A ) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d . n > 10 organoids in each experiment . B ) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel ( A ) , error bars show 1 SD ( * * * * p < 0 . 0001 ) . n > 10 organoids per condition ( top ) . Quantification of the nuclear / cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel ( C ) . n > 10 organoids per condition ( bottom ) . Statistical analysis was performed with an unpaired student ' s t - test . C

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A) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d. n > 10 organoids in each experiment. B) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel (A), error bars show 1 SD (**** p <0.0001). n > 10 organoids per condition (top). Quantification of the nuclear/cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel (C). n > 10 organoids per condition (bottom). Statistical analysis was performed with an unpaired student's t-test. C


C ) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm . n > 10 organoids in each experiment . Q - PCR analysis of the YAP - TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 ( relative expression , error bars = 1 SD ) . Statistical analysis was performed with an unpaired student ' s t - test .

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C) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm. n > 10 organoids in each experiment. Q-PCR analysis of the YAP-TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 (relative expression, error bars = 1 SD). Statistical analysis was performed with an unpaired student's t-test.


A ) Mouse intestinal organoids immunostained for YAP ( green ) . Note the mostly cytoplasmic localisation of YAP at 48h in the organoid . Irradiation with 4Gy of X - rays drives strong nuclear localisation of YAP in many cells , which is reversible by treatment with the Src family kinase inhibitor Dasatinib . The Dasatinib treated organoids exhibit increased cell death after irradiation , as indicated by pyknotic nuclei ( white arrow ) . n > 10 organoids in each experiment . This experiment employs the same control batch of 2 - 3d organoids also shown in Figure 6A .

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A) Mouse intestinal organoids immunostained for YAP (green). Note the mostly cytoplasmic localisation of YAP at 48h in the organoid. Irradiation with 4Gy of X-rays drives strong nuclear localisation of YAP in many cells, which is reversible by treatment with the Src family kinase inhibitor Dasatinib. The Dasatinib treated organoids exhibit increased cell death after irradiation, as indicated by pyknotic nuclei (white arrow). n > 10 organoids in each experiment. This experiment employs the same control batch of 2-3d organoids also shown in Figure 6A.


B ) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation . Irradiation with 14Gy of X - rays drives strong nuclear localisation of YAP along the entire crypt - villus axis . Deletion of Src within VillinCreERt Srcflox / flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation . Arrows point to villar cells with nuclear YAP localisation . Lower panels show high - mag views of a different example than those shown in upper panels .

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B) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation. Irradiation with 14Gy of X-rays drives strong nuclear localisation of YAP along the entire crypt-villus axis. Deletion of Src within VillinCreERt Srcflox/flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation. Arrows point to villar cells with nuclear YAP localisation. Lower panels show high-mag views of a different example than those shown in upper panels.


A ) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA ( black arrows point to mRNA granules in the control ) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes . Nuclear localisation of beta - catenin is readily detectable after irradiation . n = 3 samples per condition , quantification shown in the insets , error bars = 1 SD . Statistical analysis was performed with an unpaired student ' s t - test . Similar results were obtained at 3dpi and 5dpi .

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A) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA (black arrows point to mRNA granules in the control) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes. Nuclear localisation of beta-catenin is readily detectable after irradiation. n = 3 samples per condition, quantification shown in the insets, error bars = 1 SD. Statistical analysis was performed with an unpaired student's t-test. Similar results were obtained at 3dpi and 5dpi.


B ) Q - PCR analysis of YAP , TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy . Note the strong upregulation of TEAD4 , Ctgf , and Cyr61 . In contrast , Axin2 , TEAD2 and Sox9 mRNA expression levels are only moderately altered , confirming that Wnt signalling remains active upon irradiation - induced YAP - TEAD activation . n = 4 biological replicates per time point . Statistical analysis was performed with unpaired t - test compared to the control 0Gy condition , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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B) Q-PCR analysis of YAP, TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy. Note the strong upregulation of TEAD4, Ctgf, and Cyr61. In contrast, Axin2, TEAD2 and Sox9 mRNA expression levels are only moderately altered, confirming that Wnt signalling remains active upon irradiation-induced YAP-TEAD activation. n = 4 biological replicates per time point. Statistical analysis was performed with unpaired t-test compared to the control 0Gy condition, * p<0.05, ** p<0.01, ***p<0.001.


C ) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation . Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 ( n = 4 animals in each condition ) .

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C) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation. Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 (n= 4 animals in each condition).


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 )

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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 ) and for cell 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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 ) 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 .

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


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

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


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

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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 - 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 .

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


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

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


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

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


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

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


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

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


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

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


( 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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(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 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 ) .

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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 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 ) .

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

(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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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(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 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 .

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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 ∆ 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 .

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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 and Western blot analysis with the indicated antibodies of total yeast lysates from WT cells and the indicated

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


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

O O O 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

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 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 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 .

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

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 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 ) .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR 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. (C) SEC13 WT and sec13-4 cells were preincubated for 30 minutes at 37°C before CHX was added.


( 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 .

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(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 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 .

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(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 ∆ ) 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 .

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(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 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 .

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 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

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 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 .

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-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

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 ∆ ) 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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

(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 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 .

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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 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 .

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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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

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(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 ) 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 − / − 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 .

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(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 ) 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 .

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(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 ) 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 .

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(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 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 .

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(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 cells were treated with or without valinomycin for 40 hr . Total cell lysates were analyzed by immunoblotting .

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(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 − / − 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 .

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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 − / − , 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 .

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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 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 .

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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 − / − 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 .

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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 cells . Cells were subjected to immunostaining with anti - HA and 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.