Mice were treated by 2 % dextran sodium sulfate ( DSS ) in drinking water for 7 days . ( I ) The frequency of CD11b + Ly6C + and CD11b + Ly6G + cells in colonic lamina propria ( cLP ) of COPS8fl / fl and or COPS8ΔIEC mice with DSS - induced colitis . The column graph represents percentage of Ly6C + and Ly6G + cells , presented as mean ± SEM from three biological replicates per genotype . * P < 0 . 05 , NS - non - significant using Student ' s t - test . ( J ) Representative FACS plots and percentage of intracellular staining of Forkhead box protein P3 ( FOXP3 ) , Interferon gamma ( IFN - γ ) and IL - 17A in CD3 + CD4 + T cells from colonic lamina propria of COPS8fl / fl or COPS8ΔIEC mice with DSS - induced colitis . The column graph represents percentage of Treg + and ratio of Th1 / Th17 cells , represented as mean ± SEM from three biological replicates per genotype . * P < 0 . 05 using Student ' s t - test .

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Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (I) The frequency of CD11b+Ly6C+ and CD11b+Ly6G+ cells in colonic lamina propria (cLP) of COPS8fl/fl and or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Ly6C+ and Ly6G+ cells, presented as mean ± SEM from three biological replicates per genotype. *P<0.05, NS- non-significant using Student's t-test. (J) Representative FACS plots and percentage of intracellular staining of Forkhead box protein P3 (FOXP3), Interferon gamma (IFN-γ) and IL-17A in CD3+CD4+ T cells from colonic lamina propria of COPS8fl/fl or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Treg+ and ratio of Th1/Th17 cells, represented as mean ± SEM from three biological replicates per genotype. *P<0.05 using Student's t-test.


( H ) Flow cytometric analysis of CD45 + infiltrating immune cells in the lamina propria of the ileum . Data are represented as mean ± SEM from three biological replicates per genotype . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(H) Flow cytometric analysis of CD45+ infiltrating immune cells in the lamina propria of the ileum. Data are represented as mean ± SEM from three biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA.


( I ) Real - time quantitative reverse transcription polymerase chain reaction ( qRT - PCR ) analysis of the expression of genes encoding inflammatory cytokines in the ileum . COPS8fl / fl and COPS8ΔIEC mice were either untreated ( phosphate buffered saline ( PBS ) ) or treated with the antibiotic cocktail ( Abx ) for 3 months . Data are mean ± SEM from three biological replicates . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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

(I) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression of genes encoding inflammatory cytokines in the ileum. COPS8fl/fl and COPS8ΔIEC mice were either untreated (phosphate buffered saline (PBS)) or treated with the antibiotic cocktail (Abx) for 3 months. Data are mean ± SEM from three biological replicates. **P<0.01, NS- non-significant using one-way ANOVA.


( B ) Representative image showing changes in colon morphology and length following DSS - induced colitis with concurrent treatment with MBELNs COPS8fl / fl and COPS8ΔIEC mice . Representative data from seven biological replicates per genotype .

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(B) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs COPS8fl/fl and COPS8ΔIEC mice. Representative data from seven biological replicates per genotype.


Hematoxylin and eosin staining ( HE ) to show histological changes Scale bar

Hematoxylin and eosin staining (HE) to show histological changes Scale bar 200μm


( F ) Expression of anti - microbial peptide ( AMPs ) in the colon of COPS8fl / fl and COPS8ΔIEC mice while being treated with MBELNs by oral administration . Data are mean ± SEM from three biological replicates per group . * P < 0 . 05 , * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(F) Expression of anti-microbial peptide (AMPs) in the colon of COPS8fl/fl and COPS8ΔIEC mice while being treated with MBELNs by oral administration. Data are mean ± SEM from three biological replicates per group. *P<0.05, **P<0.01, NS- non-significant using one-way ANOVA.


( I - J ) Transcriptional expression of nuclear factor kappa B ( NF - κB ) ( I ) and inhibitor of nuclear factor kappa - β kinase ( IKK - β ) ( J ) mRNA using Real - time quantitative reverse transcription Polymerase chain reaction ( qRT - PCR ) in colonic epithelial cells upon treatment with MBELNs in addition to DSS . Data are mean ± SEM from three biological replicates per group . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(I-J) Transcriptional expression of nuclear factor kappa B (NF-κB) (I) and inhibitor of nuclear factor kappa-β kinase (IKK-β) (J) mRNA using Real-time quantitative reverse transcription Polymerase chain reaction (qRT-PCR) in colonic epithelial cells upon treatment with MBELNs in addition to DSS. Data are mean ± SEM from three biological replicates per group. **P<0.01, NS- non-significant using one-way ANOVA.


( A ) Western blots showing expression of AhR in MC38 cells induced by different fractions ( protein , lipid and RNA ) derived from Mulberry bark derived exosome - like nanoparticles ( MBELNs ) . Data shown are from three biological replicates .

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(A) Western blots showing expression of AhR in MC38 cells induced by different fractions (protein, lipid and RNA) derived from Mulberry bark derived exosome-like nanoparticles (MBELNs). Data shown are from three biological replicates.


( B ) The effect of different size protein fractions of mulberry bark derived exosome - like nanoparticles ( MBELNs ) on the expression of aryl hydrocarbon receptor ( AhR ) demonstrated in Western blots . Data shown are from three biological replicates .

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

(B) The effect of different size protein fractions of mulberry bark derived exosome-like nanoparticles (MBELNs) on the expression of aryl hydrocarbon receptor (AhR) demonstrated in Western blots. Data shown are from three biological replicates.


( F ) SPR experiment showing recombinant HSPA8 protein interaction with recombinant hAhR on a SPR chip . Data shown are from three biological replicates .

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(F) SPR experiment showing recombinant HSPA8 protein interaction with recombinant hAhR on a SPR chip. Data shown are from three biological replicates.


( d ) NIH3T3 cells stably expressing PS - CFP2 proteins were exposed to a 405 nm light for the indicated times and photoconverting was monitored by imaging cells in the green and blue channels .

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(d) NIH3T3 cells stably expressing PS-CFP2 proteins were exposed to a 405 nm light for the indicated times and photoconverting was monitored by imaging cells in the green and blue channels.


( e , f ) Mouse fibroblasts ( NIH3T3 ) stably expressing PS - CFP2 or KFERQ - PS - CFP2 were photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . ( e ) Representative images in both channels . ( f ) Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence ( white bars ) or absence ( gray bars ) of serum . Values are mean + s . e . of four different experiments with > 50 cells counted per experiment . ( * ) Differences with PS - CFP2 in the green channel ( P = 0 . 005 , ANOVA - Bonferroni ) . ( § ) Differences with samples in the presence of serum ( P = 0 . 03 , ANOVA - Bonferroni ) . ( g ) Green fluorescence intensity at excitation 450 nm and emission 510 - 550 nm . Fluorescence intensity in each sample was normalized by the number of cells present in the sample and the percentage of intensity at time 0 still remaining after 16 h of incubation in the presence ( white bars ) or absence ( gray bars ) of serum was calculated for each condition . Values are expressed relative to the decay observed in cells maintained in the presence of serum and are means + s . e . of 3 independent experiments . Differences with serum ( * ) were significant for P = 0 . 04 ( t - test ) . Scale bars , 5 μm .

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(e, f) Mouse fibroblasts (NIH3T3) stably expressing PS-CFP2 or KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. (e) Representative images in both channels. (f) Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (white bars) or absence (gray bars) of serum. Values are mean+s.e. of four different experiments with >50 cells counted per experiment. (*) Differences with PS-CFP2 in the green channel (P=0.005, ANOVA-Bonferroni). (§) Differences with samples in the presence of serum (P=0.03, ANOVA-Bonferroni). (g) Green fluorescence intensity at excitation 450 nm and emission 510-550 nm. Fluorescence intensity in each sample was normalized by the number of cells present in the sample and the percentage of intensity at time 0 still remaining after 16 h of incubation in the presence (white bars) or absence (gray bars) of serum was calculated for each condition. Values are expressed relative to the decay observed in cells maintained in the presence of serum and are means+s.e. of 3 independent experiments. Differences with serum (*) were significant for P=0.04 (t-test). Scale bars, 5 μm.


( a - c ) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ - PS - CFP2 maintained in the absence ( black circles ) or presence ( white squares ) of serum for the indicated times . ( a ) Representative images of cells maintained in serum - free media . Quantification of the number of puncta per cell ( b ) and green fluorescence intensity ( c ) . Values are expressed as percentage of fluorescence at time 0 and half - lives ( t1 / 2 ) are indicated . Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum ( * ) were significant for P = 0 . 048 ( 16 h ) and P = 0 . 04 ( 24 h ) ( t - test ) .

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(a-c) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ-PS-CFP2 maintained in the absence (black circles) or presence (white squares) of serum for the indicated times. (a) Representative images of cells maintained in serum-free media. Quantification of the number of puncta per cell (b) and green fluorescence intensity (c). Values are expressed as percentage of fluorescence at time 0 and half-lives (t1/2) are indicated. Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum (*) were significant for P=0.048 (16 h) and P=0.04 (24 h) (t-test).


( d ) The same cells imaged in the cyan channel , before and at the indicated times after photoconversion ( arrow ) by exposure to 405 nm light . Top : Representative images of the cyan fluorescence . Bottom : Quantification of the intensity of the cyan fluorescence . Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1 . Values are mean + s . e . of 3 different experiments with > 10 cells counted per experiment . Scale bars , 5 μm .

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(d) The same cells imaged in the cyan channel, before and at the indicated times after photoconversion (arrow) by exposure to 405 nm light. Top: Representative images of the cyan fluorescence. Bottom: Quantification of the intensity of the cyan fluorescence. Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1. Values are mean+s.e. of 3 different experiments with >10 cells counted per experiment. Scale bars, 5 μm.


( a ) Localization of photoconverted KFERQ - PS - CFP2 ( green ) in LysoTracker - stained compartments ( red ) in mouse fibroblasts maintained in the presence or absence of serum .

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(a) Localization of photoconverted KFERQ-PS-CFP2 (green) in LysoTracker-stained compartments (red) in mouse fibroblasts maintained in the presence or absence of serum.


( b ) Percentage of co - localization of KFERQ - PS - CFP2 with the indicated proteins . Values are mean + s . e . of three different experiments with > 50 cells counted per experiment .

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(b) Percentage of co-localization of KFERQ-PS-CFP2 with the indicated proteins. Values are mean+s.e. of three different experiments with >50 cells counted per experiment.


( c ) Co - localization of photoconverted KFERQ - PS - CFP2 ( green ) with LAMP - 1 , LAMP - 2 or LAMP - 2A .

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(c) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LAMP-1, LAMP-2 or LAMP-2A.


( d ) Immunofluorescence with antibodies that recognize PS - CFP2 ( green ) and hsc70 ( red ) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction .

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(d) Immunofluorescence with antibodies that recognize PS-CFP2 (green) and hsc70 (red) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction.


( e , f ) Co - localization of photoconverted KFERQ - PS - CFP2 ( green ) with LC3 , CD - M6PR , Rab5 ( e ) and ubiquitin ( f ) in mouse fibroblasts maintained in serum - free media . Scale bars , 5 μm .

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(e, f) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LC3, CD-M6PR, Rab5 (e) and ubiquitin (f) in mouse fibroblasts maintained in serum-free media. Scale bars, 5 μm.


( a ) Mouse fibroblasts knockeddown for LAMP - 2A and transfected with KFERQ - PS - CFP2 were photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Representative images of cells also immunostained for LAMP - 1 are shown .

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(a) Mouse fibroblasts knockeddown for LAMP-2A and transfected with KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Representative images of cells also immunostained for LAMP-1 are shown.


( b , c ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were photoconverted and maintained for 16 h in media supplemented ( + ) or not ( − ) with serum in the presence of 3 - methyladenine ( 3 - MA ) to inhibit macroautophagy . ( b ) Representative images of the 3 - MA - treated cells . ( c ) Quantification of the number of green fluorescence puncta per cell in 3 - MA treated or not cells maintained in the presence ( white bars ) or absence ( black bars ) of serum . Values are mean + s . e . of 3 different experiments with > 50 cells counted per experiment . No significant differences were detectable between samples supplemented or not with 3MA . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(b, c) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were photoconverted and maintained for 16 h in media supplemented (+) or not (−) with serum in the presence of 3-methyladenine (3-MA) to inhibit macroautophagy. (b) Representative images of the 3-MA-treated cells. (c) Quantification of the number of green fluorescence puncta per cell in 3-MA treated or not cells maintained in the presence (white bars) or absence (black bars) of serum. Values are mean+s.e. of 3 different experiments with >50 cells counted per experiment. No significant differences were detectable between samples supplemented or not with 3MA. Scale bars, 5 μm.


( a ) Immunoblot with the antibody that recognizes PS - CFP2 of homogenates ( Hom ) , cytosol ( Cyt ) and lysosomal membranes ( MB ) and matrices ( Mtx ) isolated from mouse fibroblasts stably expressing PS - CFP2 or KFERQ - PS - CFP2 proteins 16 h after serum removal .

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(a) Immunoblot with the antibody that recognizes PS-CFP2 of homogenates (Hom), cytosol (Cyt) and lysosomal membranes (MB) and matrices (Mtx) isolated from mouse fibroblasts stably expressing PS-CFP2 or KFERQ-PS-CFP2 proteins 16 h after serum removal.


( b ) Immunoblot for the indicated proteins of homogenates ( Hom ) , mitochondria ( Mit ) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ - PS - CFP2 maintained for 16 h in media supplemented ( + ) or not ( − ) with serum . ( c ) Quantification of the amount of KFERQ - PS - CFP2 associated to lysosomes ( matrix and membrane ) performed in blots as the one shown in ( b ) . Values are expressed as percentage of the total KFERQ - PS - CFP2 in the cell and are mean + s . e . of three different experiments . Difference in the lysosomal associated KFERQ - PS - CFP2 between serum + and serum − condition ( * ) were significant for P = 0 . 033 ( t - test ) .

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(b) Immunoblot for the indicated proteins of homogenates (Hom), mitochondria (Mit) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ-PS-CFP2 maintained for 16 h in media supplemented (+) or not (−) with serum. (c) Quantification of the amount of KFERQ-PS-CFP2 associated to lysosomes (matrix and membrane) performed in blots as the one shown in (b). Values are expressed as percentage of the total KFERQ-PS-CFP2 in the cell and are mean+s.e. of three different experiments. Difference in the lysosomal associated KFERQ-PS-CFP2 between serum+ and serum− condition (*) were significant for P=0.033 (t-test).


( d ) Changes in the intracellular content of KFERQ - PS - CFP2 on serum removal . Values are expressed as percentage of KFERQ - PS - CFP2 present in serum - supplemented cells and area mean + s . e . of three different experiments . Values are mean + s . e . of three different experiments and differences are significant ( * ) for P = 0 . 007 ( t - test ) .

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(d) Changes in the intracellular content of KFERQ-PS-CFP2 on serum removal. Values are expressed as percentage of KFERQ-PS-CFP2 present in serum-supplemented cells and area mean+s.e. of three different experiments. Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.007 (t-test).


( e - g ) Effect of inhibition of macroautophagy by treatment with 3 - methyladenine ( 3 - MA ) on the lysosomal content of KFERQ - PS - CFP2 in cells maintained in serum - free media for 16 h . ( e ) Immunoblot for the indicated proteins of the same fractions as described in ( b ) . The amount of KFERQ - PS - CFP2 associated to lysosomes ( f ) and the changes in the intracellular content of KFERQ - PS - CFP2 were calculated as in ( c , d ) . Values are mean + s . e . of three different experiments and differences are significant ( * ) for P = 0 . 032 ( t - test ) .

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

(e-g) Effect of inhibition of macroautophagy by treatment with 3-methyladenine (3-MA) on the lysosomal content of KFERQ-PS-CFP2 in cells maintained in serum-free media for 16 h. (e) Immunoblot for the indicated proteins of the same fractions as described in (b). The amount of KFERQ-PS-CFP2 associated to lysosomes (f) and the changes in the intracellular content of KFERQ-PS-CFP2 were calculated as in (c, d). Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.032 (t-test).


( a , b ) Human neuroblastoma ( SH - SY5Y ) , hepatoma ( HuH - 7 ) and breast adenocarcinoma ( MCF - 7 ) cell lines were transiently transfected with KFERQ - PS - CFP2 , photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Images show representative cells after immunostaining for LAMP - 2 ( red ) . ( b ) Quantification of the number of green fluorescent puncta per cell under basal conditions ( black bars ) and after serum removal ( gray bars ) . Values of NIH3T3 are included as reference . Values are mean + s . e . of three - to - five different experiments with > 50 cells counted per experiment . Differences with ( * ) NIH3T3 or ( § ) with cell supplemented with serum are significant for P0 . 05 ( range 0 . 002 - 0 . 03 , ANOVA - Bonferroni ) .

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(a, b) Human neuroblastoma (SH-SY5Y), hepatoma (HuH-7) and breast adenocarcinoma (MCF-7) cell lines were transiently transfected with KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Images show representative cells after immunostaining for LAMP-2 (red). (b) Quantification of the number of green fluorescent puncta per cell under basal conditions (black bars) and after serum removal (gray bars). Values of NIH3T3 are included as reference. Values are mean+s.e. of three-to-five different experiments with >50 cells counted per experiment. Differences with (*) NIH3T3 or (§) with cell supplemented with serum are significant for P0.05 (range 0.002-0.03, ANOVA-Bonferroni).


( c ) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ - PS - CFP2 , photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Left : representative image in the green channel . Right : Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence ( black bars ) or absence ( gray bars ) of serum . Values are mean + s . e . of three different experiments with > 50 cells counted per experiment . ( P = 0 . 002 , ANOVA - Bonferroni ) .

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(c) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: representative image in the green channel. Right: Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (black bars) or absence (gray bars) of serum. Values are mean+s.e. of three different experiments with >50 cells counted per experiment. (P=0.002, ANOVA-Bonferroni).


( d ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were plated in 384 - well plates , maintained in the presence ( white bar ) or absence ( black bars ) of serum and subjected to treatment with 3 - methyladenine ( 3 - MA ) , the proteasome inhibitor MG - 132 or H2O2 . In both ( d , e ) , after fixation , images were subjected to high content image analysis and the percent of responders was calculated in > 200 cells per condition . Values are mean + s . e . of results in three different experiments . * P0 . 001 ( range 0 . 0002 - 0 . 0007 , t - test ) . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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

(d) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence (white bar) or absence (black bars) of serum and subjected to treatment with 3-methyladenine (3-MA), the proteasome inhibitor MG-132 or H2O2. In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.


( e ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were plated in 384 - well plates , maintained in the presence of serum supplemented media and leave untreated ( white bar ) or subjected to the indicated treatments ( black bars ) . In both ( d , e ) , after fixation , images were subjected to high content image analysis and the percent of responders was calculated in > 200 cells per condition . Values are mean + s . e . of results in three different experiments . * P0 . 001 ( range 0 . 0002 - 0 . 0007 , t - test ) . Scale bars , 5 μm .

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(e) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence of serum supplemented media and leave untreated (white bar) or subjected to the indicated treatments (black bars). In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.


( b ) Mouse fibroblasts ( NIH3T3 ) stably expressing KFERQ - PA - mCherry1 were photoactivated and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Left : Representative images . Nuclei are staining with DAPI . Right : Quantification of the number of red fluorescent puncta per cell . Values are mean + s . e . of three different experiments with > 20 cells counted per experiment . * P = 0 . 001 , ANOVA - Bonferroni .

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(b) Mouse fibroblasts (NIH3T3) stably expressing KFERQ-PA-mCherry1 were photoactivated and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: Representative images. Nuclei are staining with DAPI. Right: Quantification of the number of red fluorescent puncta per cell. Values are mean+s.e. of three different experiments with >20 cells counted per experiment. *P=0.001, ANOVA-Bonferroni.


( c ) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ - PA - mCherry1 at the indicated times after photoactivation using FACS analysis . Cells were maintained in media supplemented ( white squares ) or not ( black circles ) with serum , and fluorescence by the MFI was calculated on events gated on living cells . Values are expressed as percentage of the intensity at time 0 and are mean + s . e . of 3 - 4 individual experiments .

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(c) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ-PA-mCherry1 at the indicated times after photoactivation using FACS analysis. Cells were maintained in media supplemented (white squares) or not (black circles) with serum, and fluorescence by the MFI was calculated on events gated on living cells. Values are expressed as percentage of the intensity at time 0 and are mean+s.e. of 3-4 individual experiments.


( d ) Representative FACS experiment showing the effect of serum removal at 6 h ( top ) and 42 h ( bottom ) . PS , photoswitching . Scale bars , 5 μm .

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(d) Representative FACS experiment showing the effect of serum removal at 6 h (top) and 42 h (bottom). PS, photoswitching. Scale bars, 5 μm.


( a ) PPARγ mRNA expression in sperm from control and MSUS mice . Control n = 15 , MSUS n = 13 , two - tailed Student ' s t - test , P = 0 . 029 , t = 2 . 30 , df = 26 .

(a) PPARγ mRNA expression in sperm from control and MSUS mice. Control n = 15, MSUS n = 13, two-tailed Student's t-test, P = 0.029, t = 2.30, df = 26.


( c ) Relative luciferase luminescence in transfected GC - 1 cells exposed to serum from control or MSUS mice . Numbers correspond to serum from individual animals applied to different cell culture wells . Control n = 17 , MSUS n = 22 , two - tailed Student ' s t - test , P = 0 . 042 , t = 2 . 1 , df = 37 .

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(c) Relative luciferase luminescence in transfected GC-1 cells exposed to serum from control or MSUS mice. Numbers correspond to serum from individual animals applied to different cell culture wells. Control n = 17, MSUS n = 22, two-tailed Student's t-test, P = 0.042, t = 2.1, df = 37.


( b ) Adult weight in the offspring of tesaglitazar - injected males ( Tesa - inj , n = 16 ) compared to the offspring of vehicle - injected males ( Vehicle - inj , n = 23 ) . Two - tailed Student ' s t - test , P = 0 . 019 , t = 2 . 44 , df = 37 .

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(b) Adult weight in the offspring of tesaglitazar-injected males (Tesa-inj, n = 16) compared to the offspring of vehicle-injected males (Vehicle-inj, n = 23). Two-tailed Student's t-test, P = 0.019, t = 2.44, df = 37.


( c ) Glucose level in the offspring of Tesa - inj ( n = 14 ) and Vehicle - inj ( n = 21 ) males during a glucose tolerance test . Repeated measures ANOVA , treatment effect P = 0 . 0083 , F ( 1 , 33 ) = 7 . 877 , time effect P < 0 . 0001 , F ( 4 , 132 ) = 347 . 7 , interaction P = 0 . 0776 , F ( 4 , 132 ) = 2 . 155 Conc . ; concentration .

O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Glucose level in the offspring of Tesa-inj (n = 14) and Vehicle-inj (n = 21) males during a glucose tolerance test. Repeated measures ANOVA, treatment effect P = 0.0083, F (1, 33) = 7.877, time effect P < 0.0001, F (4, 132) = 347.7, interaction P = 0.0776, F (4, 132) = 2.155 Conc.; concentration.


( a Differentially expressed transposable elements in sperm from Tesa - inj and MSUS males . Data represent genes with P < 0 . 05 and similar fold change . Fold change in heat map represents log2 ( fold change ) respective to the corresponding control group . Total overlap is presented in Extended Data Fig . 11 .

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(a Differentially expressed transposable elements in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.


b ) mRNAs / lincRNAs in sperm from Tesa - inj and MSUS males . Data represent genes with P < 0 . 05 and similar fold change . Fold change in heat map represents log2 ( fold change ) respective to the corresponding control group . Total overlap is presented in Extended Data Fig . 11 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mRNAs/lincRNAs in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.


( c , d ) ( c ) Volcano plot and ( d ) heat map of differentially expressed ( FDR < 0 . 05 ) mRNA / lincRNA in sperm from Tesa - inj males . Dashed black lines in ( c ) represent y = 1 . 3 , equivalent to FDR = 0 . 05 , and x = ± 0 . 03 , equivalent to FC = 1 . 23 and 0 . 81 . Black dots represent top candidates , which all have FC > 2 or FC < 0 . 5 , and FDR < 0 . 05 . Teal dots represent non - significant genes .

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(c,d) (c) Volcano plot and (d) heat map of differentially expressed (FDR < 0.05) mRNA/lincRNA in sperm from Tesa-inj males. Dashed black lines in (c) represent y = 1.3, equivalent to FDR = 0.05, and x = ±0.03, equivalent to FC = 1.23 and 0.81. Black dots represent top candidates, which all have FC > 2 or FC < 0.5, and FDR < 0.05. Teal dots represent non-significant genes.


( b ) Weight in adult male MSUS offspring and the offspring of males injected with MSUS serum compared to respective control groups . MSUS offspring n = 22 , Control offspring n = 24 , one - tailed Student ' s t - test ( data reproduced ) P = 0 . 045 , t = 1 . 734 , df = 44 . MSUS serum - injected offspring n = 30 , Control serum - injected offspring n = 31 , two - tailed Mann - Whitney U = 334 . 5 , P = 0 . 059 .

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(b) Weight in adult male MSUS offspring and the offspring of males injected with MSUS serum compared to respective control groups. MSUS offspring n = 22, Control offspring n = 24, one-tailed Student's t-test (data reproduced) P = 0.045, t = 1.734, df = 44. MSUS serum-injected offspring n = 30, Control serum-injected offspring n = 31, two-tailed Mann-Whitney U = 334.5, P = 0.059.


( c , d ) Blood glucose levels in MSUS offspring and the offspring of MSUS serum - injected males following a 30 - min restraint challenge . MSUS offspring n = 13 , Control offspring n = 12 , repeat measures ANOVA , treatment effect P = 0 . 016 , F ( 1 , 23 ) = 6 . 704 , time effect P < 0 . 0001 , F ( 3 , 69 ) = 53 . 13 , interaction P = 0 . 027 , F ( 3 , 69 ) = 3 . 25 , at 15 - min adjusted P = 0 . 0015 , t = 3 . 693 , df = 92 . MSUS serum - injected offspring n = 17 , and Control serum - injected offspring n = 14 , repeat measures ANOVA , treatment effect P = 0 . 023 , F ( 1 , 14 ) = 6 . 493 , time effect P < 0 . 0001 , F ( 3 , 42 ) = 48 . 4 , interaction P = 0 . 29 , F ( 3 , 42 ) = 1 . 29 . Conc . , concentration .

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

(c,d) Blood glucose levels in MSUS offspring and the offspring of MSUS serum-injected males following a 30-min restraint challenge. MSUS offspring n = 13, Control offspring n = 12, repeat measures ANOVA, treatment effect P = 0.016, F (1, 23) = 6.704, time effect P < 0.0001, F (3, 69) = 53.13, interaction P = 0.027, F (3, 69) = 3.25, at 15-min adjusted P = 0.0015, t = 3.693, df = 92. MSUS serum-injected offspring n = 17, and Control serum-injected offspring n = 14, repeat measures ANOVA, treatment effect P = 0.023, F (1,14) = 6.493, time effect P < 0.0001, F (3, 42) = 48.4, interaction P = 0.29, F (3, 42) = 1.29. Conc., concentration.


I Protein complexes involving aggregators . Heatmaps show the absolute difference in solubility change after heat shock between each complex member . Protein complexes with at least 75 % of its members quantified and containing at least 60 % of its members as aggregators are shown . ' DNA repair complex ' = ' DNA repair complex NEIL2 - PNK - Pol ( beta ) - LigIII ( alpha ) - XRCC1 ' .

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I Protein complexes involving aggregators. Heatmaps show the absolute difference in solubility change after heat shock between each complex member. Protein complexes with at least 75% of its members quantified and containing at least 60% of its members as aggregators are shown. 'DNA repair complex' = 'DNA repair complex NEIL2-PNK-Pol(beta)-LigIII(alpha)-XRCC1'.


A Line graphs showing solubility after heat shock ( HS ) and during different time points of recovery . Each line corresponds to one protein . Orange lines show the mean solubility .

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A Line graphs showing solubility after heat shock (HS) and during different time points of recovery. Each line corresponds to one protein. Orange lines show the mean solubility.


C Different disaggregation profiles for aggregating proteins . Solubility line graphs as in A shown for aggregators with the bottom 10 % , middle 80 % and top 10 % of disaggregation rates . Proteins with bottom 10 % of disaggregation rates and related to DNA repair are highlighted with red . Proteins with top10 % disaggregation rates and directly related to transcription are highlighted with blue .

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C Different disaggregation profiles for aggregating proteins. Solubility line graphs as in A shown for aggregators with the bottom 10%, middle 80% and top 10% of disaggregation rates. Proteins with bottom 10% of disaggregation rates and related to DNA repair are highlighted with red. Proteins with top10% disaggregation rates and directly related to transcription are highlighted with blue.


D Protein localization upon heat shock and during recovery as analyzed by immunofluorescence microscopy . HeLa cells were fixed and immunostained with antibodies against indicated target proteins ( green ) at different conditions [ control , after heat shock ( 10 minutes at 44 ° C ) and after five hours of recovery from the heat shock ] . DNA staining ( Hoechst ) is shown in blue .

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D Protein localization upon heat shock and during recovery as analyzed by immunofluorescence microscopy. HeLa cells were fixed and immunostained with antibodies against indicated target proteins (green) at different conditions [control, after heat shock (10 minutes at 44°C) and after five hours of recovery from the heat shock]. DNA staining (Hoechst) is shown in blue.


E - H Comparison of disaggregation rates with different protein characteristics enriched in aggregators ( presented in Fig 2B - C and Fig 2E - G ) . Volcano plot presenting correlation coefficients and Benjamini - Hochberg adjusted p - values ( E ; horizontal dashed line shows a p - value of 0 . 05 ) . Scatterplots comparing disaggregation slopes and solubility change after heat shock ( F ) , fraction of intrinsically disordered regions ( G ) and fraction of ( random ) coil - like secondary structure ( H ) . In F - H , scatter plots are shown for correlations with a p - value lower than 0 . 05 . Correlation coefficients ( r ) with p - values are shown for Spearman ' s rank - order correlation .

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E-H Comparison of disaggregation rates with different protein characteristics enriched in aggregators (presented in Fig 2B-C and Fig 2E-G). Volcano plot presenting correlation coefficients and Benjamini-Hochberg adjusted p-values (E; horizontal dashed line shows a p-value of 0.05). Scatterplots comparing disaggregation slopes and solubility change after heat shock (F), fraction of intrinsically disordered regions (G) and fraction of (random) coil-like secondary structure (H). In F-H, scatter plots are shown for correlations with a p-value lower than 0.05. Correlation coefficients (r) with p-values are shown for Spearman's rank-order correlation.


A - B Accumulation of newly synthesized proteins after mock shock ( A ) and heat shock ( B ) . Line graphs showing the accumulation of signal intensity ( log2 - transformed ratios to pre - shock control ) in the soluble fraction . Orange lines show the mean ratio . C Amount of newly synthesized proteins after heat shock compared to mock shock . Line graphs show the heat shock to mock shock ratio during recovery . Orange line show the mean ratio . Data information : All proteomics data shown in A - D and F is quantified with at least two unique peptides from at least two biological replicates . Proteomics data shown for newly synthesized proteins ( heavy ) . HS = heat shock . MS = mock shock .

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A-B Accumulation of newly synthesized proteins after mock shock (A) and heat shock (B). Line graphs showing the accumulation of signal intensity (log2-transformed ratios to pre-shock control) in the soluble fraction. Orange lines show the mean ratio. C Amount of newly synthesized proteins after heat shock compared to mock shock. Line graphs show the heat shock to mock shock ratio during recovery. Orange line show the mean ratio. Data information: All proteomics data shown in A-D and F is quantified with at least two unique peptides from at least two biological replicates. Proteomics data shown for newly synthesized proteins (heavy). HS = heat shock. MS = mock shock.


A - B Accumulation of newly synthesized proteins after mock shock ( A ) and heat shock ( B ) . Line graphs showing the accumulation of signal intensity ( log2 - transformed ratios to pre - shock control ) in the soluble fraction . Orange lines show the mean ratio .

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A-B Accumulation of newly synthesized proteins after mock shock (A) and heat shock (B). Line graphs showing the accumulation of signal intensity (log2-transformed ratios to pre-shock control) in the soluble fraction. Orange lines show the mean ratio.


F Comparison of heat shock - induced protein synthesis and mRNA synthesis after heat shock ( Vihervaara et al , 2017 ) . Ten most upregulated proteins in the proteomics analysis are labelled and highlighted in blue ; heat shock proteins are labelled red . HSPA1A and HSPA1B could not be distinguished from one another in the proteomics analysis and are plotted as they would have the same intensity ( indicated with a vertical dashed line ) .

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F Comparison of heat shock-induced protein synthesis and mRNA synthesis after heat shock (Vihervaara et al, 2017). Ten most upregulated proteins in the proteomics analysis are labelled and highlighted in blue; heat shock proteins are labelled red. HSPA1A and HSPA1B could not be distinguished from one another in the proteomics analysis and are plotted as they would have the same intensity (indicated with a vertical dashed line).


B : Volcano plot for the score for thermal stability . Significant ( see ' Materials and Methods ' ) heat shock - induced stabilization and destabilization are indicated with blue and red , respectively . Proteins that aggregate in heat shock ( as defined in Figure 2A ) are shown in black - aggregators with the strongest stabilization are annotated . Members of stress - activated MAPK pathway highlighted in green - alternative names for MAP - kinases are shown in parenthesis . Boxplot on top compares stability scores of aggregators ( as defined in Figure 2A and other proteins ) . The p - value is for non - parametric Wilcoxon test .

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B: Volcano plot for the score for thermal stability. Significant (see 'Materials and Methods') heat shock-induced stabilization and destabilization are indicated with blue and red, respectively. Proteins that aggregate in heat shock (as defined in Figure 2A) are shown in black - aggregators with the strongest stabilization are annotated. Members of stress-activated MAPK pathway highlighted in green - alternative names for MAP-kinases are shown in parenthesis. Boxplot on top compares stability scores of aggregators (as defined in Figure 2A and other proteins). The p-value is for non-parametric Wilcoxon test.


D Thermal stability changes in protein complex members . Scores for thermal stability shown for proteins from 26S proteasome ( separately for 19S regulatory and 20S core particles ) and CCT chaperonin complex . Boxplots indicate median , first and third quartiles with whiskers extended to 1 . 5 times the interquartile range out from each quartile . Data used in stability score calculation is from two ( mock shock ) and three ( heat shock ) biological replicates .

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D Thermal stability changes in protein complex members. Scores for thermal stability shown for proteins from 26S proteasome (separately for 19S regulatory and 20S core particles) and CCT chaperonin complex. Boxplots indicate median, first and third quartiles with whiskers extended to 1.5 times the interquartile range out from each quartile. Data used in stability score calculation is from two (mock shock) and three (heat shock) biological replicates.


( b ) Total RNA sequencing of cenRNAs in yeasts . Left ; read counts per million tracks for CEN16 in wildtype ( black , top ) and DAD1E50D humanized ( red , bottom ) yeasts . Right ; heatmap of cenRNAs transcripts per million counts across all chromosomes in wildtype and DAD1E50D humanized strains . Centromere VI is marked with an asterisk to indicate that the plasmid encoding the histone plasmid also encodes a CEN6 sequence , which reads likely

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(b) Total RNA sequencing of cenRNAs in yeasts. Left; read counts per million tracks for CEN16 in wildtype (black, top) and DAD1E50D humanized (red, bottom) yeasts. Right; heatmap of cenRNAs transcripts per million counts across all chromosomes in wildtype and DAD1E50D humanized strains. Centromere VI is marked with an asterisk to indicate that the plasmid encoding the histone plasmid also encodes a CEN6 sequence, which reads likely emanant


( c ) RT - PCRs of the indicated RNAs . Lane 1 is genomic DNA , lane 2 is wildtype shuffle strain ( yDT67 ) , lane 3 is BY4742 , lane 4 is DAD1E50D humanized , lane 5 is scc4D65Y humanized , lanes 6 - 9 are the same as 2 - 5 , but without reverse transcriptase added , lane 10 is no DNA .

(c) RT-PCRs of the indicated RNAs. Lane 1 is genomic DNA, lane 2 is wildtype shuffle strain (yDT67), lane 3 is BY4742, lane 4 is DAD1E50D humanized, lane 5 is scc4D65Y humanized, lanes 6-9 are the same as 2-5, but without reverse transcriptase added, lane 10 is no DNA.


( d ) Example images from strains with fluorescent tags on the microtubule organizing center ( Spc110 - GFP ) and the kinetochore ( Nuf2 - RFP ) . Images were taken from cultures in mid - log phase of growth .

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(d) Example images from strains with fluorescent tags on the microtubule organizing center (Spc110-GFP) and the kinetochore (Nuf2-RFP). Images were taken from cultures in mid-log phase of growth.


( e ) DASH mutants increase histone - humanization in the scc4D65Y background . Humanization rates of the scc4D65Y mutant with and without DAD1E50D or DAM1N80Y . Significance of the mean difference in 5 - FOAR frequency was determined with the Mann - Whitney test . Each dot represents one biological replicate ( n = 8 ) , central band represents the median , box extends from the 25th to 75th percentiles , and whiskers represent the minimum and maximum .

(e) DASH mutants increase histone-humanization in the scc4D65Y background. Humanization rates of the scc4D65Y mutant with and without DAD1E50D or DAM1N80Y. Significance of the mean difference in 5-FOAR frequency was determined with the Mann-Whitney test. Each dot represents one biological replicate (n = 8), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.


( f ) Ploidy analysis by flow cytometry in the scc4D65Y humanized mutants from panel d . Flow cytometry data from humanized lineages yHs5 and yHs7 are shown for aneuploid and euploid controls , respectively .

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(f) Ploidy analysis by flow cytometry in the scc4D65Y humanized mutants from panel d. Flow cytometry data from humanized lineages yHs5 and yHs7 are shown for aneuploid and euploid controls, respectively.


( a ) Growth assays of wild type , ipl1 - 2 mutants , and ipl1 - 2 kinetochore double mutants at permissive ( 24ºC ) and non - permissive temperature ( 32ºC ) . Ten - fold serial dilutions of ~ 1 . 0 OD600 yeast cultures were spotted onto YPD solid media and grown at the indicated temperatures for four and three days , respectively .

(a) Growth assays of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at permissive (24ºC) and non-permissive temperature (32ºC). Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto YPD solid media and grown at the indicated temperatures for four and three days, respectively.


( b ) Flow cytometry analysis of wild type , ipl1 - 2 mutants , and ipl1 - 2 kinetochore double mutants at the permissive ( 24 ˚ C ) and non - permissive temperature ( 30ºC ) . Yeast cultures were grown to logarithmic phase at 24 ˚ C and divided and grown at 24 ˚ C and 30 ˚ C for 6 hours before cells were collected and processed for DNA content analysis using Sytox Green stain .

(b) Flow cytometry analysis of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at the permissive (24˚C) and non-permissive temperature (30ºC). Yeast cultures were grown to logarithmic phase at 24˚C and divided and grown at 24˚C and 30˚C for 6 hours before cells were collected and processed for DNA content analysis using Sytox Green stain.


( c ) Growth assays of wild type , mad3 ∆ mutants , and mad3 ∆ DAD1E50D double mutants on YPD with and without 30 µg / ml benomyl . Ten - fold serial dilutions of ~ 1 . 0 OD600 yeast cultures were spotted onto the indicated solid media and grown at 30 ˚ C for three days .

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(c) Growth assays of wild type, mad3∆ mutants, and mad3∆ DAD1E50D double mutants on YPD with and without 30 µg/ml benomyl. Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto the indicated solid media and grown at 30˚C for three days.


( b ) Histone - humanization assay for various dam1N80X mutants . 5 - FOA is used to counter - select the yeast histone plasmid , forcing growth with the human histone plasmid . Yeast were serially diluted from a starting culture of 1 . 0 OD600 .

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(b) Histone-humanization assay for various dam1N80X mutants. 5-FOA is used to counter-select the yeast histone plasmid, forcing growth with the human histone plasmid. Yeast were serially diluted from a starting culture of 1.0 OD600.


( f ) Rupture force assay from experiments at three concentrations of DASH complex incubated with the beads . Briefly , 3 . 5 pM of beads were incubated with the indicated concentrations of DASH complex ( representing a ratio of DASH complex to beads of 714 : 1 , 1428 : 1 , and 2860 : 1 ) . These DASH - decorated beads were then washed before suspending in a reaction mix containing free tubulin and 5 nM free DASH complex . Two way ANOVA tests of the mean difference in rupture force are shown with multiple test corrections ( false discovery rate ) . Each dot represents an individual rupture force measurement ( n ≥ 21 ; exact number shown below each box between parentheses ) , central band represents the median , box extends from the 25th to 75th percentiles , and whiskers represent the minimum and maximum .

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(f) Rupture force assay from experiments at three concentrations of DASH complex incubated with the beads. Briefly, 3.5 pM of beads were incubated with the indicated concentrations of DASH complex (representing a ratio of DASH complex to beads of 714:1, 1428:1, and 2860:1). These DASH-decorated beads were then washed before suspending in a reaction mix containing free tubulin and 5 nM free DASH complex. Two way ANOVA tests of the mean difference in rupture force are shown with multiple test corrections (false discovery rate). Each dot represents an individual rupture force measurement (n ≥ 21; exact number shown below each box between parentheses), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.


( g ) Survival curves for each DASH complex are shown at the indicated concentrations . Log - rank tests were performed to test whether the survival curves were significantly different from one another . Error bars represent the standard error and are displayed as the shaded area .

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(g) Survival curves for each DASH complex are shown at the indicated concentrations. Log-rank tests were performed to test whether the survival curves were significantly different from one another. Error bars represent the standard error and are displayed as the shaded area.


( a ) Sporulation efficiency after five days in sporulation medium at 25 ˚ C . Example micrographs are shown to the left for both wildtype and DAD1E50D SK1 strains . Right , quantification of average sporulation efficiency from all micrographs . Two tailed unpaired t test of the mean difference in sporulation efficiency . Each dot represents the fraction of cells sporulated in a single field of view ( technical replicates ; n ≥ 15 ) , central band represents the median , box extends from the 25th to 75th percentiles , and whiskers represent the minimum and maximum .

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(a) Sporulation efficiency after five days in sporulation medium at 25 ˚C. Example micrographs are shown to the left for both wildtype and DAD1E50D SK1 strains. Right, quantification of average sporulation efficiency from all micrographs. Two tailed unpaired t test of the mean difference in sporulation efficiency. Each dot represents the fraction of cells sporulated in a single field of view (technical replicates; n ≥15), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.


( b ) Spore viability assay . Individual tetrads were micromanipulated on a agar surface and spores were dissected and arrayed . Genotype was determined by PCR genotyping of the DAD1 locus . Quantification from all tetrad dissections performed , total are shown below . The one - tailed probability of observing 51 % spore viability in DAD1E50D SK1 over a total of 139 tetrads is p < 0 . 000001 , assuming a spore viability of ~ 90 % from wild type SK1 .

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(b) Spore viability assay. Individual tetrads were micromanipulated on a agar surface and spores were dissected and arrayed. Genotype was determined by PCR genotyping of the DAD1 locus. Quantification from all tetrad dissections performed, total are shown below. The one-tailed probability of observing 51% spore viability in DAD1E50D SK1 over a total of 139 tetrads is p < 0.000001, assuming a spore viability of ~90% from wild type SK1.


B , C Relative abundance of sgRNAs in cell populations with high versus low signal were visualized as log2 fold chances for basal as well as activated autophagy conditions for ( B ) p62 or ( C ) NDP52 . Entire data is reported in Dataset EV1

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B, C Relative abundance of sgRNAs in cell populations with high versus low signal were visualized as log2 fold chances for basal as well as activated autophagy conditions for (B) p62 or (C) NDP52. Entire data is reported in Dataset EV1


D - F Validation of TMEM41B . H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non - targeting ( NT ) control , treated with 500 nM AZD8055 , 50nM Bafilomycin A1 ( BafA1 ) or DMSO vehicle control for 24 h , and analyzed by immunoblotting . ( E , F ) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells ( E ) , or as flux by calculating the ratio in BafA1 - treated cells versus vehicle control ( F ) . Data are presented as mean ± SD ( n = 6 independent experiments ) with paired t - test values

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D-F Validation of TMEM41B. H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non-targeting (NT) control, treated with 500 nM AZD8055, 50nM Bafilomycin A1 (BafA1) or DMSO vehicle control for 24 h, and analyzed by immunoblotting. (E, F) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells (E), or as flux by calculating the ratio in BafA1-treated cells versus vehicle control (F). Data are presented as mean ± SD (n=6 independent experiments) with paired t-test values


G LAMP1 and p62 were co - stained and imaged using an LSM700 confocal microscope . Arrowheads point at p62 - filled and empty LAMP1 - positive organelles . Scale bar : 5 µm

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G LAMP1 and p62 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at p62-filled and empty LAMP1-positive organelles. Scale bar: 5 µm


H LAMP1 - positive organelles were manually quantified for containing a p62 - filled or empty lumen . A total of 50 - 60 LAMP1 - positive organelles were analyzed in each experiment . Data are presented as mean ± SD ( n = 3 independent experiments ) with paired t - test values

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H LAMP1-positive organelles were manually quantified for containing a p62-filled or empty lumen. A total of 50-60 LAMP1-positive organelles were analyzed in each experiment. Data are presented as mean ± SD (n=3 independent experiments) with paired t-test values


I HeLa cells were transduced with Cas9 and TMEM41B or NT sgRNAs followed by infection with luciferase - expressing S . typhimurium . Luciferase readings were taken up to 8 . 5 h post - infection . Results from one representative experiment are presented as mean ± SD ( n = 8 technical replicates )

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I HeLa cells were transduced with Cas9 and TMEM41B or NT sgRNAs followed by infection with luciferase-expressing S. typhimurium. Luciferase readings were taken up to 8.5 h post-infection. Results from one representative experiment are presented as mean ± SD (n=8 technical replicates)


A H4 Cas9 cells were infected with sgRNAs targeting ATG7 , TMEM41B or NT control , treated with 500 nM AZD8055 or vehicle control for 24 h , and analyzed by immunoblotting . B Ratio of LC3 - II to LC3 - I band intensities are depicted as mean ± SD ( n = 6 independent experiments ) with Wilcoxon test values

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A H4 Cas9 cells were infected with sgRNAs targeting ATG7, TMEM41B or NT control, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting. B Ratio of LC3-II to LC3-I band intensities are depicted as mean ± SD (n=6 independent experiments) with Wilcoxon test values


C H4 Cas9 TMEM41B and NT control cells were probed by LC3B immunostaining and imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm . D The number of LC3 puncta per cell was quantified using Yokogawa Analysis Software ( YAS ) and depicted as mean ± SD ( n = 3 technical replicates )

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C H4 Cas9 TMEM41B and NT control cells were probed by LC3B immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. D The number of LC3 puncta per cell was quantified using Yokogawa Analysis Software (YAS) and depicted as mean ± SD (n=3 technical replicates)


E LC3 and WIPI2 were co - stained and imaged using an LSM700 confocal microscope . Arrowheads point at co - localized LC3 and WIPI2 puncta . Scale bar : 5 µm

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E LC3 and WIPI2 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at co-localized LC3 and WIPI2 puncta. Scale bar: 5 µm


F The number of WIPI2 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD ( n = 5 technical replicates ) . G The total number of WIPI2 puncta per cell was quantified using the Harmony software on images acquired with an automated Operetta microscope . Data are depicted as mean ± SD ( n = 3 technical replicates )

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F The number of WIPI2 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=5 technical replicates). G The total number of WIPI2 puncta per cell was quantified using the Harmony software on images acquired with an automated Operetta microscope. Data are depicted as mean ± SD (n=3 technical replicates)


H H4 Cas9 TMEM41B and NT control cells were infected with a RFP - DFCP1 expression construct for 72h , fixed , stained with LC3 antibodies and imaged with an automated CV7000 confocal microscope . Arrowheads point at co - localized LC3 and RFP - DFCP1 puncta . Scale bar : 5 µm . I The number of RFP - DFCP1 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD ( n = 10 technical replicates )

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H H4 Cas9 TMEM41B and NT control cells were infected with a RFP-DFCP1 expression construct for 72h, fixed, stained with LC3 antibodies and imaged with an automated CV7000 confocal microscope. Arrowheads point at co-localized LC3 and RFP-DFCP1 puncta. Scale bar: 5 µm. I The number of RFP-DFCP1 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=10 technical replicates)


J mCherry - GFP - LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50nM BafA1 or vehicle control for 24 h , fixed and imaged . Scale bar : 20 μm . The number of mCherry - and GFP - positive puncta per cell was quantified using YAS and are depicted as mean ± SD ( n = 3 technical replicates )

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J mCherry-GFP-LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50nM BafA1 or vehicle control for 24 h, fixed and imaged. Scale bar: 20 μm. The number of mCherry- and GFP-positive puncta per cell was quantified using YAS and are depicted as mean ± SD (n=3 technical replicates)


A H4 Cas9 cells stably expressing NT , TMEM41B or ATG7 sgRNAs were stained with BODIPY 493 , NBD cholesterol or BODIPY FL C12 probes for 2 h at 37 ° C and imaged live with an automated CV7000 confocal microscope . Puncta area per cell was quantified using YAS and depicted as mean ± SD ( n = 4 technical replicates ) . Scale bar : 20 μm

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A H4 Cas9 cells stably expressing NT, TMEM41B or ATG7 sgRNAs were stained with BODIPY 493, NBD cholesterol or BODIPY FL C12 probes for 2 h at 37°C and imaged live with an automated CV7000 confocal microscope. Puncta area per cell was quantified using YAS and depicted as mean ± SD (n=4 technical replicates). Scale bar: 20 μm


B H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA - conjugated oleic acid or 0 . 1 % BSA as vehicle control , stained for 2 h with HCS LipidTox Green Neutral Lipid Stain and imaged with an automated Operetta microscope . Scale bar : 20 μm

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B H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA-conjugated oleic acid or 0.1% BSA as vehicle control, stained for 2 h with HCS LipidTox Green Neutral Lipid Stain and imaged with an automated Operetta microscope. Scale bar: 20 μm


C Lipid droplet size , number and mean fluorescence intensity was quantified using Harmony software . Data are presented as mean ± SD ( n = 3 technical replicates ) . An average of 1500 cells was analyzed per replicate

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C Lipid droplet size, number and mean fluorescence intensity was quantified using Harmony software. Data are presented as mean ± SD (n=3 technical replicates). An average of 1500 cells was analyzed per replicate


D H4 Cas9 TMEM41B KO and NT control cells were probed by ADRP immunostaining and imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm . E Size of ADRP droplets was quantified with YAS and depicted as mean ± SD ( n = 3 technical replicates )

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D H4 Cas9 TMEM41B KO and NT control cells were probed by ADRP immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. E Size of ADRP droplets was quantified with YAS and depicted as mean ± SD (n=3 technical replicates)


F Protein level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO and NT control cells . G ADRP band intensities were quantified and depicted as mean ± SD ( n = 4 independent experiments ) with paired t - test values

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F Protein level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO and NT control cells. G ADRP band intensities were quantified and depicted as mean ± SD (n=4 independent experiments) with paired t-test values


A H4 Cas9 TMEM41B KO and NT control cells were pulsed with the fatty acid analog Red C12 , chased in complete medium or upon serum - deprivation , and stained with MitoTracker . Cells were imaged live with an automated CV7000 confocal microscope . Scale bar : 20 μm

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A H4 Cas9 TMEM41B KO and NT control cells were pulsed with the fatty acid analog Red C12, chased in complete medium or upon serum-deprivation, and stained with MitoTracker. Cells were imaged live with an automated CV7000 confocal microscope. Scale bar: 20 μm


C - E H4 Cas9 TMEM41B KO and NT control cells were plated in XF96 plates and analyzed using a Seahorse Bioscience XF96 . ( C ) Basal oxygen consumption rates ( OCR ) and extra cellular acidification rates ( ECAR ) were measured in cells grown in complete medium . OCR / ECAR ratio is also reported . Data are presented as mean ± SEM ( n = 30 - 32 technical replicates ) from one representative experiment

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C-E H4 Cas9 TMEM41B KO and NT control cells were plated in XF96 plates and analyzed using a Seahorse Bioscience XF96. (C) Basal oxygen consumption rates (OCR) and extra cellular acidification rates (ECAR) were measured in cells grown in complete medium. OCR/ECAR ratio is also reported. Data are presented as mean ± SEM (n=30-32 technical replicates) from one representative experiment


( D ) OCR was measured in cells grown in substrate - limited medium and treated in the absence or presence of Etomoxir ( Eto ) . Oligomycin ( OA ) , FCCP ( FC ) and a mixture of rotenone and antimycin A ( RA ) was added as indicated . Data are shown as mean ± SEM ( n = 69 technical replicates )

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(D) OCR was measured in cells grown in substrate-limited medium and treated in the absence or presence of Etomoxir (Eto). Oligomycin (OA), FCCP (FC) and a mixture of rotenone and antimycin A (RA) was added as indicated. Data are shown as mean ± SEM (n= 69 technical replicates)


( E ) Endogenous FA utilization was calculating by subtracting basal normalized OCR values of Eto - treated cells from untreated cells . Data are shown as mean ± SEM ( n = 69 technical replicates )

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(E) Endogenous FA utilization was calculating by subtracting basal normalized OCR values of Eto-treated cells from untreated cells. Data are shown as mean ± SEM (n= 69 technical replicates)


A H4 Cas9 cells stably expressing N - terminally ( Myc - TMEM41B ) or C - terminally Myc - tagged TMEM41B ( TMEM41B - Myc ) were probed by calnexin ( CANX ) and Myc tag immunostaining and imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm

A H4 Cas9 cells stably expressing N-terminally (Myc-TMEM41B) or C-terminally Myc-tagged TMEM41B (TMEM41B-Myc) were probed by calnexin (CANX) and Myc tag immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm


B - D Endogenous TMEM41B locus was tagged with C - terminal Myc using CRISPR - directed homologous recombination in HeLa cells . HeLa knock - in ( KI ) cells were compared to parental HeLa ( - ) and HeLa cells stably transduced with a TMEM41B - Myc over - expression ( OE ) construct . ( B ) Confocal micrographs of cells stained with antibodies against CANX and Myc confirming ER localization of both KI and OE TMEM41B - Myc . Scale bar represents 20 μm

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B-D Endogenous TMEM41B locus was tagged with C-terminal Myc using CRISPR-directed homologous recombination in HeLa cells. HeLa knock-in (KI) cells were compared to parental HeLa (-) and HeLa cells stably transduced with a TMEM41B-Myc over-expression (OE) construct. (B) Confocal micrographs of cells stained with antibodies against CANX and Myc confirming ER localization of both KI and OE TMEM41B-Myc. Scale bar represents 20 μm


μm . ( C ) Immunoblot analysis confirming expression of both KI and OE TMEM41B -

μm. (C) Immunoblot analysis confirming expression of both KI and OE TMEM41B-Myc


( D ) PCR analysis with primers binding to TMEM41B last intron and to Myc tag confirming successful targeting of the endogenous TMEM41B locus with Myc ( band labeled with asterisk ) . M = size

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(D) PCR analysis with primers binding to TMEM41B last intron and to Myc tag confirming successful targeting of the endogenous TMEM41B locus with Myc (band labeled with asterisk). M=size marker


E H4 Cas9 cells and H4 Cas9 cells stably expressing Myc - TMEM41B or TMEM41B - Myc were lysed and subjected to anti - Myc IP . Eluates were analyzed by mass spectrometry . Enrichment of proteins in IPs from Myc - TMEM41B or TMEM41B - Myc cells versus H4 Cas9 cells is depicted as log2 fold changes . Entire data is reported in Dataset EV2

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E H4 Cas9 cells and H4 Cas9 cells stably expressing Myc-TMEM41B or TMEM41B-Myc were lysed and subjected to anti-Myc IP. Eluates were analyzed by mass spectrometry. Enrichment of proteins in IPs from Myc-TMEM41B or TMEM41B-Myc cells versus H4 Cas9 cells is depicted as log2 fold changes. Entire data is reported in Dataset EV2


( A ) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 and fused in all three possible frames with the GFP deleted of its ATG .

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(A) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 and fused in all three possible frames with the GFP deleted of its ATG.


( B ) LC - MS / MS spectra of the N - terminal part of the GFP - immunoprecipitated and LysC digested polyGA protein expressed as in ( A ) .

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(B) LC-MS/MS spectra of the N-terminal part of the GFP-immunoprecipitated and LysC digested polyGA protein expressed as in (A).


( C , D ) Immunoblotting against the GFP ( C ) or the HA tag ( D ) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3 , 20 or 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame .

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(C, D) Immunoblotting against the GFP (C) or the HA tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3, 20 or 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame.


( E ) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected with either a wild type or mutant ( AGG into CGG ) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GR frame .

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(E) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected with either a wild type or mutant (AGG into CGG) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GR frame.


( A ) Immunoblotting against the HA tag or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 and fused in all three possible frames with a HA tag .

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(A) Immunoblotting against the HA tag or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 and fused in all three possible frames with a HA tag.


( B ) LC - MS / MS spectra of the N - terminal part of the HA - immunoprecipitated and LysC digested polyPG protein expressed as in ( A ) .

(B) LC-MS/MS spectra of the N-terminal part of the HA-immunoprecipitated and LysC digested polyPG protein expressed as in (A).


( C , D ) Immunoblotting against the GFP ( C ) or the HA tag ( D ) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3 , 10 or 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to the GFP in the PG frame .

(C, D) Immunoblotting against the GFP (C) or the HA tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3, 10 or 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to the GFP in the PG frame.


( A ) Cell viability ( TO - PRO - 3 FACS staining ) of GT1 - 7 neuronal cells transfected for 24 hours with either wild type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA , PG or GR frame .

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(A) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells transfected for 24 hours with either wild type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.


( B ) Immunoblotting against the GFP , endogenous C9ORF72 or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA .

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(B) Immunoblotting against the GFP, endogenous C9ORF72 or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA.


( C ) Immunoblotting against the HA tag , endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours .

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(C) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.


( D ) Immunoblotting against the HA tag , endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA tag in the GR frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours .

(D) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA tag in the GR frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.


( E ) Cell viability ( TO - PRO - 3 FACS staining ) of GT1 - 7 neuronal cells co - transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and wild - type or mutants constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA , PG or GR frame .

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(E) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and wild-type or mutants constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.


( A ) Left panel , immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 µM of the indicated compound for 15 hours . Right panel , quantification of polyGA expression relative to the GAPDH .

(A) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyGA expression relative to the GAPDH.


( B ) Left panel , immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and treated with 10 µM of the indicated compound for 15 hours . Right panel , quantification of polyPG expression relative to the GAPDH .

(B) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyPG expression relative to the GAPDH.


( C ) Cell viability ( TO - PRO - 3 FACS staining ) of GT1 - 7 neuronal cells treated with 1 , 3 or 10 µM of promethazine and co - transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame .

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(C) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells treated with 1, 3 or 10 µM of promethazine and co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.


( A ) Bcl - xL was cotransfected into HeLa cells with empty vector ( IP negative control ) , Beclin 1 - Flag , Beclin 1 - Flag / HA - Bim ( EL ) , Beclin 1 - Flag / HA - Puma , Beclin 1 - Flag / HA - Noxa , or Beclin 1 - Flag / Bad . Anti - Flag ( M2 ) was used for immunoprecipitation , and proteins were detected with anti - Bcl - xL , anti - HA , or anti - Bad , anti - Flag ( Rabbit ) .

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(A) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), Beclin 1-Flag, Beclin 1-Flag/HA-Bim(EL), Beclin 1-Flag/HA-Puma, Beclin 1-Flag/HA-Noxa, or Beclin 1-Flag/Bad. Anti-Flag (M2) was used for immunoprecipitation, and proteins were detected with anti-Bcl-xL, anti-HA, or anti-Bad, anti-Flag (Rabbit).


( B ) HA - Bim ( wild - type ) was cotransfected into Bax / Bak double - knockout ( DKO ) mouse embryonic fibroblasts ( MEFs ) with empty vector ( IP negative control ) or Beclin 1 - Flag . Immunoprecipitations were performed as in ( A ) .

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(B) HA-Bim (wild-type) was cotransfected into Bax/Bak double-knockout (DKO) mouse embryonic fibroblasts (MEFs) with empty vector (IP negative control) or Beclin 1-Flag. Immunoprecipitations were performed as in (A).


( C ) Beclin 1 - Flag was cotransfected into Bax / Bak DKO MEFs with empty vector ( IP negative control ) or HA - Bim ( wild - type ) . Anti - HA was used for immunoprecipitation . ∗ , antibody heavy chain .

(C) Beclin 1-Flag was cotransfected into Bax/Bak DKO MEFs with empty vector (IP negative control) or HA-Bim (wild-type). Anti-HA was used for immunoprecipitation. ∗, antibody heavy chain.


( D ) HeLa cell lysates were subjected to GFP antibody ( negative control ) and anti - Bim antibody immunoprecipitation . Proteins were detected with anti - Beclin 1 and anti - Bim . ∗ ∗ , antibody heavy chain ; ∗ , antibody light chain .

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(D) HeLa cell lysates were subjected to GFP antibody (negative control) and anti-Bim antibody immunoprecipitation. Proteins were detected with anti-Beclin 1 and anti-Bim. ∗∗, antibody heavy chain; ∗, antibody light chain.


( E ) Bcl - xL was cotransfected into HeLa cells with empty vector ( IP negative control ) , wild - type HA - Bim ( EL ) , or HA - Bim L152E F159E [ Bim ( EL ) EE ] . Anti - Bim was used for immunoprecipitation . ∗ , antibody light chain .

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(E) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), wild-type HA-Bim(EL), or HA-Bim L152E F159E [Bim(EL)EE]. Anti-Bim was used for immunoprecipitation. ∗, antibody light chain.


( F ) HeLa cells were transfected with vector , wild - type HA - Bim , or HA - BimEE . Lysates were probed with the indicated antibodies . HeLa cells treated with staurosporine ( STS ) for 5 hr were a positive control . Note that wild - type Bim causes apoptosis , which reduces its levels compared to BimEE .

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(F) HeLa cells were transfected with vector, wild-type HA-Bim, or HA-BimEE. Lysates were probed with the indicated antibodies. HeLa cells treated with staurosporine (STS) for 5 hr were a positive control. Note that wild-type Bim causes apoptosis, which reduces its levels compared to BimEE.


( G ) Vector , wild - type Bim ( EL ) , or Bim ( EL ) L152E F159E ( BimEE ) was transfected into HeLa cells . Transfection efficiency was > 90 % . After 20 hr , cell viability was determined by measurement pf ATP levels . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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(G) Vector, wild-type Bim(EL), or Bim(EL) L152E F159E (BimEE) was transfected into HeLa cells. Transfection efficiency was >90%. After 20 hr, cell viability was determined by measurement pf ATP levels. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( H ) HeLa cells were transfected with vector / GFP ( 3 : 1 ) , wild - type HA - Bim / GFP ( 3 : 1 ) , or HA - BimEE / GFP ( 3 : 1 ) ( to ensure that Bim - transfected cells also contain GFP ) . After 16 hr , cells were stained with Annexin V - APC and analyzed by FACS . The percentage of APC and GFP double - positive cells / GFP - positive cells are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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(H) HeLa cells were transfected with vector/GFP (3:1), wild-type HA-Bim/GFP (3:1), or HA-BimEE/GFP (3:1) (to ensure that Bim-transfected cells also contain GFP). After 16 hr, cells were stained with Annexin V-APC and analyzed by FACS. The percentage of APC and GFP double-positive cells/GFP-positive cells are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( I ) Bim ( EL ) / Bcl - xL were cotransfected into HeLa cells with empty vector ( IP negative control ) or Beclin 1 - Flag ; Bim ( EL ) - EE / Bcl - xL were also cotransfected into HeLa cells with Beclin 1 - Flag . Anti - Flag ( M2 ) was used for immunoprecipitation . Data are shown as mean ± SD . NS , not significant .

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(I) Bim(EL)/Bcl-xL were cotransfected into HeLa cells with empty vector (IP negative control) or Beclin 1-Flag; Bim(EL)-EE/Bcl-xL were also cotransfected into HeLa cells with Beclin 1-Flag. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. NS, not significant.


( J ) BimEL - EE ( EL ) / vector ( IP negative control ) , BimEL - EE ( EL ) / Beclin 1 - Flag , BimL - EE ( L ) / Beclin 1 - Flag , and BimS - EE ( S ) / Beclin 1 - Flag were transfected into HeLa cells . Anti - Flag was used for immunoprecipitation . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant .

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(J) BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag, BimL-EE (L)/Beclin 1-Flag, and BimS-EE (S)/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant.


( A ) Bim − / − and Bim + / + MEFs were cultured in 6 - well dishes . Cells were treated with vehicle or Bafilomycin A1 ( Baf ) for 4 hr . Blots were probed with the indicated antibodies . LC3 - II / tubulin in wild - type control MEFs is set as 1 . The relative value of LC3 - II / tubulin in Bim - knockout MEFs is shown ( n = 5 ) . Data are mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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(A) Bim−/− and Bim+/+ MEFs were cultured in 6-well dishes. Cells were treated with vehicle or Bafilomycin A1 (Baf) for 4 hr. Blots were probed with the indicated antibodies. LC3-II/tubulin in wild-type control MEFs is set as 1. The relative value of LC3-II/tubulin in Bim-knockout MEFs is shown (n = 5). Data are mean ± SD. ∗∗∗p < 0.001.


( B ) HeLa cells were transfected with control siRNA and Bim siRNA . After 48 hr , cells were treated with vehicle or Baf for 4 hr . Blots were probed with the indicated antibodies and analyzed as described in ( A ) . ∗ ∗ ∗ p < 0 . 001 .

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(B) HeLa cells were transfected with control siRNA and Bim siRNA. After 48 hr, cells were treated with vehicle or Baf for 4 hr. Blots were probed with the indicated antibodies and analyzed as described in (A). ∗∗∗p < 0.001.


( C ) Bim − / − and Bim + / + MEFs were transfected with GFP - LC3 . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

(C) Bim−/− and Bim+/+ MEFs were transfected with GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.


( D ) HeLa cells were transfected with control plasmid ( pMKO ) / GFP - LC3 or pMKO - BimshRNA / GFP - LC3 ( to ensure that GFP - LC3 - positive cells also contain BimshRNA or control plasmid , the ratio of pMKO or pMKO - BimshRNA / GFP - LC3 is 3 : 1 ) . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

(D) HeLa cells were transfected with control plasmid (pMKO)/GFP-LC3 or pMKO-BimshRNA/GFP-LC3 (to ensure that GFP-LC3-positive cells also contain BimshRNA or control plasmid, the ratio of pMKO or pMKO-BimshRNA/GFP-LC3 is 3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( E ) HeLa cells were transfected with control plasmid ( pMKO ) or pMKO - BimshRNA . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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(E) HeLa cells were transfected with control plasmid (pMKO) or pMKO-BimshRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( F ) HeLa cells were transfected with control plasmid ( pMKO ) / GFP - PX or pMKO - BimshRNA / GFP - PX ( 3 : 1 ) . The percentages of cells with GFP - vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

(F) HeLa cells were transfected with control plasmid (pMKO)/GFP-PX or pMKO-BimshRNA/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.