( C ) Western Blot analysis CREBH protein stability after HRD1 over - expression . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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(C) Western Blot analysis CREBH protein stability after HRD1 over-expression. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Western Blot analysis of hepatic CREBH and HRD1 protein levels in WT and HRD1 LKO mice . The mice were fasted 16 hours and refed for another 4 hours .

O O O O O O O O O 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) Western Blot analysis of hepatic CREBH and HRD1 protein levels in WT and HRD1 LKO mice. The mice were fasted 16 hours and refed for another 4 hours.


( E ) Western Blot analysis interaction of CREBH and HRD1 after immunoprecipitates of Flag - agarose in transfected HEK293T .

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(E) Western Blot analysis interaction of CREBH and HRD1 after immunoprecipitates of Flag-agarose in transfected HEK293T.


( F ) Endogenous interaction between CREBH and HRD1 in liver .

(F) Endogenous interaction between CREBH and HRD1 in liver.


( G ) Western Blot analysis ubiquitination of CREBH after immunoprecipitates of Flag - agarose in transfected HEK293T .

(G) Western Blot analysis ubiquitination of CREBH after immunoprecipitates of Flag-agarose in transfected HEK293T.


( H ) Hepatic CREBH ubiquitination level in the WT and L - HRD1 KO

(H) Hepatic CREBH ubiquitination level in the WT and L-HRD1 KO mice


( I ) Relative protein levels of CREBH , HRD1 and ubiquitination of CREBH . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

(I) Relative protein levels of CREBH, HRD1 and ubiquitination of CREBH. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( J ) Primary hepatocytes were isolated from WT and HRD1 LKO mice and Western Blot analysis CREBH protein stability .

(J) Primary hepatocytes were isolated from WT and HRD1 LKO mice and Western Blot analysis CREBH protein stability.


( L ) Interactions of full - length and C - terminal of HRD1 with CREBH were measured by Co - IP . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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

(L) Interactions of full-length and C-terminal of HRD1 with CREBH were measured by Co-IP. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( B ) Interactions of full - length and activated form of CREBH with HRD1 were measured by Co - IP .

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

(B) Interactions of full-length and activated form of CREBH with HRD1 were measured by Co-IP.


( C , D ) Ubiquitination of CREBH and CREBH K294R were measured after co - transfected C - terminal of HRD1 .

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(C, D) Ubiquitination of CREBH and CREBH K294R were measured after co-transfected C-terminal of HRD1.


( E ) Western Blot analysis CREBH or CREBH - K294R protein stability after HRD1 protein over - expression . Data information : The data are representative of three independent experiments ( mean ± s . d .

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(E) Western Blot analysis CREBH or CREBH-K294R protein stability after HRD1 protein over-expression. Data information: The data are representative of three independent experiments (mean ± s.d.


( F ) Western Blot analysis of the CREBH WT or K48 only ubiquitination level after HRD1 C - terminal co - expression .

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(F) Western Blot analysis of the CREBH WT or K48 only ubiquitination level after HRD1 C-terminal co-expression.


( H ) Western Blot analysis of the CREBH K6 , K11 , K27 , K29 and K33 only ubiquitination level after HRD1 co - expression .

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

(H) Western Blot analysis of the CREBH K6, K11, K27, K29 and K33 only ubiquitination level after HRD1 co-expression.


( I ) Western Blot analysis of the CREBH WT , K27R ubiquitination level after HRD1 full - length co - expression .

(I) Western Blot analysis of the CREBH WT, K27R ubiquitination level after HRD1 full-length co-expression.


( J ) Western Blot analysis of the CREBH WT , K27 and K48 only ubiquitination level after HRD1 C - terminal co - expression .

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

(J) Western Blot analysis of the CREBH WT, K27 and K48 only ubiquitination level after HRD1 C-terminal co-expression.


( B ) Hepatic CREBH protein levels 5 weeks after AAV - shCrebh injection . ( n = 5 for each group ) .

(B) Hepatic CREBH protein levels 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( C ) Hepatic Fgf21 mRNA in the WT and L - HRD1 KO mice 5 weeks after AAV - shCrebh injection . ( n = 5 for each group ) .

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(C) Hepatic Fgf21 mRNA in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( D ) Serum FGF21 protein levels in the WT and L - HRD1 KO mice 5 weeks after AAV - shCrebh injection . ( n = 5 for each group ) .

(D) Serum FGF21 protein levels in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( E ) Body height and tibia length of the WT and L - HRD1 KO mice 5 weeks after AAV - shCrebh injection ( n = 5 for each group ) .

(E) Body height and tibia length of the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection (n=5 for each group).


( F ) Hepatic Igf1 , Cyp2d9 and Cyp4a12 mRNA in the WT and L - HRD1 KO mice 5 weeks after AAV - shCrebh injection . ( n = 5 for each group ) .

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(F) Hepatic Igf1, Cyp2d9 and Cyp4a12 mRNA in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( G , H ) Percentage of the mated and estrus cycle of the WT and L - HRD KO 5 weeks after AAV - shCrebh injection . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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

(G, H) Percentage of the mated and estrus cycle of the WT and L-HRD KO 5 weeks after AAV-shCrebh injection. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( A ) Examples of the body sizes of the WT , L - HRD KO and HRD1 / FGF21 DKO mice . ( B , C ) Body weight and body height of the WT , L - HRD KO and HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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

(A) Examples of the body sizes of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (B, C) Body weight and body height of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Tibia lengths of the WT , L - HRD KO and HRD1 / FGF21 DKO mice fed with normal chow diet . ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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

(D) Tibia lengths of the WT, L-HRD KO and HRD1/FGF21 DKO mice fed with normal chow diet. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( E ) Hepatic Hsd3b5 , Cyp2d9 and Cyp4a12 mRNA in the WT , L - HRD1 KO and HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

(E) Hepatic Hsd3b5, Cyp2d9 and Cyp4a12 mRNA in the WT, L-HRD1 KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( F ) Estrus cycle of L - HRD KO and HRD1 / FGF21 DKO mice at the age of 4 months . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

(F) Estrus cycle of L-HRD KO and HRD1/FGF21 DKO mice at the age of 4 months. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( G ) Percentage of the Activity , feeding and water intake of the WT , L - HRD KO and HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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

(G) Percentage of the Activity, feeding and water intake of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( b ) 2D - [ 1H ; 15N ] - TROSY spectrum of 400 µM 2H , 13C , 15N - labeled TREM2 - TMH ( residues R161 - H206 ) in DPC micelles at 37 ° C . Assigned resonances are labeled .

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(b) 2D-[1H;15N]-TROSY spectrum of 400 µM 2H,13C,15N-labeled TREM2-TMH (residues R161-H206) in DPC micelles at 37 °C. Assigned resonances are labeled.


( c ) Secondary chemical shifts plotted against the residue number indicates an α - helical region between residues 173 and 198 . The used TREM2 - TMH construct along with the predicted transmembrane helix content ( aa 175 - 195 ) is also indicated .

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(c) Secondary chemical shifts plotted against the residue number indicates an α-helical region between residues 173 and 198. The used TREM2-TMH construct along with the predicted transmembrane helix content (aa 175-195) is also indicated.


( d ) Circular dichroism ( CD ) spectrum of TREM2 - TMH in DPC micelles indicates an α - helical secondary structure content of ~ 50 % .

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(d) Circular dichroism (CD) spectrum of TREM2-TMH in DPC micelles indicates an α-helical secondary structure content of ~50%.


( e ) 3D - 15N - edited NOESY spectra show that the transmembrane helix is interrupted at residues 190 - 192 . Dashed red lines indicate sequential amide NOE contacts that are characteristic for an α - helical secondary structure .

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(e) 3D-15N-edited NOESY spectra show that the transmembrane helix is interrupted at residues 190-192. Dashed red lines indicate sequential amide NOE contacts that are characteristic for an α-helical secondary structure.


( a , b ) Overlay of 2D - [ 1H , 15N ] - TROSY spectra of 2H , 15N - labeled TREM2 - TMH in DPC micelles ( black ) and in complex with ( a ) unlabeled DAP12 ( red ) or ( b ) TREM2 - TMH lacking its positively charged lysine residue ( K186A , red ) .

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(a, b) Overlay of 2D-[1H,15N]-TROSY spectra of 2H,15N-labeled TREM2-TMH in DPC micelles (black) and in complex with (a) unlabeled DAP12 (red) or (b) TREM2-TMH lacking its positively charged lysine residue (K186A, red).


( c ) Chemical shift perturbations ( CSPs ) between the two spectra in panels ( a ) or ( b ) . The α - helical secondary structure as determined for wild - type TREM2 - TMH is indicated above and the position of the K186A mutation is labeled in green .

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(c) Chemical shift perturbations (CSPs) between the two spectra in panels (a) or (b). The α-helical secondary structure as determined for wild-type TREM2-TMH is indicated above and the position of the K186A mutation is labeled in green.


( f ) For TREM2 - K186A - TMH a ~ 7 % increase in α - helical secondary structure can be detected by CD spectroscopy .

(f) For TREM2-K186A-TMH a ~7% increase in α-helical secondary structure can be detected by CD spectroscopy.


( g ) A continuous α - helical secondary structure in the kink region ( aa 189 - 192 ) was probed by sequential amide NOE contacts in TREM2 - K186A - TMH . Red dots indicate the chemical shift of intra - residual ( i ) amide resonance and red broken lines the inter - residual connectivity ( i ± 1 ) .

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(g) A continuous α-helical secondary structure in the kink region (aa 189-192) was probed by sequential amide NOE contacts in TREM2-K186A-TMH. Red dots indicate the chemical shift of intra-residual (i) amide resonance and red broken lines the inter-residual connectivity (i±1).


( a , b ) Generalized order parameter S2 for backbone amide resonances derived from NMR relaxation experiments with wild - type TREM2 - TMH ( a ) and the K186A variant ( b ) , indicative of motions in the nanosecond to picosecond time scale . S2 can adopt values between 1 ( rigid ) and 0 ( unrestricted motion ) . Error bars have been obtained by a Monte - Carlo - simulation - based error estimation and motional model selection procedure implemented in the software Relax

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

(a,b) Generalized order parameter S2 for backbone amide resonances derived from NMR relaxation experiments with wild-type TREM2-TMH (a) and the K186A variant (b), indicative of motions in the nanosecond to picosecond time scale. S2 can adopt values between 1 (rigid) and 0 (unrestricted motion). Error bars have been obtained by a Monte-Carlo-simulation-based error estimation and motional model selection procedure implemented in the software Relax


( a , b ) Generalized order parameter S2 for backbone amide resonances derived from NMR relaxation experiments with wild - type TREM2 - TMH ( a ) and the K186A variant ( b ) , indicative of motions in the nanosecond to picosecond time scale . S2 can adopt values between 1 ( rigid ) and 0 ( unrestricted motion ) . Error bars have been obtained by a Monte - Carlo - simulation - based error estimation and motional model selection procedure implemented in the software

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


( a ) Paramagnetic relaxation enhancement ( PRE ) with a free - radical - modified stearic fatty acid in DPC micelles . Low values indicate location inside whereas values close to one suggest a location outside the detergent micelle . Orange bars : TREM2 - TMH wt , green bars : K186A variant . The N - terminal residues of TREM2 - TMH - wt are located deeper inside the micelles as in the K186A case , whereas the C - terminal end is positioned at the outside for wild - type and inside for the K186A variant . Negative bars indicate missing assignments .

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

(a) Paramagnetic relaxation enhancement (PRE) with a free-radical-modified stearic fatty acid in DPC micelles. Low values indicate location inside whereas values close to one suggest a location outside the detergent micelle. Orange bars: TREM2-TMH wt, green bars: K186A variant. The N-terminal residues of TREM2-TMH-wt are located deeper inside the micelles as in the K186A case, whereas the C-terminal end is positioned at the outside for wild-type and inside for the K186A variant. Negative bars indicate missing assignments.


( b ) NOE - cross peaks between backbone amides of TREM2 - TMH wt ( top ) or the K186A variant ( bottom ) and selected regions of DPC ( see color coding of the structure of the DPC molecule on the right ) . The K186A variant shows strong contacts to the hydrophobic aliphatic regions of the detergent throughout the transmembrane helix whereas no such contacts can be seen at the kink site of the wild - type helix , indicating a location outside of the micelle for this part .

O O O O O 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 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

(b) NOE-cross peaks between backbone amides of TREM2-TMH wt (top) or the K186A variant (bottom) and selected regions of DPC (see color coding of the structure of the DPC molecule on the right). The K186A variant shows strong contacts to the hydrophobic aliphatic regions of the detergent throughout the transmembrane helix whereas no such contacts can be seen at the kink site of the wild-type helix, indicating a location outside of the micelle for this part.


( b ) Immunoblot detection of ICD accumulation of TREM2 wildtype , K186A , K186P , and K186L variants mediated by γ - secretase cleavage upon overnight incubation of membrane preparations at 37 ° C . ICD formation is blocked by 1 µM of the transition state analogue L - 685 , 458 . Note that inhibition of ICD formation from wt TREM2 was less efficient . Due to the charge within the TMH wt TREM2 is less efficiently transported to the cell surface ( Lanier et al , 1998 ) and therefore retained within the endoplasmic reticulum , where other proteases not related to γ - secretase and therefore not inhabitable by L - 685 , 458 may cleave . In vitro generation of the ICD of APP ( AICD ) was used as a positive control . Immunoblot detection of mature nicastrin and the presenilin 1 NTF , which results from presenilin endoproteolysis necessary to activate γ - secretase ( Thinakaran et al , 1996 ) , proves comparable amounts of γ - secretase in the individual assays .

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

(b) Immunoblot detection of ICD accumulation of TREM2 wildtype, K186A, K186P, and K186L variants mediated by γ-secretase cleavage upon overnight incubation of membrane preparations at 37 °C. ICD formation is blocked by 1 µM of the transition state analogue L-685,458. Note that inhibition of ICD formation from wt TREM2 was less efficient. Due to the charge within the TMH wt TREM2 is less efficiently transported to the cell surface (Lanier et al, 1998) and therefore retained within the endoplasmic reticulum, where other proteases not related to γ-secretase and therefore not inhabitable by L-685,458 may cleave. In vitro generation of the ICD of APP (AICD) was used as a positive control. Immunoblot detection of mature nicastrin and the presenilin 1 NTF, which results from presenilin endoproteolysis necessary to activate γ-secretase (Thinakaran et al, 1996), proves comparable amounts of γ-secretase in the individual assays.


( c ) MALDI MS analysis of the ε - cleavage sites of wt and mutant TREM2 C - terminal fragments . Mass spectrometry reveals ε - cleavage C - terminal of A192 for the wt CTF . MS determination of the ε - cleavage site of TREM2 CTF K186A reveals major and minor ε - cleavage sites C - terminal of A195 and L193 , respectively . The exact same cleavage pattern is observed for the K186L mutant . MS analysis of the cleavage pattern of the K186P mutant reveals major and minor ε - cleavage sites C - terminal of L193 and S191 . Minor peaks that could not be assigned are indicated by an asterisk .

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(c) MALDI MS analysis of the ε-cleavage sites of wt and mutant TREM2 C-terminal fragments. Mass spectrometry reveals ε-cleavage C-terminal of A192 for the wt CTF. MS determination of the ε-cleavage site of TREM2 CTF K186A reveals major and minor ε-cleavage sites C-terminal of A195 and L193, respectively. The exact same cleavage pattern is observed for the K186L mutant. MS analysis of the cleavage pattern of the K186P mutant reveals major and minor ε-cleavage sites C-terminal of L193 and S191. Minor peaks that could not be assigned are indicated by an asterisk.


( d , e ) Chemically induced unfolding of TREM2 - TMH wild - type ( d ) and the K186A variant ( e ) demonstrates that the less dynamic variant is more stable than the wild - type protein .

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(d, e) Chemically induced unfolding of TREM2-TMH wild-type (d) and the K186A variant (e) demonstrates that the less dynamic variant is more stable than the wild-type protein.


( f ) Water exchange peak as observed in a 3D - 15N - edited - [ 1H , 1H ] - NOESY experiment which is only absent if the amide moiety is present in a stable hydrogen bond .

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(f) Water exchange peak as observed in a 3D-15N-edited-[1H,1H]-NOESY experiment which is only absent if the amide moiety is present in a stable hydrogen bond.


A - C Viability counts of the indicated V . parahaemolyticus ( A - B ) or E . coli ( C ) prey strains before ( 0 h ) and after ( 4 h ) co - incubation with the indicated V . parahaemolyticus attackers on media containing 3 % NaCl at 30 ° C . In A and B , media also contain 0 . 1 % arabinose to induce expression from plasmids . In A , prey strains contain either an empty plasmid ( pEmpty ) or a plasmid for arabinose - inducible expression of VP1389 ( pVP1389 ) . In B and C , prey strains contain an empty plasmid that provides a selection marker , and the attackers are derivatives of a ∆ hns mutant ( parental ) . In B , the attackers contain an empty plasmid , or plasmids for the arabinose - inducible expression of VP1388 ( pVP1388 ) or VP1390 ( pVP1390 ) . Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above . A significant difference was considered as P < 0 . 05 . DL , assay detection limit . ∆ hcp1 was used as a T6SS1 - control strain . The experiment was performed at least three times with similar results ; results of a representative experiment are shown .

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A-C Viability counts of the indicated V. parahaemolyticus (A-B) or E. coli (C) prey strains before (0 h) and after (4 h) co-incubation with the indicated V. parahaemolyticus attackers on media containing 3% NaCl at 30°C. In A and B, media also contain 0.1% arabinose to induce expression from plasmids. In A, prey strains contain either an empty plasmid (pEmpty) or a plasmid for arabinose-inducible expression of VP1389 (pVP1389). In B and C, prey strains contain an empty plasmid that provides a selection marker, and the attackers are derivatives of a ∆hns mutant (parental). In B, the attackers contain an empty plasmid, or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit. ∆hcp1 was used as a T6SS1- control strain. The experiment was performed at least three times with similar results; results of a representative experiment are shown.


A VP1388 binds VP1390 . Immunoprecipitation using α - FLAG antibodies from E . coli cells co - expressing the indicated C - terminal FLAG - and Myc - tagged proteins from arabinose - inducible plasmids .

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A VP1388 binds VP1390. Immunoprecipitation using α-FLAG antibodies from E. coli cells co-expressing the indicated C-terminal FLAG- and Myc-tagged proteins from arabinose-inducible plasmids.


B VP1388 and VgrG1 co - precipitate with VP1390 . Immunoprecipitation using α - FLAG antibodies from V . parahaemolyticus ∆ hns / ∆ hcp1 / ∆ vp1390 derivatives harboring plasmids for the arabinose - inducible expression of FLAG - tagged sfGFP or VP1390 . Cells were grown in MLB media supplemented with chloramphenicol to maintain the plasmids , and 0 . 1 % arabinose . Endogenous VP1388 and VgrG1 were detected using α - VP1388 and α - VgrG1 antibodies , respectively .

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B VP1388 and VgrG1 co-precipitate with VP1390. Immunoprecipitation using α-FLAG antibodies from V. parahaemolyticus ∆hns/∆hcp1/∆vp1390 derivatives harboring plasmids for the arabinose-inducible expression of FLAG-tagged sfGFP or VP1390. Cells were grown in MLB media supplemented with chloramphenicol to maintain the plasmids, and 0.1% arabinose. Endogenous VP1388 and VgrG1 were detected using α-VP1388 and α-VgrG1 antibodies, respectively.


C Expression ( cells ) and secretion ( media ) of VP1388 , VP1390 , and VgrG1 from the indicated V . parahaemolyticus ∆ hns - derived strains harboring an empty plasmid ( pEmpty ) or plasmids for the arabinose - inducible expression of VP1388 ( pVP1388 ) or VP1390 ( pVP1390 ) . Samples were grown in media containing 3 % NaCl and supplemented with 0 . 1 % arabinose at 30 ° C . RNA polymerase β ( RNAp ) was used as a non - secreted protein loading control . Arrows denote the bands at the expected size of VP1388 or VP1390 , as indicated in the appropriate panels .

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C Expression (cells) and secretion (media) of VP1388, VP1390, and VgrG1 from the indicated V. parahaemolyticus ∆hns-derived strains harboring an empty plasmid (pEmpty) or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Samples were grown in media containing 3% NaCl and supplemented with 0.1% arabinose at 30°C. RNA polymerase β (RNAp) was used as a non-secreted protein loading control. Arrows denote the bands at the expected size of VP1388 or VP1390, as indicated in the appropriate panels.


A Toxicity of periplasm - targeted proteins in E . coli . E . coli strains containing plasmids for the arabinose - inducible expression of sfGFP ( used as a control ) , VP1388 or VP1390 fused to an N - terminal PelB signal peptide ( perisfGFP , periVP1388 , and periVP1390 , respectively ) were spotted at 10 - fold serial dilutions onto LB agar plates supplemented with kanamycin ( to maintain plasmids ) and either 0 . 2 % glucose , to repress protein expression , or 0 . 001 % arabinose , to induce protein expression . Plates were incubated over - night at 30 ° C or 37 ° C , as indicated .

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A Toxicity of periplasm-targeted proteins in E. coli. E. coli strains containing plasmids for the arabinose-inducible expression of sfGFP (used as a control), VP1388 or VP1390 fused to an N-terminal PelB signal peptide (perisfGFP, periVP1388, and periVP1390, respectively) were spotted at 10-fold serial dilutions onto LB agar plates supplemented with kanamycin (to maintain plasmids) and either 0.2% glucose, to repress protein expression, or 0.001% arabinose, to induce protein expression. Plates were incubated over-night at 30°C or 37°C, as indicated.


B VP1389 interacts with VP1390 . Co - immunoprecipitation of FLAG - tagged VP1390 or BC3020 using Myc - tagged VP1389 when co - expressed in V . parahaemolyticus Δvp1389 ( input ) . Precipitated proteins ( output ) were detected by immunobotting using α - Myc and α - FLAG antibodies . Source data are available online for this figure .

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B VP1389 interacts with VP1390. Co-immunoprecipitation of FLAG-tagged VP1390 or BC3020 using Myc-tagged VP1389 when co-expressed in V. parahaemolyticus Δvp1389 (input). Precipitated proteins (output) were detected by immunobotting using α-Myc and α-FLAG antibodies. Source data are available online for this figure.


C VP1390 induces cell lysis in E . coli . Time - lapse microscopy of E . coli cells expressing periplasm - targeted sfGFP , VP1388 , or VP1390 ( perisfGFP , periVP1388 , and periVP1390 , respectively ) from an arabinose - inducible vector , grown on LB agarose pads supplemented with kanamycin ( to maintain the plasmid ) , 0 . 2 % arabinose ( to induce expression ) , and propidium iodide ( PI ; pink ) . Merging of the phase contrast and PI channels are shown . Scale bar = 2 µm .

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C VP1390 induces cell lysis in E. coli. Time-lapse microscopy of E. coli cells expressing periplasm-targeted sfGFP, VP1388, or VP1390 (perisfGFP, periVP1388, and periVP1390, respectively) from an arabinose-inducible vector, grown on LB agarose pads supplemented with kanamycin (to maintain the plasmid), 0.2% arabinose (to induce expression), and propidium iodide (PI; pink). Merging of the phase contrast and PI channels are shown. Scale bar = 2 µm.


Time - lapse microscopy of competition between V . parahaemolyticus ∆ hns ( T6SS + / VP1390 + ) , ∆ hns / ∆ hcp1 ( T6SS - / VP1390 + ) , or ∆ hns / ∆ vp1390 ( T6SS + / VP1390 - ) attackers and V . parahaemolyticus ∆ vp1389 prey that express GFP . Attacker and prey were mixed ( 2 : 1 ratio ) and spotted on LB agarose pads supplemented with propidium iodide ( PI ; pink ) . Merging of the phase contrast , GFP ( green ) , and PI ( pink ) channels , as well as the PI channel alone are shown . Scale bar = 5 µm . The experiment was performed three times with similar results ; results of a representative experiment are shown .

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

Time-lapse microscopy of competition between V. parahaemolyticus ∆hns (T6SS+/VP1390+), ∆hns/∆hcp1 (T6SS-/VP1390+), or ∆hns/∆vp1390 (T6SS+/VP1390-) attackers and V. parahaemolyticus ∆vp1389 prey that express GFP. Attacker and prey were mixed (2:1 ratio) and spotted on LB agarose pads supplemented with propidium iodide (PI; pink). Merging of the phase contrast, GFP (green), and PI (pink) channels, as well as the PI channel alone are shown. Scale bar = 5 µm. The experiment was performed three times with similar results; results of a representative experiment are shown.


Aci2 and Pa were both grown in monoculture in the two - resource environment . The same data is shown in both plots . The left plot shows the average of eight technical replicates on a log scale , while the right plot shows each of the replicates on a linear scale . Overlain on the left plot are growth rate fits of gAci2 = 0 . 88hr - 1 and gPa = 0 . 67hr - 1 . Data Information : Data in A were collected at 400nm .

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

Aci2 and Pa were both grown in monoculture in the two-resource environment. The same data is shown in both plots. The left plot shows the average of eight technical replicates on a log scale, while the right plot shows each of the replicates on a linear scale. Overlain on the left plot are growth rate fits of gAci2 = 0.88hr-1 and gPa = 0.67hr-1. Data Information: Data in A were collected at 400nm.


Aci2 was grown with alanine and glutamate supplied at 25 different ratios from 1 : 16 to 16 : 1 with the total supply kept constant at 0 . 1 % w / Data Information : Data were collected at 600nm .

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Aci2 was grown with alanine and glutamate supplied at 25 different ratios from 1:16 to 16:1 with the total supply kept constant at 0.1%w/ Data Information: Data were collected at 600nm.


The population size at which Aci2 ' s diauxic shift occurred is linearly correlated to the alanine supply , indicating that Aci2 initially consumes almost entirely alanine . Data Information : Data were collected at 600nm .

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

The population size at which Aci2's diauxic shift occurred is linearly correlated to the alanine supply, indicating that Aci2 initially consumes almost entirely alanine. Data Information: Data were collected at 600nm.


Instantaneous growth rates were extracted from the Aci2 monoculture data A variation in Aci2 ' s lag time is clearly visible in this representation . Data Information : Data were collected at 600nm .

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Instantaneous growth rates were extracted from the Aci2 monoculture data A variation in Aci2's lag time is clearly visible in this representation. Data Information: Data were collected at 600nm.


The same monoculture experiments were performed for Pa . The appearance of Pa having a long initial lag is primarily due to the growth that it needs to accomplish before its population size becomes significant on a linear scale as well as a small optical density artifact Data Information : Data were collected at 600nm .

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The same monoculture experiments were performed for Pa. The appearance of Pa having a long initial lag is primarily due to the growth that it needs to accomplish before its population size becomes significant on a linear scale as well as a small optical density artifact Data Information: Data were collected at 600nm.


Pa ' s population size at the onset of its diauxic shift correlates to the supply concentration of whichever resource is supplied in a more limiting amount , indicating coutilization . Data Information : Data were collected at 600nm .

Pa's population size at the onset of its diauxic shift correlates to the supply concentration of whichever resource is supplied in a more limiting amount, indicating coutilization. Data Information: Data were collected at 600nm.


Instantaneous growth rates extracted from the Pa monoculture data . Data Information : Data were collected at 600nm .

Instantaneous growth rates extracted from the Pa monoculture data. Data Information: Data were collected at 600nm.


Diauxic lag times were fit from the monoculture data and are plotted as circles . Lag times could not be fit for conditions with too little growth on the remaining glutamate . The lag times used in the modeling are shown as lines through the data .

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Diauxic lag times were fit from the monoculture data and are plotted as circles. Lag times could not be fit for conditions with too little growth on the remaining glutamate. The lag times used in the modeling are shown as lines through the data.


In order to validate the modeling and test its predictive power , we repeated the competition experiments between Aci2 and Pa across a 54 - condition grid of resource supply ratios and dilution factors Shown is the fraction Pa on Days 1 , 3 , 5 , and 7 for eight total replicates of each condition ( two biological replicates , each with four technical replicates started at different initial species fractions We used the model to predict steady - state population fractions for each condition . We calculated mean final population fractions from the experimental data and compared these fractions to the model predictions .

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In order to validate the modeling and test its predictive power, we repeated the competition experiments between Aci2 and Pa across a 54-condition grid of resource supply ratios and dilution factors Shown is the fraction Pa on Days 1, 3, 5, and 7 for eight total replicates of each condition (two biological replicates, each with four technical replicates started at different initial species fractions We used the model to predict steady-state population fractions for each condition. We calculated mean final population fractions from the experimental data and compared these fractions to the model predictions.


Monoculture experiments were performed to determine species ' growth rates and lag times for each two - resource environment . For each pair of species , coculture competition experiments were performed in each two - resource environment , and qualitative competitive outcomes were determined There were 44 competitions in which both species could be assigned growth rates and lag times Each point represents one competition . Pie charts summarize the competitive outcomes for cases in which the slow - grower was the fast - switcher ( above the Δtlag = 0 line ) and in which the slow - grower was not the slow - switcher ( on and below Δtlag = 0 ) .

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Monoculture experiments were performed to determine species' growth rates and lag times for each two-resource environment. For each pair of species, coculture competition experiments were performed in each two-resource environment, and qualitative competitive outcomes were determined There were 44 competitions in which both species could be assigned growth rates and lag times Each point represents one competition. Pie charts summarize the competitive outcomes for cases in which the slow-grower was the fast-switcher (above the Δtlag = 0 line) and in which the slow-grower was not the slow-switcher (on and below Δtlag = 0).


( A ) Time course of rapid internalization of fluorescent α - synuclein fibrils by CADcells ( for characterization of α - synuclein assemblies , see Figure EV 1A ) . Internalization was measured by recording ATTO - 550 - positive neuron - like cells by flow cytometry . Percentage of ATTO - 550 - positive cells was quantified ( mean ± s . e . m ) ( left panel ) and representative histograms of ATTO - 550 - positive cells are showed on the right panel ( a . u . arbitrary units ) . Similarly , α - synuclein fibrils internalization was also confirmed by fluorescent microscopy ( see Figure EV 1B ) .

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(A) Time course of rapid internalization of fluorescent α-synuclein fibrils by CADcells (for characterization of α-synuclein assemblies, see Figure EV 1A). Internalization was measured by recording ATTO-550-positive neuron-like cells by flow cytometry. Percentage of ATTO-550-positive cells was quantified (mean ±s.e.m) (left panel) and representative histograms of ATTO-550-positive cells are showed on the right panel (a.u. arbitrary units). Similarly, α-synuclein fibrils internalization was also confirmed by fluorescent microscopy (see Figure EV 1B).


B ) Representative images of donor ( upper panel ) and acceptor cells ( lower panel ) after 24 h co - culture . Donor cells were loaded with α - synuclein fibrils prior to co - culture with GFP - transfected acceptor cells ; in red : α - synuclein fibrils , in green : acceptor cells and in blue : nuclei . Scale bar represents 10 μm . ( n = 3 independent experiments ) . A larger field where donor and acceptor cells are shown is presented in Figure EV 3A . ( C ) Percentage of donor and acceptor cells containing α - synuclein fibrils after co - culture as in B : all acceptor cells received α - synuclein fibrils . ( D ) Quantification of the number of α - synuclein fibrils in donor and acceptor cells after co - culture as in B . Donor cells contain around 70 α - synuclein fibrils puncta ( median ) while acceptor cells contain 38 α - synuclein fibrils puncta , respectively ( * * * * , p < 0 . 0001 by two - tailed Mann Whitney test ) . ( E ) Quantification of the average size of α - synuclein fibrillar foci in donor and acceptor cells after co - culture as in B . ( * * * * , p < 0 . 0001 by two - tailed Mann Whitney test ) . In Figure EV 3 is shown an example of α - synuclein fibrillar puncta detection in an acceptor cell . After detection , the number and the size of foci were determined using the ICY software .

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B) Representative images of donor (upper panel) and acceptor cells (lower panel) after 24 h co-culture. Donor cells were loaded with α-synuclein fibrils prior to co-culture with GFP-transfected acceptor cells; in red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bar represents 10 μm. (n = 3 independent experiments). A larger field where donor and acceptor cells are shown is presented in Figure EV 3A.(C) Percentage of donor and acceptor cells containing α-synuclein fibrils after co-culture as in B: all acceptor cells received α-synuclein fibrils.(D) Quantification of the number of α-synuclein fibrils in donor and acceptor cells after co-culture as in B. Donor cells contain around 70 α-synuclein fibrils puncta (median) while acceptor cells contain 38 α-synuclein fibrils puncta, respectively (****, p < 0.0001 by two-tailed Mann Whitney test).(E) Quantification of the average size of α-synuclein fibrillar foci in donor and acceptor cells after co-culture as in B. (****, p < 0.0001 by two-tailed Mann Whitney test). In Figure EV 3 is shown an example of α-synuclein fibrillar puncta detection in an acceptor cell. After detection, the number and the size of foci were determined using the ICY software.


( A ) Representative confocal images showing ChFP - α - syn transfected cells ( in red ) cultured alone ( upper ) or co - cultured with donor cells containing fluorescent Alexa - 488 α - synuclein fibrils ( in green ) for 72 h ( bottom ) . The arrow is pointing out representative α - synuclein fibrils Alexa - 488 co - localized with ChFP - α - syn puncta . Scale bar represents 10 μm . Nuclei are stained with DAPI ( blue ) ( n = 3 independent experiments ) . ( B ) Quantification of the number of ChFP - α - synuclein puncta in ChFP - α - syn acceptor cells showed a significant increase in ChFP - α - synuclein puncta number when cells were co - cultured with donor cells containing α - synuclein fibrils ( * * * , p < 0 . 001 by two - tailed Mann Whitney test ) . ( C ) Quantification of the percentage of co - localization of α - synuclein fibrils Alexa - 488 and ChFP - α - syn puncta . Data shows mean ± s . e . m . After co - culture , 11 % of the transferred α - synuclein fibrils Alexa - 488 co - localized with ChFP - α - syn

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(A) Representative confocal images showing ChFP-α-syn transfected cells (in red) cultured alone (upper) or co-cultured with donor cells containing fluorescent Alexa-488 α-synuclein fibrils (in green) for 72 h (bottom). The arrow is pointing out representative α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta. Scale bar represents 10 μm. Nuclei are stained with DAPI (blue) (n=3 independent experiments).(B) Quantification of the number of ChFP-α-synuclein puncta in ChFP-α-syn acceptor cells showed a significant increase in ChFP-α-synuclein puncta number when cells were co-cultured with donor cells containing α-synuclein fibrils (***, p < 0.001 by two-tailed Mann Whitney test).(C) Quantification of the percentage of co-localization of α-synuclein fibrils Alexa-488 and ChFP-α-syn puncta. Data shows mean ± s.e.m. After co-culture, 11% of the transferred α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta


( A ) Representative images of donor cells ( upper panel ) and acceptor cells ( bottom panel ) of the co - culture system explained in Figure EV 2E ; in red : α - synuclein fibrils ATTO - 550 , in green : α - synuclein fibrils Alexa - 488 and in blue : nuclei . Scale bar represents 10 μm ( n = 3 independent experiments ) . In insets , arrows point to co - localized ATTO - 550 and Alexa - 488 α - synuclein fibrils puncta whereas arrowheads point to puncta of α - synuclein Alexa - 488 fibrils that do not co - localize with fibrillar ATTO - 550 α - synuclein .

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(A) Representative images of donor cells (upper panel) and acceptor cells (bottom panel) of the co-culture system explained in Figure EV 2E; in red: α-synuclein fibrils ATTO-550, in green: α-synuclein fibrils Alexa-488 and in blue: nuclei. Scale bar represents 10 μm (n=3 independent experiments). In insets, arrows point to co-localized ATTO-550 and Alexa-488 α-synuclein fibrils puncta whereas arrowheads point to puncta of α-synuclein Alexa-488 fibrils that do not co-localize with fibrillar ATTO-550 α-synuclein.


( B ) Quantification of the percentage of Alexa - 488 α - synuclein fibrils co - localizing with α - ATTO - 550 α - synuclein fibrils and the reverse co - localization in discrete puncta in donor and acceptor cells as in A . Both measurements revealed high co - localization of the two fluorophores in donor cells ( white bar ) , but less in acceptor cells .

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(B) Quantification of the percentage of Alexa-488 α-synuclein fibrils co-localizing with α- ATTO-550 α-synuclein fibrils and the reverse co-localization in discrete puncta in donor and acceptor cells as in A. Both measurements revealed high co-localization of the two fluorophores in donor cells (white bar), but less in acceptor cells.


( C ) The amount of fibrils taken up by CAD cells exposed to 1µM fibrils , of fibrils remaining in the cells and exported into the medium after 24h incubation were quantified by a filter trap assay . Data are mean ± s . d ( n = 3 independent measurements , filtered in duplicate ) . The standard fluorescence curve for increasing ATTO550 - α - synucleinfibrils concentrations is given .

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(C) The amount of fibrils taken up by CAD cells exposed to 1µM fibrils, of fibrils remaining in the cells and exported into the medium after 24h incubation were quantified by a filter trap assay. Data are mean ± s.d (n=3 independent measurements, filtered in duplicate). The standard fluorescence curve for increasing ATTO550-α-synucleinfibrils concentrations is given.


( D ) Representative images of GFP - transfected acceptor cells that were either ( i ) co - cultured with donor cells ( upper panel , Co - culture ) , ( ii ) cultured with the conditioned medium of donor cells ( middle panel , CM ) or ( iii ) physically separated from donor cells using a filter ( bottom panel , Filter ) . Prior to culture , donor cells were loaded with ATTO - 550 α - synuclein fibrils . In red : α - synuclein fibrils , in green : acceptor cells and in blue : nuclei . Scale bar represents 10 μm ( n = 3 independent experiments ) . ( E ) Quantification of the percentage of acceptor cells containing α - synuclein fibrils from images such as those presented in C . When acceptor cells were cultured with the conditioned medium from donor cells previously treated with α - synucleinfibrils ( not diluted , concentrated or diluted ) or co - cultured with a filter , the percentage of acceptor cells containing α - synuclein puncta was low . Data shows mean ± s . e . m ( * * * , p < 0 . 001 ; by two - tailed Student ' s t - test ) . ( F ) Quantification of the number of puncta of α - synuclein fibrils per acceptor cell from D . While in co - culture condition the number of α - synuclein puncta in acceptor cells is on average 35 ( median ) , this number was on average 1 puncta per acceptor cells in CM and filter conditions , respectively ( * * * , p < 0 . 001 by two - tailed Mann Whitney test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of GFP-transfected acceptor cells that were either (i) co-cultured with donor cells (upper panel, Co-culture), (ii) cultured with the conditioned medium of donor cells (middle panel, CM) or (iii) physically separated from donor cells using a filter (bottom panel, Filter). Prior to culture, donor cells were loaded with ATTO-550 α-synuclein fibrils. In red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bar represents 10 μm (n = 3 independent experiments).(E) Quantification of the percentage of acceptor cells containing α-synuclein fibrils from images such as those presented in C. When acceptor cells were cultured with the conditioned medium from donor cells previously treated with α-synucleinfibrils (not diluted, concentrated or diluted) or co-cultured with a filter, the percentage of acceptor cells containing α-synuclein puncta was low. Data shows mean ± s.e.m (***, p < 0.001; by two-tailed Student's t-test).(F) Quantification of the number of puncta of α-synuclein fibrils per acceptor cell from D. While in co-culture condition the number of α-synuclein puncta in acceptor cells is on average 35 (median), this number was on average 1 puncta per acceptor cells in CM and filter conditions, respectively (***, p < 0.001 by two-tailed Mann Whitney test).


( A ) Representative images of control cells ( upper panel ) or cells treated with α - synucleinfibrilsATTO - 550 ( bottom panel ) for 16 hours ; in red : α - synucleinfibrils and in green : WGAAlexa - 488 ( plasma membrane dye marker ) , blue : DAPI . Scale bar represents 10 μm ( n = 3 independent experiments ) . ( B ) Relative percentage of TNT - connected cells after α - synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment . Data are mean ± s . e . m ( * * * , p < 0 . 001 by paired , two - tailed Student ' s t - test ) .

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(A) Representative images of control cells (upper panel) or cells treated with α-synucleinfibrilsATTO-550 (bottom panel) for 16 hours; in red: α-synucleinfibrils and in green: WGAAlexa-488 (plasma membrane dye marker), blue: DAPI. Scale bar represents 10 μm (n = 3 independent experiments).(B) Relative percentage of TNT-connected cells after α-synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment. Data are mean ± s.e.m (***, p < 0.001 by paired, two-tailed Student's t-test).


( C ) Confocal images showing one TNT connecting two cells loaded with α - synucleinfibrilsATTO - 550 ( in red ) 16 hours after co - culture and stained with WGAAlexa - 488 ( in blue ) . The image represents a Z - projection of the several middle stacks where the TNT is located ( top panel ) . Insets show 3D reconstructions of the TNT using ICY software ( middle panel : merge ; bottom panel : red channel ) . Several α - synucleinfibrils ( red ) are present in the lumen of the TNT ( WGAAlexa - 488 in white ) . Scale bars represent 10 μm ( confocal images ) and 4 μm ( insets ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images showing one TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 (in red) 16 hours after co-culture and stained with WGAAlexa-488 (in blue). The image represents a Z-projection of the several middle stacks where the TNT is located (top panel). Insets show 3D reconstructions of the TNT using ICY software (middle panel: merge; bottom panel: red channel). Several α-synucleinfibrils (red) are present in the lumen of the TNT (WGAAlexa-488 in white). Scale bars represent 10 μm (confocal images) and 4 μm (insets).


( D ) Confocal image showing TNT connecting a donor cell containing α - synucleinfibrils and GFP transfected acceptor cell ( top panel ) strongly suggesting α - synucleinfibrils transfer from donor to acceptor cell . Insets show 3D reconstructions of TNT containing α - synucleinfibrils in the lumen of the TNT ; in red : α - synucleinfibrils , in green : GFP transfected acceptor cells in blue : WGAAlexa - 488 . Scale bars represent 10 μm and 3 μm for the insets .

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(D) Confocal image showing TNT connecting a donor cell containing α-synucleinfibrils and GFP transfected acceptor cell (top panel) strongly suggesting α-synucleinfibrils transfer from donor to acceptor cell. Insets show 3D reconstructions of TNT containing α-synucleinfibrils in the lumen of the TNT; in red: α-synucleinfibrils, in green: GFP transfected acceptor cells in blue: WGAAlexa-488. Scale bars represent 10 μm and 3 μm for the insets.


( E ) Relative percentage of TNTs in cells containing α - synucleinfibrils untransfected ( control ) or transfected with Myosin 10 ( Myosin 10 ) . Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs .

O O O O O O O O O O O O O O O O O O O O O O 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) Relative percentage of TNTs in cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs.


( F ) Quantification of the average number of ATTO - 550α - synucleinfibrils per acceptor cell after 24 hours co - culture with donor cells containing α - synucleinfibrils untransfected ( control ) or transfected with Myosin 10 ( Myosin 10 ) . Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α - synuclein puncta in acceptor cells ; n = 3 independent experiments ( * * , p < 0 . 01 by two - tailed Mann Whitney test ) .

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(F) Quantification of the average number of ATTO-550α-synucleinfibrils per acceptor cell after 24 hours co-culture with donor cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α-synuclein puncta in acceptor cells; n = 3 independent experiments (**, p < 0.01 by two-tailed Mann Whitney test).


( G ) Quantification of the relative proportion of TNTs in co - culture of donor cells containing α - synuclein puncta and GFP acceptor cells under sub - confluent or sparse culture conditions . Data are mean ± s . e . m ; n = 3 independent experiments ( * , p < 0 . 05 by paired , two - tailed Student ' s t - test ) . ( H ) Quantification of the percentage of acceptor cells containing α - synuclein puncta of the co - culture in G . n = 3 independent experiments ( * , p < 0 . 05 by two - tailed Mann Whitney test ) . Condition impairing TNT formation ( i . e . , sparse condition ) induces a drastic decrease of TNT number and α - synucleinfibrils transfer compared to condition allowing TNT formation ( sub - confluent condition ) .

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(G) Quantification of the relative proportion of TNTs in co-culture of donor cells containing α-synuclein puncta and GFP acceptor cells under sub-confluent or sparse culture conditions. Data are mean ± s.e.m; n = 3 independent experiments (*, p < 0.05 by paired, two-tailed Student's t-test).(H) Quantification of the percentage of acceptor cells containing α-synuclein puncta of the co-culture in G. n = 3 independent experiments (*, p < 0.05 by two-tailed Mann Whitney test). Condition impairing TNT formation (i.e., sparse condition) induces a drastic decrease of TNT number and α-synucleinfibrils transfer compared to condition allowing TNT formation (sub-confluent condition).


( A ) Subcellular localization of α - synucleinfibrils in donor cells after 24 hours co - culture . The left panels show representative images of co - localization of α - synuclein puncta with early endosomes ( EEA1 , top panel ) , recycling endosomes ( Vamp3 , middle panel ) and lysosomal vesicles ( Lamp1 , bottom panel ) ( see arrows for co - localized spots ) ; in red : α - synucleinfibrils , in green : EEA1 , Vamp3 or Lamp1 and in blue : nuclei . Scale bar represents 10 μm . On the right , the bar graph represents the percentage of α - synucleinpuncta co - localized with endo - lysosomal vesicles in A . Data are mean ± s . e . m . from three independent experiments .

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(A) Subcellular localization of α-synucleinfibrils in donor cells after 24 hours co-culture. The left panels show representative images of co-localization of α-synuclein puncta with early endosomes (EEA1, top panel), recycling endosomes (Vamp3, middle panel) and lysosomal vesicles (Lamp1, bottom panel) (see arrows for co-localized spots); in red: α-synucleinfibrils, in green: EEA1, Vamp3 or Lamp1 and in blue: nuclei. Scale bar represents 10 μm. On the right, the bar graph represents the percentage of α-synucleinpuncta co-localized with endo-lysosomal vesicles in A. Data are mean ± s.e.m. from three independent experiments.


( B ) Confocal images showing a TNT connecting two cells loaded with α - synucleinfibrilsATTO - 550 immunostained with Lamp1 ( in green ) . Labelling of TNTs is performed using a plasma membrane dye ( WGAAlexa - 488 , in blue ) . The image represents a Z - projection of the several middle stacks where the TNT is located . The merged image with WGA - Alexa 633 ( blue ) , the orthogonal view and insets showing 3D reconstructions of the TNT ( with WGA in white ) are in the right panel . Several α - synuclein fibrillar foci ( in red ) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles ( in green ) . Of note , only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia . Scale bars represent 10 μm ( confocal images ) and 3 μm ( insets ) . The merge of α - synucleinfibrils ( red ) and Lamp1 ( green ) channels and the respective single channels are showed on the left panel .

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(B) Confocal images showing a TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 immunostained with Lamp1 (in green). Labelling of TNTs is performed using a plasma membrane dye (WGAAlexa-488, in blue). The image represents a Z-projection of the several middle stacks where the TNT is located. The merged image with WGA-Alexa 633 (blue), the orthogonal view and insets showing 3D reconstructions of the TNT (with WGA in white) are in the right panel. Several α-synuclein fibrillar foci (in red) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles (in green). Of note, only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia. Scale bars represent 10 μm (confocal images) and 3 μm (insets). The merge of α-synucleinfibrils (red) and Lamp1 (green) channels and the respective single channels are showed on the left panel.


( C ) The top panels show representative images of GFP - transfected acceptor cells after 24 h co - culture with donor cells containing α - synucleinfibrilsATTO - 550 . Immunofluorescence was performed with endo - lysosomal organelle markers ( top panel : EEA1 , middle panel : Vamp3 and Lamp1 : bottom panel ) and revealed that α - synucleinfibrils co - localized with lysosomal vesicles ( see arrow ) ; in red : α - synucleinfibrils , in green : organelle markers and in blue : nuclei . Scale bar represents 10 μm , arrows pinpoint assemblies co - localizing with organelle markers . The bar graph at the bottom shows the quantification of the percentage of co - localization of α - synucleinfibrils with organelle markers , revealing that α - synuclein puncta co - localized mostly with lysosomal vesicles . Data show mean ± s . e . m . from three independent experiments .

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(C) The top panels show representative images of GFP-transfected acceptor cells after 24 h co-culture with donor cells containing α-synucleinfibrilsATTO-550. Immunofluorescence was performed with endo-lysosomal organelle markers (top panel: EEA1, middle panel: Vamp3 and Lamp1: bottom panel) and revealed that α-synucleinfibrils co-localized with lysosomal vesicles (see arrow); in red: α-synucleinfibrils, in green: organelle markers and in blue: nuclei. Scale bar represents 10 μm, arrows pinpoint assemblies co-localizing with organelle markers. The bar graph at the bottom shows the quantification of the percentage of co-localization of α-synucleinfibrils with organelle markers, revealing that α-synuclein puncta co-localized mostly with lysosomal vesicles. Data show mean ± s.e.m. from three independent experiments.


( D ) Representative confocal pictures of control cells loaded with ATTO - 550α - synucleinfibrils and LysoTracker Deep Red ( top panel ) , and of donor ( middle panel ) and acceptor GFP - transfected acceptor cells ( bottom panel , GFP - vector not shown ) cells , loaded with α - synucleinfibrilsATTO - 550 and LysoTracker Deep Red ( middle panel ) and co - cultured for 24 hours ; in red : α - synucleinfibrils , in green : LysoTracker Deep Red and in blue : nuclei . Scale bar represents 10 μm , arrows in inset pinpoint fibrils co - localizing with LysoTracker positive vesicles . ( E ) The percentage of co - localization of LysoTracker positive vesicles with α - synucleinfibrils revealed that 80 % of lysosomal vesicles that transferred from donor to acceptor cells contained α - synucleinfibrils thus demonstrating direct transfer of α - synucleinfibrils from donor to acceptor cells in majority inside lysosomal vesicles derived from donor cells . Data show mean ± s . e . m . from three independent experiments .

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(D) Representative confocal pictures of control cells loaded with ATTO-550α-synucleinfibrils and LysoTracker Deep Red (top panel), and of donor (middle panel) and acceptor GFP-transfected acceptor cells (bottom panel, GFP-vector not shown) cells, loaded with α-synucleinfibrilsATTO-550 and LysoTracker Deep Red (middle panel) and co-cultured for 24 hours; in red: α-synucleinfibrils, in green: LysoTracker Deep Red and in blue: nuclei. Scale bar represents 10 μm, arrows in inset pinpoint fibrils co-localizing with LysoTracker positive vesicles.(E) The percentage of co-localization of LysoTracker positive vesicles with α-synucleinfibrils revealed that 80% of lysosomal vesicles that transferred from donor to acceptor cells contained α-synucleinfibrils thus demonstrating direct transfer of α-synucleinfibrils from donor to acceptor cells in majority inside lysosomal vesicles derived from donor cells. Data show mean ± s.e.m. from three independent experiments.


( A ) Representative images of control ( upper panel ) and α - synuclein loaded neurons ( middle , 0 . 5µM , and bottom , 1µM , panels ) after 16 h . In red : α - synuclein fibrils , in green : MAP - 2 and in blue : nuclei . Scale bar represents 10 μm .

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(A) Representative images of control (upper panel) and α-synuclein loaded neurons (middle, 0.5µM, and bottom, 1µM, panels) after 16 h. In red: α-synuclein fibrils, in green: MAP-2 and in blue: nuclei. Scale bar represents 10 μm.


( B ) Representative images of donor ( upper panels ) and acceptor neurons ( bottom panels ) after 72 hours in co - culture . Donor neurons were loaded with α - synuclein fibrils prior to co - culture with CTG - labelled acceptor neurons and then cells were labelled either with MAP - 2 ( first and third panels ) or Lamp1 ( second and fourth panels ) . In red : α - synuclein fibrils , in green : CTG , in white : MAP - 2 / Lamp1 and in blue : nuclei . Scale bar represents 5 μm . ( C ) The graph bar shows the percentage of donor ( white bar ) and acceptor ( black bar ) neurons containing α - synuclein puncta . Data show mean ± s . e . m . ( D ) The box plot depicts the number of puncta in donor ( white ) and acceptor ( grey ) neurons . ( E ) Box plot showing the distribution of the average size of α - synuclein puncta in donor ( white ) and acceptor ( grey ) neurons . ( F ) The graph bar represents the percentage of α - synuclein puncta co - localized with lysosomes in donor ( white ) and acceptor ( black ) neurons . Data show mean ± s . e . m . Graphs in C , D , E and F correspond to data from 3 independent experiments analysed after 72 hours of co - culture ( * * * * , p < 0 . 0001 compared to donor by two - tailed Mann Whitney test ) .

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(B) Representative images of donor (upper panels) and acceptor neurons (bottom panels) after 72 hours in co-culture. Donor neurons were loaded with α-synuclein fibrils prior to co-culture with CTG-labelled acceptor neurons and then cells were labelled either with MAP-2 (first and third panels) or Lamp1 (second and fourth panels). In red: α-synuclein fibrils, in green: CTG, in white: MAP-2/ Lamp1 and in blue: nuclei. Scale bar represents 5 μm.(C) The graph bar shows the percentage of donor (white bar) and acceptor (black bar) neurons containing α-synuclein puncta. Data show mean ± s.e.m.(D) The box plot depicts the number of puncta in donor (white) and acceptor (grey) neurons.(E) Box plot showing the distribution of the average size of α-synuclein puncta in donor (white) and acceptor (grey) neurons.(F) The graph bar represents the percentage of α-synuclein puncta co-localized with lysosomes in donor (white) and acceptor (black) neurons. Data show mean ± s.e.m. Graphs in C, D, E and F correspond to data from 3 independent experiments analysed after 72 hours of co-culture (****, p < 0.0001 compared to donor by two-tailed Mann Whitney test).


( B ) Representative images of acceptor neurons that were either ( i ) co - cultured with donor neurons ( upper panel , Co - culture ) , ( ii ) physically separated from donor cells ( middle panel , No contact ) or ( iii ) cultured with the conditioned medium of donor neurons ( bottom panel , CM ) . In red : α - synucleinfibrils , in white : acceptor neurons and in blue : nuclei . Scale bar represents 10 μm .

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(B) Representative images of acceptor neurons that were either (i) co-cultured with donor neurons (upper panel, Co-culture), (ii) physically separated from donor cells (middle panel, No contact) or (iii) cultured with the conditioned medium of donor neurons (bottom panel, CM). In red: α-synucleinfibrils, in white: acceptor neurons and in blue: nuclei. Scale bar represents 10 μm.


( C ) The graph bar shows the percentage of acceptor cells containing α - synuclein puncta from images such as those presented in B . * * * * , p < 0 . 001 compared to the co - culture condition by two - tailed Student ' s t - test . Data show mean ± s . e . m from 3 independent experiments . ( D ) The box plot shows the number of α - synuclein puncta in acceptor neurons in co - culture ( white ) , in CM ( grey ) and in the No contact condition ( light grey ) . * * * * , p < 0 . 001 compared to the co - culture condition by two - tailed Mann Whitney test ( n = 3 independent experiments ) . .

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(C) The graph bar shows the percentage of acceptor cells containing α-synuclein puncta from images such as those presented in B. ****, p < 0.001 compared to the co-culture condition by two-tailed Student's t-test. Data show mean ± s.e.m from 3 independent experiments.(D) The box plot shows the number of α-synuclein puncta in acceptor neurons in co-culture (white), in CM (grey) and in the No contact condition (light grey). ****, p < 0.001 compared to the co-culture condition by two-tailed Mann Whitney test (n=3 independent experiments)..


( E ) Western blot analysis of α - synuclein indicates the presence of some α - synuclein in the CM of neurons loaded with the fibrils , after 72h compared to control medium and to lysate of cells directly exposed to the fibrils . α - tubulin was used as loading control .

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(E) Western blot analysis of α-synuclein indicates the presence of some α-synuclein in the CM of neurons loaded with the fibrils, after 72h compared to control medium and to lysate of cells directly exposed to the fibrils. α-tubulin was used as loading control.


( A ) Representative images of donor ( upper panel ) and acceptor cells ( bottom panel ) after 24 hours in co - culture . Donor neurons were loaded with α - synucleinfibrils prior to co - culture with H2B - GFP - acceptor CAD cells . In red : α - synuclein puncta , in green : acceptor CADs , in white donor neurons and in blue : nuclei . The images of donor cells are representative Z - stack projections of the lower slices where these cells are located , and the images corresponding to acceptor CADs are Z - stack projections of the upper slices , covering the whole cell body . Scale bar represents 10 μm . ( B ) Box plots showing the number ( left side ) and average size ( right side ) of α - synuclein puncta in acceptor CAD cells after 24 hours in co - culture with donor neurons . ( n = 3 independent experiments ) .

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

(A) Representative images of donor (upper panel) and acceptor cells (bottom panel) after 24 hours in co-culture. Donor neurons were loaded with α-synucleinfibrils prior to co-culture with H2B-GFP-acceptor CAD cells. In red: α-synuclein puncta, in green: acceptor CADs, in white donor neurons and in blue: nuclei. The images of donor cells are representative Z-stack projections of the lower slices where these cells are located, and the images corresponding to acceptor CADs are Z-stack projections of the upper slices, covering the whole cell body. Scale bar represents 10 μm.(B) Box plots showing the number (left side) and average size (right side) of α-synuclein puncta in acceptor CAD cells after 24 hours in co-culture with donor neurons. (n=3 independent experiments).


( C ) Representative images showing a TNT detected between CAD cells and neurons after 24 hours in co - culture . In green : MAP - 2 , in white : WGA , in red : α - synuclein puncta and in blue nuclei . The merge image in the bottom panel shows in detail the TNT connecting the cells , and the insets and the 3D reconstruction of the neuron ( green ) and the CAD cell ( white ) show the presence of α - synuclein puncta inside the TNT .

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

(C) Representative images showing a TNT detected between CAD cells and neurons after 24 hours in co-culture. In green: MAP-2, in white: WGA, in red: α-synuclein puncta and in blue nuclei. The merge image in the bottom panel shows in detail the TNT connecting the cells, and the insets and the 3D reconstruction of the neuron (green) and the CAD cell (white) show the presence of α-synuclein puncta inside the TNT.


( E ) Expression levels of LSD1 in MGC - 803 , MKN - 45 , HGC - 27 , BGC - 823 , and NCI - N87 cell lines and their corresponding sEVs . The samples with equal amounts of proteins were loaded ( n = 3 biological replicates ; mean ± SEM ; compared with the NCI - N87 ; * P = 0 . 0432 ( cell / MGC - 803 ) , * P = 0 . 0300 ( cell / HGC - 27 ) , * * P = 0 . 0306 ( cell / BGC - 823 ) , * * P = 0 . 0018 ( sEVs / MGC - 803 ) , * * P = 0 . 0029 ( sEVs / HGC - 27 ) , and * * * P = 0 . 0003 ( sEVs / BGC - 823 ) ; two - tailed unpaired Student ' s t - test ; GAPDH was used as a loading control for cell lysis ; CD9 was used as a loading control for sEV lysis ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression levels of LSD1 in MGC-803, MKN-45, HGC-27, BGC-823, and NCI-N87 cell lines and their corresponding sEVs. The samples with equal amounts of proteins were loaded (n = 3 biological replicates; mean ± SEM; compared with the NCI-N87; *P = 0.0432 (cell/MGC-803), *P = 0.0300 (cell/HGC-27), **P = 0.0306 (cell/BGC-823), **P = 0.0018 (sEVs/MGC-803), **P = 0.0029 (sEVs/HGC-27), and ***P = 0.0003 (sEVs/BGC-823); two-tailed unpaired Student's t-test; GAPDH was used as a loading control for cell lysis; CD9 was used as a loading control for sEV lysis).


( J ) Expression levels of LSD1 , CD63 , and CD9 in sEVs with indicated treatment .

(J) Expression levels of LSD1, CD63, and CD9 in sEVs with indicated treatment.


( A ) Confocal microscopy image analysis of sEV fusion to MGC - 803 cells . The MGC - 803 ( left side ) and MKN - 45 ( right side ) cells were treated with sEVs derived from MGC - 803 cells ( Con sEVs ) or LSD1 knockout ( KO ) MGC - 803 cells ( KO sEVs ) and stained with PKH26 for 12 h . Additionally , the cell membrane was stained with Dio , while the nuclei were stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar = 100 µm .

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(A) Confocal microscopy image analysis of sEV fusion to MGC-803 cells. The MGC-803 (left side) and MKN-45 (right side) cells were treated with sEVs derived from MGC-803 cells (Con sEVs) or LSD1 knockout (KO) MGC-803 cells (KO sEVs) and stained with PKH26 for 12 h. Additionally, the cell membrane was stained with Dio, while the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 100 µm.


( B ) Immunofluorescence confocal microscopy analysis of LSD1 ( green ) in LSD1 KO MGC - 803 cells ( left panel ) and LSD1 KO MKN - 45 cells ( right panel ) incubated with 20 μg / mL Con sEVs and KO sEVs for 12 h . The cell membrane was stained with PKH26 , while the nuclei were stained with DAPI . Scale bar = 100 µm .

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

(B) Immunofluorescence confocal microscopy analysis of LSD1 (green) in LSD1 KO MGC-803 cells (left panel) and LSD1 KO MKN-45 cells (right panel) incubated with 20 μg/mL Con sEVs and KO sEVs for 12 h. The cell membrane was stained with PKH26, while the nuclei were stained with DAPI. Scale bar = 100 µm.


( F ) Expression levels of LSD1 , Nanog , OCT4 , SOX2 , and CD44 in LSD1 KO MGC - 803 cells .

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(F) Expression levels of LSD1, Nanog, OCT4, SOX2, and CD44 in LSD1 KO MGC-803 cells.


( N ) Immunoprecipitation of Kme1 / 2 on SOX2 ( WT indicates HEK293T cells co - transfected with LSD1 - WT and SOX2 - WT ; Mut indicates HEK293T cells co - transfected with LSD1 - WT and SOX2 - Mut ; WT , wild type ; Mut , mutant ) .

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

(N) Immunoprecipitation of Kme1/2 on SOX2 (WT indicates HEK293T cells co-transfected with LSD1-WT and SOX2-WT; Mut indicates HEK293T cells co-transfected with LSD1-WT and SOX2-Mut; WT, wild type; Mut, mutant).


( C ) Expression levels of LSD1 , OCT4 , and SOX2 in MGC - 803 and MKN - 45 cells incubated with 20 μg / mL sEVs from MGC - 803 or LSD1 KO MGC - 803 cells for 48 h . ( D ) Quantification of the results of ( C ) ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0385 ( LSD1 ) , * P = 0 . 0160 ( OCT4 ) , and * * * P = 0 . 0006 ( SOX2 ) for MGC - 803 ; * P = 0 . 0299 ( LSD1 ) , * P = 0 . 0147 ( OCT4 ) , and * P = 0 . 0258 ( SOX2 ) for MKN - 45 ; two - tailed unpaired Student ' s t - test ) .

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

(C) Expression levels of LSD1, OCT4, and SOX2 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 48 h. (D) Quantification of the results of (C) (n = 3 biological replicates; mean ± SEM; *P = 0.0385 (LSD1), *P = 0.0160 (OCT4), and ***P = 0.0006 (SOX2) for MGC-803; *P = 0.0299 (LSD1), *P = 0.0147 (OCT4), and *P = 0.0258 (SOX2) for MKN-45; two-tailed unpaired Student's t-test).


( F ) Sphere formation assay results of MGC - 803 ( upper panel ) and MKN - 45 ( bottom panel ) cells incubated with 20 μg / mL sEVs from HEK293T cells ( left panel ) , WT - LSD1 sEVs ( middle panel ) , and LSD1 K661A sEVs ( right panel ) for 7 days . Scale bar = 100 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0021 ( MGC - 803 ) and * * P = 0 . 0020 ( MKN - 45 ) ; two - tailed unpaired Student ' s t - test ) .

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(F) Sphere formation assay results of MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from HEK293T cells (left panel), WT-LSD1 sEVs (middle panel), and LSD1 K661A sEVs (right panel) for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0021 (MGC-803) and **P = 0.0020 (MKN-45); two-tailed unpaired Student's t-test).


( F ) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay . Scale bar = 50 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0024 , * * P = 0 . 0088 , and * P = 0 . 0342 ; two - tailed unpaired Student ' s t - test ) .

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(F) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay. Scale bar = 50 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0024, **P = 0.0088, and *P = 0.0342; two-tailed unpaired Student's t-test).


( I and J ) Expression levels of LSD1 in sEVs isolated from the plasma . CD9 and CD63 were used as markers of sEVs . CD9 was used as a loading control for sEV lysis ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0256 ( patient 1 ) , * * P = 0 . 0068 ( patient 2 ) , * * P = 0 . 0013 ( patient 3 ) , * * P = 0 . 0031 ( patient 4 ) , * * * P = 0 . 0009 ( patient 5 ) , * * P = 0 . 0012 ( patient 6 ) , * * * P = 0 . 0007 ( patient 7 ) , * * * P = 0 . 0010 ( patient 8 ) , * * * P = 0 . 0001 ( patient 9 ) , and * P = 0 . 0461 ( patient 10 ) ; two - tailed unpaired Student ' s t - test ) .

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(I and J) Expression levels of LSD1 in sEVs isolated from the plasma. CD9 and CD63 were used as markers of sEVs. CD9 was used as a loading control for sEV lysis (n = 3 biological replicates; mean ± SEM; *P = 0.0256 (patient 1), **P = 0.0068 (patient 2), **P = 0.0013 (patient 3), **P = 0.0031 (patient 4), ***P = 0.0009 (patient 5), **P = 0.0012 (patient 6), ***P = 0.0007 (patient 7), ***P = 0.0010 (patient 8), ***P = 0.0001 (patient 9), and *P = 0.0461 (patient 10); two-tailed unpaired Student's t-test).


( K ) Sphere formation assay results of MGC - 803 cells treated with sEVs as indicated for 7 days . Scale bar = 100 µm ( n = 3 biological replicates ; mean ± SEM ; * * P = 0 . 0019 and * * P = 0 . 0050 ; two - tailed unpaired Student ' s t - test ) .

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(K) Sphere formation assay results of MGC-803 cells treated with sEVs as indicated for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0019 and **P = 0.0050; two-tailed unpaired Student's t-test).


Proliferation assay results of MGC - 803 ( A ) , MKN - 45 ( B ) , NCI - N87 ( C ) cells treated with oxaliplatin along with indicated treatments ( n = 3 biological replicates , mean ± standard error of mean ( SEM ) ; * P = 0 . 0204 ( MGC - 803 ) , * P = 0 . 0498 ( MKN - 45 ) , * P = 0 . 0473 ( NCI - N87 ) , and * P = 0 . 0019 ( LSD1 KO MGC - 803 ) ; two - tailed unpaired Student ' s t - test ) .

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

Proliferation assay results of MGC-803 (A), MKN-45 (B), NCI-N87 (C) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *P = 0.0204 (MGC-803), *P = 0.0498 (MKN-45), *P = 0.0473 (NCI-N87), and *P = 0.0019 (LSD1 KO MGC-803); two-tailed unpaired Student's t-test).


Representative images of tumors ( E ) and the tumor weight ( F ) of mice treated with oxaliplatin in the presence or absence of sEVs ( n = 3 biological replicates ; mean ± SEM ; * P = 0 . 0488 ; two - tailed unpaired Student ' s t - test ) .

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

Representative images of tumors (E) and the tumor weight (F) of mice treated with oxaliplatin in the presence or absence of sEVs (n = 3 biological replicates; mean ± SEM; *P = 0.0488; two-tailed unpaired Student's t-test).


( a ) Expression levels of Tcfeb mRNA in tissues isolated from 24 - h - fasted ( 24 h starved ) 6 - week - old mice . Values are expressed as fold change relative to Tcfeb expression in mice fed ad libitum ( Fed ) . Bars represent mean ± s . d . for n = 5 mice ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 .

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

(a) Expression levels of Tcfeb mRNA in tissues isolated from 24-h-fasted (24 h starved) 6-week-old mice. Values are expressed as fold change relative to Tcfeb expression in mice fed ad libitum (Fed). Bars represent mean ± s.d. for n = 5 mice; *P≤0.05; **P≤0.01; ***P≤0.001.


( b ) Representative β - gal staining of liver and kidney frozen sections isolated from fed and 24 - h - fasted heterozygous Tcfeb - β - gal mice .

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

(b) Representative β-gal staining of liver and kidney frozen sections isolated from fed and 24-h-fasted heterozygous Tcfeb- β-gal mice.


c ) Time - course expression analysis of TFEB in wild - type HeLa cells , MEFs and hepatocytes after the addition of starvation media ( time 0 ) . Data represent mean ± s . d . for n = 3 independent experiments .

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

c) Time-course expression analysis of TFEB in wild-type HeLa cells, MEFs and hepatocytes after the addition of starvation media (time 0). Data represent mean ± s.d. for n = 3 independent experiments.


( d ) Expression levels of transfected hTFEB - FLAG , Tcfeb - β - gal fusion transcript and endogenous Tcfeb mRNAs in MEFs isolated from control ( + / + ) and heterozygous Tcfeb - β - gal / + mice . Data represent mean ± s . d . for n = 3 mice ; * P ≤ 0 . 05 .

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

(d) Expression levels of transfected hTFEB-FLAG, Tcfeb- β-gal fusion transcript and endogenous Tcfeb mRNAs in MEFs isolated from control (+/+) and heterozygous Tcfeb- β-gal/+ mice. Data represent mean ± s.d. for n = 3 mice; *P≤0.05.


( e ) Time - course expression analysis of TFEB mRNA in control ( Ctrl ) or TFEB - overexpressing HeLa cells during fasting and re - feeding . Primers specific for the endogenous TFEB were used . Data represent mean ± s . d . for n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-course expression analysis of TFEB mRNA in control (Ctrl) or TFEB-overexpressing HeLa cells during fasting and re-feeding. Primers specific for the endogenous TFEB were used. Data represent mean ± s.d. for n = 3.


( f ) Time - course expression analysis of Tcfeb mRNA in heterozygous Tcfeb - β - gal / + or control MEFs during fasting and re - feeding . Specific primers for the endogenous Tcfeb were used . Data represent mean ± s . d . for n = 3 mice .

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

(f) Time-course expression analysis of Tcfeb mRNA in heterozygous Tcfeb- β-gal/+ or control MEFs during fasting and re-feeding. Specific primers for the endogenous Tcfeb were used. Data represent mean ± s.d. for n = 3 mice.


( g ) Expression analysis of Tcfeb mRNA in heterozygous Tcfeb - β - gal / + or control hepatocytes in fed and after 24 h fasting . Data represent mean ± s . d . for n = 3 mice .

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

(g) Expression analysis of Tcfeb mRNA in heterozygous Tcfeb- β-gal/+ or control hepatocytes in fed and after 24 h fasting. Data represent mean ± s.d. for n = 3 mice.


( h ) ChIP analysis from liver of mice fed ad libitum or 24 h fasted . The CLEAR elements in the first intron of Tcfeb genomic DNA are shown as numbered black rectangles as indicated in Supplementary Table S1 . Red rectangles represent exons , and the ATG indicates the first codon ( from the mouse Tcfeb isoform b ) . The histogram shows the amount of immunoprecipitated DNA as detected by quantitative PCR assay . Values were normalized to the input and plotted as relative enrichment over a mock control . Data represent mean ± s . d of 3 independent experiments .

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(h) ChIP analysis from liver of mice fed ad libitum or 24 h fasted. The CLEAR elements in the first intron of Tcfeb genomic DNA are shown as numbered black rectangles as indicated in Supplementary Table S1. Red rectangles represent exons, and the ATG indicates the first codon (from the mouse Tcfeb isoform b). The histogram shows the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Data represent mean ± s.d of 3 independent experiments.


( a ) ChIP analysis from liver of mice fed ad libitum ( fed ) or 24 h - fasted ( starved ) . CLEAR sites in the promoter region of Pgc - 1α are indicated by squares . Numbers indicate the distance ( bp ) of the binding element from the start codon . Bar graphs show the amount of immunoprecipitated DNA as detected by quantitative PCR assay . Values were normalized to the input and plotted as relative enrichment over a mock control . Bar graphs represent mean ± s . d of 3 independent experiments , * P ≤ 0 . 05 ; * * P ≤ 0 . 01 .

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(a) ChIP analysis from liver of mice fed ad libitum (fed) or 24 h-fasted (starved). CLEAR sites in the promoter region of Pgc-1α are indicated by squares. Numbers indicate the distance (bp) of the binding element from the start codon. Bar graphs show the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Bar graphs represent mean ± s.d of 3 independent experiments, *P≤0.05; **P≤0.01.


( c ) Luciferase activity was measured after transfecting increasing amounts of TFEB - FLAG in combination with Pgc - 1α WT or Pgc - 1α DEL plasmids . Bar graphs represent mean ± s . d . of n = 3independent experiments , * * * * P ≤ 0 . 0001 compared with mock transfected cells .

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(c) Luciferase activity was measured after transfecting increasing amounts of TFEB-FLAG in combination with Pgc-1α WT or Pgc-1α DEL plasmids. Bar graphs represent mean ± s.d. of n = 3independent experiments, ****P≤0.0001 compared with mock transfected cells.


( d ) Luciferase activity was measured in cells stably overexpressing TFEB cultured in normal and starved media . Bar graphs represent mean ± s . d . of n = 3independent experiments * * P ≤ 0 . 01 compared with mock transfected cells .

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

(d) Luciferase activity was measured in cells stably overexpressing TFEB cultured in normal and starved media. Bar graphs represent mean ± s.d. of n = 3independent experiments **P≤0.01 compared with mock transfected cells.


( e ) Quantification of mRNA levels of PGC1α in liver and hepatocytes from control , HDAd - TFEB and Tcfeb - LiKO ( liver KO ) mice treated as indicated . Bar graphs show mean ± s . d . for n = 4 mice . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 .

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

(e) Quantification of mRNA levels of PGC1α in liver and hepatocytes from control, HDAd-TFEB and Tcfeb-LiKO (liver KO) mice treated as indicated. Bar graphs show mean ± s.d. for n = 4 mice. *P≤0.05; **P≤0.01.


( f ) Expression analysis of TFEB target genes in liver of mice with indicated genotypes . Bar graphs show mean ± s . d . for n = 3 mice . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 .

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

(f) Expression analysis of TFEB target genes in liver of mice with indicated genotypes. Bar graphs show mean ± s.d. for n = 3 mice. *P≤0.05;**P≤0.01; ***P≤0.001.