ (dark gray) or Orm2-K25,33R mutants (light grey) were measured using LC-MS and quantified using spiked non-yeast ceramides as internal standard. Data are normalized to Cer44:0;4 levels of WT cells (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.


( B ) Autoradiogram of sphingolipid extracts from [ 3H ] serine radiolabelled WT tul1 ∆ mutants were separated by thin layer chromatography . IPC , Inositolphosphorylceramide ; MIPC , mannosylinositolphosphorylceramide ; MIP2C , mannosyldiinositolphosphorylceramide . X indicates an unknown lipid species that is insensitive to myriocin treatment .

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

(B) Autoradiogram of sphingolipid extracts from [3H]serine radiolabelled WT cells and tul1∆ mutants were separated by thin layer chromatography. IPC, Inositolphosphorylceramide; MIPC, mannosylinositolphosphorylceramide; MIP2C, mannosyldiinositolphosphorylceramide. X indicates an unknown lipid species that is insensitive to myriocin treatment.


( C ) SDS - PAGE Western blot with indicated antibodies of input elution ( with FLAG peptide ) from native anti - FLAG co - immunoprecipitations from expressing FLAG - Orm2 - WT or FLAG - Orm2 - K25 , 33R . Control expressed untagged Orm2 WT . asterisks label unspecific cross - reactions of respective antibodies .

(C) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-WT or FLAG-Orm2-K25,33R. Control cells expressed untagged Orm2 WT. The asterisks label unspecific cross-reactions of the respective antibodies.


SDS - PAGE or Phos - tag - PAGE Western blot with indicated antibodies of total lysates ( D ) from WT expressing FLAG - Orm2 or FLAG - Orm2 - K25 , 33R asterisks label unspecific cross - reactions of respective antibodies .

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

SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (D) from WT cells expressing FLAG-Orm2 or FLAG-Orm2-K25,33R The asterisks label unspecific cross-reactions of the respective antibodies.


SDS - PAGE or Phos - tag - PAGE Western blot with indicated antibodies of total lysates ( E ) from WT tul1 ∆ mutants expressing FLAG - Orm2 , FLAG - Orm2 - 3A or FLAG - Orm2 - 3D with or without Myriocin treatment ( 1 . 5 µM ) for one hour .

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

SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (E) from WT cells and tul1∆ mutants expressing FLAG-Orm2, FLAG-Orm2-3A or FLAG-Orm2-3D with or without Myriocin treatment (1.5 µM) for one hour.


( A ) Left panel : In tor1 - 1 avo3 ∆ 1274 - 1430 rapamycin specifically inhibits TORC2 but not TORC1 ( Gaubitz et al . , 2015 ) . tor1 - 1 avo3 ∆ 1274 - 1430 were treated with cycloheximide ( CHX ) rapamycin or vehicle ( methanol ) for indicated times . Right panel : WT or Orm2 mutant were treated with CHX for indicated times . of FLAG - Orm2 or of indicated FLAG - Orm2 mutants from total lysates were analyzed by SDS - PAGE Western blotting with indicated antibodies .

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

(A) Left panel: In tor1-1 avo3∆1274-1430 cells rapamycin specifically inhibits TORC2 but not TORC1 (Gaubitz et al., 2015). tor1-1 avo3∆1274-1430 cells were treated with cycloheximide (CHX) and rapamycin or vehicle (methanol) for the indicated times. Right panel: WT or Orm2 mutant cells were treated with CHX for the indicated times. The protein levels of FLAG-Orm2 or of the indicated FLAG-Orm2 mutants from total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.


( B ) Graphs display FLAG - Orm2 determined by densitometric of Western blots in lysates of WT indicated mutants 0 , 90 180 min after addition of CHX . Each experiment was repeated at least three times . FLAG - Orm2 were normalized to Pgk1 loading controls , each timepoint was related to t = 0 min ( set to 1 ) . Data are presented as mean ± standard deviation .

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

(B) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times. FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each timepoint was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.


( C ) Denaturing immunoprecipitation ( IP ) of FLAG - Orm2 , FLAG - Orm2 - 3A FLAG - Orm2 - 3D . control expresses untagged Orm2 . Input elutions ( with FLAG peptides ) were analyzed by SDS - PAGE Western blot with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Denaturing immunoprecipitation (IP) of FLAG-Orm2, FLAG-Orm2-3A and FLAG-Orm2-3D. The control strain expresses untagged Orm2. Input and elutions (with FLAG peptides) were analyzed by SDS-PAGE and Western blot with the indicated antibodies.


( D ) Epifluorescence phase contrast microscopy of living tul1 ∆ mutants expressing GFP - Orm2 , GFP - Orm2 - 3A or GFP - Orm2 - 3D ( green ) with dsRed - HDEL ( red ) . Arrowheads point to GFP - Orm2 accumulations outside endoplasmic reticulum ( ER ) . Scale bars = 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

(D) Epifluorescence and phase contrast microscopy of living tul1∆ mutants expressing GFP-Orm2, GFP-Orm2-3A or GFP-Orm2-3D (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER). Scale bars = 5 µm.


( E ) SDS - PAGE Western blot with indicated antibodies of input elution ( with FLAG peptide ) from native anti - FLAG co - immunoprecipitations from expressing FLAG - Orm2 - K25 , 33R , FLAG - Orm2 - K25 , 33R - 3A or FLAG - Orm2 - K25 , 33R - 3D . control expresses untagged Orm2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-K25,33R, FLAG-Orm2-K25,33R-3A or FLAG-Orm2-K25,33R-3D. The control strain expresses untagged Orm2.


( F ) of long chain bases ( LCB ) C18 - dihydrosphingosine ( C18 - DHS ) C18 - phytosphingosine ( C18 - PHS ) of two major ceramide ( Cer ) species in lipid extracts from WT , tul1 ∆ , Orm2 - K25 , 33R , Orm2 - 3A Orm2 - 3D mutants were measured using LC - MS quantified using spiked non - yeast LCBs / ceramides as internal standard . Data are normalized to WT of most abundant LCB / ceramide species ( set to 1 ) presented as mean ± standard deviation from three independent experiments . Pairwise statistical significance was assessed by Student ' s t - . p values are given above respective bars .

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

(F) The levels of the long chain bases (LCB) C18-dihydrosphingosine (C18-DHS) and C18-phytosphingosine (C18-PHS) and of two major ceramide (Cer) species in lipid extracts from WT, tul1∆, Orm2-K25,33R, Orm2-3A and Orm2-3D mutants were measured using LC-MS and quantified using spiked non-yeast LCBs/ceramides as internal standard. Data are normalized to WT levels of the most abundant LCB/ceramide species (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.


( A ) Total lysates prepared from indicated HCT116 lines were analyzed by immunoblotting . For comparison , different amounts of ( 1 : 3 ) were applied . An asterisk indicated non - specific crossreactive bands .

(A) Total cell lysates prepared from the indicated HCT116 cell lines were analyzed by immunoblotting. For comparison, different amounts of proteins (1:3 ratio) were applied. An asterisk indicated non-specific crossreactive bands.


( B ) indicated lines were analyzed by immunofluorescence microscopy using anti - Cytochrome c antibody for mitochondria anti - PMP70 antibody for peroxisome . are displayed as z - stacks of 6 confocal slices . Magnified are also shown for mitochondrial morphologies . Scale bars , 10 μm . ( C ) of mitochondrial morphologies in ( B ) . Percentages of harboring fragmented , tubular , or elongated mitochondria are shown . Tubular elongated denote normal tubular mitochondria seen in WT highly connected mitochondrial network , respectively . error bars represent ± SD from three independent replicates . Over 50 were counted in each of three replicate wells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 indicated cell lines were analyzed by immunofluorescence microscopy using anti-Cytochrome c antibody for mitochondria and anti-PMP70 antibody for peroxisome staining. Images are displayed as z-stacks of 6 confocal slices. Magnified images are also shown for mitochondrial morphologies. Scale bars, 10 μm. (C) Quantification of mitochondrial morphologies in (B). Percentages of cells harboring fragmented, tubular, or elongated mitochondria are shown. Tubular and elongated denote normal tubular mitochondria seen in WT cells and highly connected mitochondrial network, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each of three replicate wells.


( A ) indicated lines stably expressing YFP - LC3 mCherry - Parkin were treated with valinomycin for 3 hr subjected to confocal immunofluorescence microscopy with anti - TOMM20 antibody . Scale bars , 10 μm . ( B ) of mCherry - Parkin translocation to mitochondria after 3 hr of valinomycin treatment . Partial or complete translocation to mitochondria in each was scored as separate . Partial complete denote that Parkin translocates to some of or mitochondria , respectively . error bars represent ± SD from three independent experiments . Over 100 were counted in each of three separate wells . ( C ) YFP - LC3 morphologies in ( A ) were quantified . Percentages of harboring diffuse , punctate or accumulated YFP - LC3 are shown . error bars represent ± SD from three independent replicates . Over 100 were counted in each replicate . For criteria of LC3 , see Figure 2 - figure supplement 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The indicated cell lines stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to confocal immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial or complete translocation to mitochondria in each cell was scored as separate phenotypes. Partial and complete denote that Parkin translocates to some of or all mitochondria, respectively. The error bars represent ±SD from three independent experiments. Over 100 cells were counted in each of three separate wells. (C) YFP-LC3 morphologies in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate. For the criteria of LC3 morphology, see Figure 2-figure supplement 1.


( D ) YFP - LC3 mCherry - Parkin stably expressing TBC1D15 − / − in absence or presence of HA - tagged TBC1D15 WT or Δ221 - 250 mutant were treated with valinomycin for 3 hr . were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( E ) YFP - LC3 of in ( D ) was quantified . error bars represent ± SD from three independent replicates . Over 50 were counted in each well .

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

(D) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the absence or presence of HA-tagged TBC1D15 WT or Δ221-250 mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (E) The YFP-LC3 morphology of cells in (D) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( F G ) YFP - LC3 mCherry - Parkin stably expressing WT ( F ) TBC1D15 − / − ( G ) were treated with valinomycin for 3 hr then subjected to immunoelectron microscopy with anti - GFP antibody . square in panel a shows enlarged areas in panel b Scale bars , 500 nm .

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

(F and G) YFP-LC3 and mCherry-Parkin stably expressing WT (F) and TBC1D15−/− (G) cells were treated with valinomycin for 3 hr and then subjected to immunoelectron microscopy with anti-GFP antibody. The square in panel a shows enlarged areas in panel b Scale bars, 500 nm.


( A ) indicated stably expressing YFP - LC3 mCherry - Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti - Cytochrome c antibody . Confocal were acquired as z - stacks comprising 6 sequential sections with 0 . 8 μm z - intervals . Scale bars , 10 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

(A) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti-Cytochrome c antibody. Confocal images were acquired as z-stacks comprising 6 sequential sections with 0.8 μm z-intervals. Scale bars, 10 μm.


( B ) TBC1D15 − / − prepared as in ( A ) were subjected to immunoelectron microscopy with anti - GFP antibody . arrowheads indicate YFP - LC3 - labeled tubules . High magnification image is shown in lower panel . Scale bars , 500 nm .

(B) TBC1D15−/− cells prepared as in (A) were subjected to immunoelectron microscopy with anti-GFP antibody. White arrowheads indicate YFP-LC3-labeled tubules. High magnification image is shown in the lower panel. Scale bars, 500 nm.


( C ) indicated prepared as in ( A ) were subjected to immunostaining with anti - Tubulin antibody . YFP - LC3 Tubulin are merged in right panels . Magnified are shown for TBC1D15 − / − . Scale bars , 10 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

(C) The indicated cells prepared as in (A) were subjected to immunostaining with anti-Tubulin antibody. YFP-LC3 and Tubulin staining are merged in the right panels. Magnified images are shown for TBC1D15−/− cells. Scale bars, 10 μm.


( D ) indicated stably expressing YFP - LC3 mCherry - Parkin were treated with valinomycin in presence or absence of nocodazole for 3 hr . YFP - LC3 mCherry - Parkin are merged in right panels . Scale bars , 10 μm . ( E ) YFP - LC3 morphologies of in ( D ) were quantified . error bars represent ± SD from three independent replicates . Over 50 were counted in each well .

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

(D) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin in the presence or absence of nocodazole for 3 hr. YFP-LC3 and mCherry-Parkin images are merged in the right panels. Scale bars, 10 μm. (E) YFP-LC3 morphologies of cells in (D) were quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( A B ) YFP - Parkin stably expressing were treated with valinomycin for indicated times . Total lysates were subjected to immunoblotting . I II denote lipidated LC3B , respectively . An asterisk indicates ubiquitinated TOMM20 . ( C ) Indicated amounts as in ( B ) were quantified . amount of without valinomycin treatment was set to 100 % . error bars represent ± SD from three independent experiments .

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

(A and B) YFP-Parkin stably expressing cells were treated with valinomycin for indicated times. Total cell lysates were subjected to immunoblotting. I and II denote cytosolic and lipidated LC3B, respectively. An asterisk indicates ubiquitinated TOMM20. (C) Indicated protein amounts as in (B) were quantified. The amount of protein without valinomycin treatment was set to 100%. The error bars represent ±SD from three independent experiments.


( D ) YFP - Parkin stably expressing WT HCT116 were treated with or without valinomycin for 40 hr . Total lysates were analyzed by immunoblotting .

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

(D) YFP-Parkin stably expressing WT HCT116 cells were treated with or without valinomycin for 40 hr. Total cell lysates were analyzed by immunoblotting.


( A ) WT , MFF − / − or DRP1 − / − stably expressing YFP - LC3 mCherry - Parkin were treated with valinomycin for 3 hr subjected to immunofluorescence microscopy with anti - TOMM20 antibody . Scale bars , 10 μm . ( B ) of mCherry - Parkin translocation to mitochondria after 3 hr of valinomycin treatment . Partial complete denote that Parkin translocates to some of mitochondria , respectively . error bars represent ± SD from three independent replicates . Over 50 were counted in each replicate . ( C ) YFP - LC3 morphologies of in ( A ) were quantified . Percentages of harboring diffuse , punctate or accumulated YFP - LC3 are shown . error bars represent ± SD from three independent replicates . Over 100 were counted in each replicate .

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(A) WT, MFF−/− or DRP1−/− cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial and complete denote that Parkin translocates to some of and all mitochondria, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate. (C) YFP-LC3 morphologies of cells in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate.


( D ) WT , FIS1 − / − , TBC1D15 − / − stably expressing YFP - LC3 were grown in starvation media . Z - stacks of confocal are shown . Scale bars , 20 μm . ( E ) number of YFP - LC3 dots in under growth or starvation conditions was quantified . error bars represent ± SD from three independent replicates . Over 50 were counted in each well .

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(D) WT, FIS1−/−, and TBC1D15−/− cells stably expressing YFP-LC3 were grown in starvation media. Z-stacks of confocal images are shown. Scale bars, 20 μm. (E) The number of YFP-LC3 dots in cells under growth or starvation conditions was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( F ) Total lysates from grown in normal or starvation media for 6 hr were subjected to immunoblotting . LC3 - I LC3 - II denote lipidated LC3B , respectively .

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(F) Total cell lysates from cells grown in normal or starvation media for 6 hr were subjected to immunoblotting. LC3-I and LC3-II denote cytosolic and lipidated LC3B, respectively.


( A ) TBC1D15 − / − those stably expressing HA - TBC1D15 WT HA - TBC1D15 ( Δ221 - 250 ) , FIS1 − / − stably expressing HA - TBC1D15 WT were subjected to immunostaining with anti - TOMM20 anti - HA antibodies . Scale bars , 10 μm .

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(A) TBC1D15−/− cells and those stably expressing HA-TBC1D15 WT and HA-TBC1D15 (Δ221-250), and FIS1−/− cells stably expressing HA-TBC1D15 WT were subjected to immunostaining with anti-TOMM20 and anti-HA antibodies. Scale bars, 10 μm.


( B ) HA - TBC1D15 WT or HA - TBC1D15 ( Δ221 - 250 ) with or without Fis1 was transiently overexpressed ( OE ) in HeLa . were subjected to immunostaining with anti - HA anti - Fis1 antibodies . Scale bars , 20 μm .

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(B) HA-TBC1D15 WT or HA-TBC1D15 (Δ221-250) with or without Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Scale bars, 20 μm.


( C ) YFP - TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed ( OE ) in HeLa . were subjected to immunostaining with anti - Fis1 anti - Cytochrome c antibodies . Scale bars , 20 μm .

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(C) YFP-TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-Fis1 and anti-Cytochrome c antibodies. Scale bars, 20 μm.


( D ) YFP or YFP - Fis1 was co - overexpressed with HA - TBC1D15 or HA - TBC1D17 in HEK293 . extracts were subjected to pull down assays with GFP - Trap . 5 % input bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) anti - GFP ( lower panel ) antibodies .

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(D) YFP or YFP-Fis1 was co-overexpressed with HA-TBC1D15 or HA-TBC1D17 in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies.


( E ) YFP , YFP - TBC1D15 , or YFP - TBC1D17 was co - overexpressed with HA - TBC1D15 ( upper panel ) or HA - TBC1D17 ( lower panel ) in HEK293 . extracts were subjected to pull down assays with GFP - Trap . 5 % input bound fractions were analyzed by immunoblotting with anti - GFP anti - HA antibodies .

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(E) YFP, YFP-TBC1D15, or YFP-TBC1D17 was co-overexpressed with HA-TBC1D15 (upper panel) or HA-TBC1D17 (lower panel) in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP and anti-HA antibodies.


( F ) HA - TBC1D15 ( Δ221 - 250 ) together with Fis1 YFP - TBC1D15 WT or YFP - TBC1D17 WT were transiently overexpressed ( OE ) in HeLa . were subjected to immunostaining with anti - HA anti - Fis1 antibodies . of HA Fis1 were merged in right panels . Scale bars , 20 μm .

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(F) HA-TBC1D15 (Δ221-250) together with Fis1 and YFP-TBC1D15 WT or YFP-TBC1D17 WT were transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Images of HA and Fis1 staining were merged in the right panels. Scale bars, 20 μm.


( A ) indicated transiently expressing YFP - GABARAPL1 ( green ) mCherry - Parkin ( red ) were treated with valinomycin for 3 hr . Scale bars , 10 μm .

(A) The indicated cells transiently expressing YFP-GABARAPL1 (green) and mCherry-Parkin (red) were treated with valinomycin for 3 hr. Scale bars, 10 μm.


( B ) YFP - TBC1D15 overexpressed in HEK293 was subjected to binding assays with GST - fused ( GABA , L1 , L2 represent GABARAP , GABARAPL1 , GABARAPL2 , respectively ) . 5 % input bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) . Coomassie brilliant blue ( CBB ) shows GST - fusion in bound fractions ( lower panel ) . ( C ) Binding carried out as in ( B ) with GST - GABARAPL1 WT ( GST - WT ) or its Y49A / L50A mutant ( GST - YL ) . Immunoblotting with anti - GFP antibody ( upper panel ) CBB ( lower panel ) are shown .

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(B) YFP-TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST-fused proteins (GABA, L1, and L2 represent GABARAP, GABARAPL1, and GABARAPL2, respectively). 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel). Coomassie brilliant blue (CBB) staining shows GST-fusion proteins in bound fractions (lower panel). (C) Binding assay carried out as in (B) with GST-GABARAPL1 WT (GST-WT) or its Y49A/L50A mutant (GST-YL). Immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel) are shown.


( D ) extracts from HEK293 overexpressed HA - TBC1D15 YFP , YFP - GABARAPL1 ( YFP - WT ) , or its Y49AL50A mutant ( YFP - YL ) were subjected to pull down assays with GFP - Trap . 5 % input bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) anti - GFP ( lower panel ) antibodies .

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(D) Cell extracts from HEK293 overexpressed HA-TBC1D15 and YFP, YFP-GABARAPL1 (YFP-WT), or its Y49AL50A mutant (YFP-YL) were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies.


( E - H ) indicated YFP - tagged TBC1D15 full - , truncated , or point - mutant or YFP - Fis1 overexpressed in HEK293 were subjected to binding assays with recombinant GST - GABARAPL1 . 5 % input bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) CBB ( lower panel ) .

(E-H) The indicated YFP-tagged TBC1D15 full-length, truncated, or point-mutant protein or YFP-Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST-GABARAPL1. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel).


( J ) LC3 - LC3 mCherry - Parkin stably expressing TBC1D15 − / − in presence of HA - tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr . were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( K ) YFP - LC3 of in ( J ) was quantified . error bars represent ± SD from three independent replicates . Over 50 were counted in each well .

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(J) LC3-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (K) The YFP-LC3 morphology of cells in (J) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( A ) Total lysates from HCT116 treated with control ( NTC ) or RAB7A siRNA were analyzed by immunoblotting .

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(A) Total cell lysates from HCT116 treated with control (NTC) or RAB7A siRNA were analyzed by immunoblotting.


( B ) indicated stably expressing YFP - LC3 ( green ) mCherry - Parkin were treated with control ( NTC ) or Rab7 _ # 5 siRNA . After 3 hr valinomycin treatment , were analyzed by immunofluorescence microscopy with anti - TOMM20 antibody ( red ) . Z - stacks of confocal are shown . Magnified are also shown . Scale bars , 10 μm . ( C ) YFP - LC3 morphologies of in ( B ) were quantified . Percentages of harboring diffuse , punctuate or accumulated / tubulated YFP - LC3 are shown . Data error bars were obtained from at least 50 in each of three independent replicates .

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(B) The indicated cells stably expressing YFP-LC3 (green) and mCherry-Parkin were treated with control (NTC) or Rab7_#5 siRNA. After 3 hr valinomycin treatment, cells were analyzed by immunofluorescence microscopy with anti-TOMM20 antibody (red). Z-stacks of confocal images are shown. Magnified images are also shown. Scale bars, 10 μm. (C) YFP-LC3 morphologies of cells in (B) were quantified. Percentages of cells harboring diffuse, punctuate or accumulated/tubulated YFP-LC3 are shown. Data and error bars were obtained from at least 50 cells in each of three independent replicates.


( D ) indicated stably expressing YFP - LC3 ( green ) , mCherry - Parkin , 2HA - Rab7 ( Red ) were treated with or without valinomycin for 3 hr analyzed by immunofluorescence microscopy with anti - HA antibody . Magnified are also shown . Scale bars , 10 μm .

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(D) The indicated cells stably expressing YFP-LC3 (green), mCherry-Parkin, and 2HA-Rab7 (Red) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti-HA antibody. Magnified images are also shown. Scale bars, 10 μm.


( E ) YFP - LC3 mCherry - Parkin stably expressing TBC1D15 − / − in presence of HA - tagged TBC1D15 WT or D397A mutant were treated with valinomycin for 3 hr . were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( F ) YFP - LC3 of in ( E ) was quantified . error bars represent ± SD from three independent replicates . Over 50 were counted in each replicate .

(E) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (F) The YFP-LC3 morphology of cells in (E) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate.


( b ) Effects of C18 - Pyr - Cer ( C18PC , lanes 2 4 ) on lipidation of LC3B ( LC3B - II ) were examined by western blotting in absence or presence of a pan - caspase inhibitor Z - VAD ( lanes 3 4 ) compared to vehicle - treated controls ( lanes 1 3 ) . Full blots can be found in Supplementary Figure 13 .

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(b) Effects of C18-Pyr-Cer (C18PC, lanes 2 and 4) on the lipidation of LC3B (LC3B-II) were examined by western blotting in the absence or presence of a pan-caspase inhibitor Z-VAD (lanes 3 and 4) and compared to vehicle-treated controls (lanes 1 and 3). Full blots can be found in Supplementary Figure 13.


( c ) Formation of double - autophagosomal vesicles ( black arrows ) in absence ( control ) or presence of C18PC was visualized by ( left right , respectively ) . Higher magnification of visualization is shown in lower panels . Scale bars , 10 μm ( top ) 500 nm ( bottom ) .

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(c) Formation of double-membrane autophagosomal vesicles (black arrows) in the absence (control) or presence of C18PC was visualized by TEM (left and right, respectively). Higher magnification of TEM visualization is shown in lower panels. Scale bars, 10 μm (top) and 500 nm (bottom).


( d ) Effects of siRNA - mediated knockdown of Atg3 or Atg7 on in absence or presence of C18PC were determined compared to effects of transfection with Scr siRNAs ( controls ) using Trypan blue exclusion .

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(d) Effects of siRNA-mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18PC were determined and compared to effects of transfection with Scr siRNAs (controls) using Trypan blue exclusion assay.


( e ) Roles of C18PC in regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild - type ( ATG5 + / + ) ATG5 − / − knockout mice with C18PC . Control were treated with vehicle .

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(e) Roles of C18PC in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild-type (ATG5+/+) and ATG5−/− knockout mice with C18PC. Control cells were treated with vehicle.


( f ) Effects of C18PC on caspase - dependent or caspase - independent were determined in MEFs isolated from Bax − / − Bak − / − or Casp3 − / − Casp7 − / − mice from wild - type ( Bax + / + Bak + / + ) or Casp3 + / − Casp7 + / − mice , used as controls . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(f) Effects of C18PC on caspase-dependent or caspase-independent cell death were determined in MEFs isolated from Bax−/− Bak−/− or Casp3−/− Casp7−/− mice and from wild-type (Bax+/+ Bak+/+) or Casp3+/− Casp7+/− mice, used as controls. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( a ) Targeting of autophagolysosomes to mitochondria in absence or presence of C18 - Pyr - Cer ( C18PC ) at 2 h in UM - SCC22A ( 22A ) was examined by visualizing colocalization of MTG ( green ) LTR ( red ) using live - imaging confocal microscopy . Scale bars , 10 μm . Enlarged are shown in boxed areas .

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(a) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18-Pyr-Cer (C18PC) at 2 h in UM-SCC22A (22A) cells was examined by visualizing the colocalization of MTG (green) and LTR (red) using live-cell imaging and confocal microscopy. Scale bars, 10 μm. Enlarged images are shown in boxed areas.


( b ) Effects of C18 - dihydro - Cer - 14 - piperidine ( dihydro - C18PC ) on OCR measured using SeaHorse alongside those of C18PC - vehicle - treated controls . Data shown are an average of at least two independent experiments performed in duplicate ± s . d . ( * P 0 . 05 ) .

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(b) Effects of C18-dihydro-Cer-14-piperidine (dihydro-C18PC) on OCR measured using the SeaHorse alongside those of C18PC- and vehicle-treated controls. Data shown are an average of at least two independent experiments performed in duplicate ± s.d. (*P 0.05).


( a ) Effects of expression of wild - type ( WT ) CerS1 its catalytically inactive V5 - tagged mutant ( Mut ) , which cannot generate C18 - ceramide , on formation of LC3B - II were determined by western blotting ( + / − tet ) . Successful induction of wild - type mutant CerS1 expression was confirmed using anti - V5 . β - actin was used as a loading control . Full blots can be found in Supplementary Figure 13 .

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(a) Effects of expression of wild-type (WT) CerS1 and its catalytically inactive V5-tagged mutant (Mut), which cannot generate C18-ceramide, on the formation of LC3B-II were determined by western blotting (+/− tet). Successful induction of wild-type and mutant CerS1 expression was confirmed using anti-V5. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.


( b ) Generation of endogenous C18 - C18 : 1 - ceramides in response to wild - type CerS1 mutant CerS1 induction was measured by LC / MS / MS .

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(b) Generation of endogenous C18- and C18:1-ceramides in response to wild-type CerS1 and mutant CerS1 induction was measured by LC/MS/MS.


( c ) Effects of wild - type CerS1 C18 - ceramide induction ( + tet ) on GFP LC3B - GFP lipidation were visualized using confocal microscopy compared to effects of noninduced controls ( − tet ) . Scale bars , 10

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(c) Effects of wild-type CerS1 and C18-ceramide induction (+ tet) on GFP and LC3B-GFP lipidation were visualized using confocal microscopy and compared to effects of noninduced controls (− tet). Scale bars, 10 μm


( d ) effects of induction of wild - type CerS1 C18 - ceramide catalytically inactive mutant CerS1 ( + tet ) in regulation of mitochondrial function were assessed by measuring OCR using SeaHorse compared to roles of noninduced controls ( − tet ) .

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(d) The effects of induction of wild-type CerS1 and C18-ceramide and catalytically inactive mutant CerS1 (+ tet) in the regulation of mitochondrial function were assessed by measuring OCR using the SeaHorse and compared to roles of noninduced controls (− tet).


( e ) Targeting mitochondria with autophagolysosomes in absence ( − tet ) or presence ( + tet ) of wild - type CerS1 C18 - ceramide was visualized by colocalization of MTG LTR using confocal microscopy . Scale bars , 10 μm .

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(e) Targeting mitochondria with autophagolysosomes in the absence (− tet) or presence (+ tet) of wild-type CerS1 and C18-ceramide was visualized by colocalization of MTG and LTR using confocal microscopy. Scale bars, 10 μm.


( f ) Effects of wild - type mutant CerS1 ( − / + tet ) on ATP generation .

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(f) Effects of wild-type and mutant CerS1 (−/+ tet) on ATP generation.


( g ) Effects of wild - type CerS1 ( − / + tet ) on ATP generation in absence ( control shRNA ) or presence of shRNA - mediated knockdown of LC3B . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(g) Effects of wild-type CerS1 (−/+ tet) on ATP generation in the absence (control shRNA) or presence of shRNA-mediated knockdown of LC3B. Data shown are an average of at least three experiments ± s.d. (*P 0.05).