( B ) IFN - γ release by NK cultured in presence of supernatants recovered from untreated DCs ( untreated ) or DCs treated O / N with LPS ( LPS ) . Where indicated IL - 18 was added to cultures .

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(B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures.


( C ) - contact dependent activation of NK by DCs depends on IL - 18 . Unstimulated or LPS - activated DCs NK were cocultured in same wells ( DC + NK + LPS ) or separated by a porous ( ( DC + LPS ) / NK ) . Where indicated IL - 18 was added to transwells ( ( DCs + LPS ) / ( NK + IL - 18 ) ) . NK alone were also cultured in presence of three selected cytokines ; IL - 2 IFN - β in upper chamber NK IL - 18 in lower chamber . IFN - γ in supernatant was then measured by ELISA after 8 hours of coculture . ( B , C ) . n ( number of independent experiments ) = 3 . Error bars depict .

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(C) Cell-contact dependent activation of NK cells by DCs depends on IL-18. Unstimulated or LPS-activated DCs and NK cells were cocultured in the same wells (DC + NK + LPS) or separated by a porous membrane ((DC+LPS)/NK). Where indicated IL-18 was added to the transwells ((DCs+LPS)/(NK+IL-18)). NK cells alone were also cultured in the presence of the three selected cytokines; IL-2 and IFN-β in the upper chamber and NK cells and IL-18 in the lower chamber. IFN-γ in the supernatant was then measured by ELISA after 8 hours of coculture. (B, C). n (number of independent experiments)=3. Error bars depict SEM.


A . IFT22 nucleotide - binding experiments . measurements using increasing amounts of TbIFT22 TbIFT22 / 74342 - 401 / 81397 - 450 incubated with mant - labeled GDP ( mant - GDP ) or non - hydrolysable GTP / ATP analogs ( mant - GMPPNP / mant - AMPPNP ) . is plotted as a function of . Data were fitted to a single - site binding equation for determination of dissociation constant ( Kd ) . Kd values standard deviations are calculated from three independent experiments .

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A. IFT22 nucleotide-binding experiments. Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22/74342-401/81397-450 core complex incubated with mant-labeled GDP (mant-GDP) or non-hydrolysable GTP / ATP analogs (mant-GMPPNP / mant-AMPPNP). The fluorescence intensity is plotted as a function of protein concentration. Data were fitted to a single-site binding equation for determination of the dissociation constant (Kd). Kd values and standard deviations are calculated from three independent experiments.


D . Top : Cartoon representation of IFT22 ( grey ) with positions of two nucleotide - binding mutants highlighted . GTP is shown in stick representation Mg2 + as a ball . D175 ( blue ) is unusual residue binding guanine base while S19 ( pink ) is a conserved residue required for coordination of Mg2 + cation is commonly mutated to an asparagine to prevent nucleotide binding . Bottom : Nucleotide - binding experiments of IFT22 nucleotide - binding mutants D175E , D175A S19N with fluorescently labeled nucleotides . Only S19N mutation ( pink ) abolished IFT22 nucleotide - binding ability completely .

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D. Top: Cartoon representation of IFT22 (grey) with positions of two nucleotide-binding mutants highlighted. GTP is shown in stick representation and Mg2+ as a ball. D175 (blue) is the unusual residue binding the guanine base while S19 (pink) is a conserved residue required for coordination of the Mg2+ cation and is commonly mutated to an asparagine to prevent nucleotide binding. Bottom: Nucleotide-binding experiments of IFT22 nucleotide-binding mutants D175E, D175A and S19N with fluorescently labeled nucleotides. Only the S19N mutation (light pink) abolished IFT22 nucleotide-binding ability completely.


C . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged TbIFT22 ( WT mutants ) untagged CrIFT25 / 27 / 74 / 81 . WT TbIFT22 is able to pull down Cr tetrameric , thus forming a chimeric IFT - B1 pentamer , while both A86R S19N mutant fail to bind .

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C. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged TbIFT22 (WT and mutants) and untagged CrIFT25/27/74/81. WT TbIFT22 is able to pull down the Cr tetrameric complex, thus forming a chimeric IFT-B1 pentamer, while both the A86R and S19N mutant fail to bind the complex.


A . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged IFT74342 - 401 / 81397 - 450 untagged IFT22 ( WT mutants ) . Pulldowns were done from lysates of co - expressed . Lanes 1 - 4 show similar total expression of different co - expressed constructs ( input samples ) . Lanes 5 - 8 show pulldown elutions . IFT22S19N ( inactive GTPase mutant ) IFT22A86R mutant ( IFT74 / 81 - binding mutant ) did not interact with IFT74342 - 401 / 81397 - 450 in pull - down experiment .

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A. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged IFT74342-401/81397-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT74342-401/81397-450 complex in the pull-down experiment.


B . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged IFT7479 - 401 / 811 - 450 untagged IFT22 ( WT mutants ) . Pulldowns were done from lysates of co - expressed . Lanes 1 - 4 show similar total expression of different co - expressed constructs ( input samples ) . Lanes 5 - 8 show pulldown elutions . IFT22S19N ( inactive GTPase mutant ) IFT22A86R mutant ( IFT74 / 81 - binding mutant ) did not interact with IFT7479 - 401 / 811 - 450 in pull - down experiment .

B. SDS-PAGE gel of a Ni2+-NTA pulldown using the His-tagged IFT7479-401/811-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT7479-401/811-450 complex in the pull-down experiment.


C . SDS - PAGE gel of a Ni2 + - NTA pull - down of full - IFT25 / 27 / 74 / 81 with His - tagged IFT22 ( WT S19N mutant ) . nucleotide - binding deficient mutant IFT22S19N interacts with IFT25 / 27 / 74 / 81 in pull - down experiment .

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C. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and S19N mutant). The nucleotide-binding deficient mutant IFT22S19N interacts with IFT25/27/74/81 in the pull-down experiment.


D . SDS - PAGE gel of a Ni2 + - NTA pull - down of full - IFT25 / 27 / 74 / 81 with His - tagged IFT22 ( WT A86R mutant ) . IFT74 / 81 - binding mutant IFT22A86R fails to pull down full - tetrameric .

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D. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and A86R mutant). The IFT74/81-binding mutant IFT22A86R fails to pull down the full-length tetrameric complex.


A . Western blot of IFT22RNAi + GFP : : IFT22S19N probed with anti - IFT22 antibody ( bottom ) with an anti - paraflagellar rod ( PFR ) as loading control ( top ) .

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A. Western blot analysis of the IFT22RNAi+GFP::IFT22S19N cell line probed with the anti-IFT22 antibody (bottom) and with an anti-paraflagellar rod (PFR) as loading control (top).


B . IFA of indicated trypanosome lines using mAb25 ( marker for axoneme , left panels ) an anti - IFT172 antibody ( marker for IFT , right panels ) . top panels show phase contrast merged with DAPI ( cyan ) that stains mitochondrial . Scale bars correspond to 5 µm .

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B. IFA of the indicated trypanosome cell lines using the mAb25 (marker for the axoneme, left panels) and an anti-IFT172 antibody (marker for IFT, right panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. Scale bars correspond to 5 µm.


C . Kymographs showing movement of GFP : : IFT22S19N in presence ( left ) or absence ( right ) of IFT22 endogenous . Note improved signal - to - noise in absence of endogenous IFT22 . P D mark proximal distal ends of flagellum , respectively . Scale bars are 5 seconds ( time , vertical ) 5 µm ( , horizontal )

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C. Kymographs showing the movement of the GFP::IFT22S19N in the presence (left) or the absence (right) of the IFT22 endogenous protein. Note the improved signal-to-noise ratio in the absence of endogenous IFT22. P and D mark the proximal and distal ends of the flagellum, respectively. Scale bars are 5 seconds (time, vertical) and 5 µm (length, horizontal)


D . Dot plot representation of flagellum in indicated lines conditions . For each condition , 100 flagella were measured statistical was performed with ANOVA . Significant differences are indicated with a star ( P < 0 .

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D. Dot plot representation of flagellum length in the indicated cell lines and conditions. For each condition, 100 flagella were measured and statistical analysis was performed with the ANOVA test. Significant differences are indicated with a star (P<0.0001


A . Western blot of IFT22RNAi + GFP : : IFT22A86R probed with anti - IFT22 antibody ( bottom ) with an anti - BiP as loading control ( top ) .

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A. Western blot analysis of the IFT22RNAi+GFP::IFT22A86R cell line probed with the anti-IFT22 antibody (bottom) and with an anti-BiP as loading control (top).


B . IFA with indicated lines using mAb25 ( marker for axoneme , central panels ) an anti - IFT172 antibody ( marker for IFT , bottom panels ) . top panels show phase contrast merged with DAPI ( cyan ) that stains mitochondrial . arrowheads indicate presence of short flagella stained with Mab25 antibody in IFT22RNAi . Scale bars correspond to 5 µm .

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B. IFA with the indicated cell lines using the mAb25 (marker for the axoneme, central panels) and an anti-IFT172 antibody (marker for IFT, bottom panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. The arrowheads indicate the presence of short flagella stained with the Mab25 antibody in the IFT22RNAi cells. Scale bars correspond to 5 µm.


C . Sections of IFT22RNAi + GFP : : IFT22rescue ( top panels ) or IFT22RNAi + GFP : : IFT22A86R ( bottom panels ) were analysed by transmission electron microscopy . Sections through flagellar pocket , transition zone flagellum are shown . Scale bars are 500 nm ( flagellar pocket sections ) or 200 nm ( transition zone flagellum sections ) . arrow indicates an endocytic vesicle budding off flagellar pocket whereas black one points at an IFT train . ( Axo = axoneme , TZ = transition zone , BB = basal )

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C. Sections of IFT22RNAi+GFP::IFT22rescue (top panels) or IFT22RNAi+GFP::IFT22A86R cells (bottom panels) were analysed by transmission electron microscopy. Sections through the flagellar pocket, the transition zone and the flagellum are shown. Scale bars are 500 nm (flagellar pocket sections) or 200 nm (transition zone and flagellum sections). The white arrow indicates an endocytic vesicle budding off the flagellar pocket whereas the black one points at an IFT train. (Axo = axoneme, TZ = transition zone, BB = basal body)


E . Naïve MCF7 MCF7aTT were exposed to indicated stress conditions for 3 h , viability was assessed by dead with trypan blue . bar graphs represent of trypan blue excluding . ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with two - stage linear step - up procedure of Benjamini , Krieger Yekutieli ) .

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E. Naïve MCF7 cells and MCF7aTT cells were exposed to the indicated stress conditions for 3 h, and cell viability was assessed by staining dead cells with trypan blue. The bar graphs represent the percentage of trypan blue excluding cells. (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).


A . Whole lysates of MCF7 MCF7aTT exposed to heat shock / or proteasome ( MG132 , 20 μM , 1h ) HSP70 ( VER - 155008 , 50 μM , 1h ) inhibitors were prepared as described in Materials Methods . Total lysate , soluble fractions insoluble fractions were stained by SYPRO Ruby ( left ) quantified ( right ) . ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with two - stage linear step - up procedure of Benjamini , Krieger Yekutieli ) .

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A. Whole lysates of MCF7 and MCF7aTT cells exposed to heat shock and/or proteasome (MG132, 20 μM, 1h) and HSP70 (VER-155008, 50 μM, 1h) inhibitors were prepared as described in Materials and Methods. Total lysate, soluble fractions and insoluble fractions were stained by SYPRO Ruby (left) and quantified (right). (means +/- SD, n = 3, numbers above the bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).


C . soluble insoluble fractions shown in ( A ) were assayed for level of K48 - linked polyubiquitin , polyubiquitylated were quantified ( blue graph , means + / - , n = 4 , numbers above bars indicate p values ) . data for bulk obtained in ( A ) were overlaid onto graph for comparison ( red , means + / - , n = 3 ) .

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C. The soluble and insoluble fractions shown in (A) were assayed for the level of K48-linked polyubiquitin, and polyubiquitylated proteins were quantified (blue line graph, means +/- SEM, n = 4, numbers above the bars indicate p values). The data for bulk protein obtained in (A) were overlaid onto the graph for comparison (red line, means +/- SEM, n = 3).


B . same experiment as described in ( A ) but with MCF7 exposed to MG132 ( 20 μM , 1 h ) or puromycin ( 100 μg / ml , 10 min ) . Means + / - , n = 2 . C . Lysate from MCF7 exposed to MG132 ( 20 μM , 1 h ) or puromycin ( 100 μg / ml , 10 min ) as indicated were subjected to immunoprecipitation with anti - puromycin antibodies , followed by assaying for co - precipitation of K48 - ubiquitylated . Actin is shown as a reference .

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B. The same experiment as described in (A) but with MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min). Means +/- SEM, n = 2. C. Lysate from MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min) as indicated were subjected to immunoprecipitation with anti-puromycin antibodies, followed by assaying for co-precipitation of K48-ubiquitylated proteins. Actin is shown as a reference.


B . Same experiment as in ( A ) with thermotolerant MCF7aTT incubated with HSP70 inhibitor VER - 155008 ( 50 μM , 1h ) or MG132 ( 20 μM , 1h ) . signal obtained for ribosomal RPS19 RPL7 are shown for reference , documenting residual polysomes despite flat UV trace . Right panels are quantifications of K48 - ub ( means + / - , n = 2 ) , black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity . bar graphs represent P / M ratios ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with two - stage linear step - up procedure of Benjamini , Krieger Yekutieli ) . C . Sucrose density gradient fractions described in ( B ) were assayed for partitioning of HSPA1 HSPH1 by immunoblotting . HSPA1 data were quantified in a graph ( means + / - , n = 4 ) . D

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

B. Same experiment as in (A) with thermotolerant MCF7aTT cells incubated with the HSP70 inhibitor VER-155008 (50 μM, 1h) or MG132 (20 μM, 1h). The signal obtained for ribosomal protein RPS19 and RPL7 are shown for reference, documenting residual polysomes despite the flat UV trace. Right panels are quantifications of K48-ub (means +/- SEM, n = 2), black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity. The bar graphs represent the P/M ratios (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli). C. Sucrose density gradient fractions described in (B) were assayed for partitioning of HSPA1 and HSPH1 by immunoblotting. HSPA1 data were quantified in a line graph (means +/- SEM, n = 4). D


D . lysate was spun through a 30 % sucrose cushion to pellet polysomes . Nascent were released from polysomes by dissociating 80S ribosomes with EDTA ( see Appendix Figures S3 ) . Soluble insoluble fractions were prepared by centrifugation assayed for level of K48 - linked polyubiquitin . of K48 - linked polyubiquitin were quantified ( means + / - SD , n = 3 ) . Immunoblots within black squares were run on same gel but irrelevant lanes were removed as indicated by lines .

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D. Cell lysate was spun through a 30% sucrose cushion to pellet polysomes. Nascent proteins were released from polysomes by dissociating 80S ribosomes with EDTA (see Appendix Figures S3). Soluble and insoluble fractions were prepared by centrifugation and assayed for the level of K48-linked polyubiquitin. The levels of K48-linked polyubiquitin were quantified (means +/- SD, n = 3). Immunoblots within black squares were run on the same gel but irrelevant lanes were removed as indicated by white lines.


D . Total lysates were prepared from naïve MCF7 thermoresistant MCF7aTT , puromycin labeled peptides were pull down as illustrated in scheme . Precipitates were assayed for presence of K48 - linked polyubiquitin , chaperones , proteasome subunits by immunoblotting .

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D. Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells, and puromycin labeled peptides were pull down as illustrated in the scheme. Precipitates were assayed for the presence of K48-linked polyubiquitin, chaperones, and proteasome subunits by immunoblotting.


E . Whole lysates of KYSE150 KYSE150HSPH1 - / - exposed to indicated conditions ( aTT scheme heat shock ) were fractionated into soluble insoluble fractions as described in Materials Methods . Insoluble fractions were assayed for level of K48 - linked polyubiquitin ( left panel ) . Total of HSPA1 HSPH1 are shown for reference . K48 ubiquitin signals were quantified displayed in a bar graph ( means + / - SD , n = 4 ) .

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E. Whole lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were fractionated into soluble and insoluble fractions as described in Materials and Methods. Insoluble fractions were assayed for the level of K48-linked polyubiquitin (left panel). Total levels of HSPA1 and HSPH1 are shown for reference. K48 ubiquitin signals were quantified and displayed in a bar graph (means +/- SD, n = 4).


F . Lysates of KYSE150 KYSE150HSPH1 - / - exposed to indicated conditions ( aTT scheme heat shock ) were obtained polysomes were isolated by centrifugation through a 30 % sucrose cushion . Whole lysates ( left panel ) polysomal pellets ( right panel ) were assayed by immunoblotting for of HSPH1 , HSPA1 proteasome subunits .

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F. Lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were obtained and polysomes were isolated by centrifugation through a 30% sucrose cushion. Whole cell lysates (left panel) and the polysomal pellets (right panel) were assayed by immunoblotting for the levels of HSPH1, HSPA1 and proteasome subunits.


A . Representative of tissue sections derived from oral squamous carcinomas ( OSCC ) esophageal squamous carcinomas ( ESCC ) stained with indicated antibodies by immunohistochemistry ( ) . Top panels show routine H & E . Black squares point to regions magnified in adjacent panels to right .

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A. Representative micrographs of tissue sections derived from oral squamous cell carcinomas (OSCC) and esophageal squamous cell carcinomas (ESCC) stained with the indicated antibodies by immunohistochemistry (IHC). Top panels show routine H&E staining. Black squares point to regions magnified in the adjacent panels to the right.


C . Kaplan - Meier plots showing correlation between HSPH1 mRNA expression level survival of with ESCC . expression data was obtained from visualized with KM Plotter ( www . kmplot . com , ( Nagy et al , 2018 ) using Pan Cancer algorithm at default settings .

C. Kaplan-Meier plots showing the correlation between HSPH1 mRNA expression level and the survival of patients with ESCC. The expression data was obtained from and visualized with KM Plotter (www.kmplot.com, (Nagy et al, 2018) using the Pan Cancer algorithm at default settings.


D . KYSE150 KYSE150HSPH1 - / - were injected into nude mice , xenograft growth was monitored over time ( left panel ; mean volume + / - , n = 4 with 5 per n ) . Mean weights at end of experiments were determined ( means + / - SD , n = 4 with 5 per n , number above bar graph represent p values , * p < 0 . 05 , * * p < 0 . 01 ( calculated with two - stage linear step - up procedure of Benjamini , Krieger Yekutieli ) ) .

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D. KYSE150 and KYSE150HSPH1-/- cells were injected into nude mice, and xenograft tumor growth was monitored over time (left panel; mean tumor volume +/- SEM, n = 4 with 5 animals per n). Mean tumor weights at the end of the experiments were determined (means +/- SD, n = 4 with 5 animals per n, number above the bar graph represent p values, * p<0.05, **p<0.01 (calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli)).


( A ) Representative of WT , Mfn1 KO Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria - targeted RFP ( mt - RFP ) . Scale bar : 5 μm .

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(A) Representative images of WT, Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria-targeted RFP (mt-RFP). Scale bar: 5 μm.


( A ) Representative of WT Mfn2 KO MEFs co - transfected with plasmids encoding mitochondria - targeted RFP ER - targeted GFP . Scale bar : 5 μm .

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(A) Representative images of WT and Mfn2 KO MEFs co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP. Scale bar: 5 μm.


( B ) After 48 h , were fixed degree of colocalization between ER mitochondria on red green pixel overlap was analyzed using Mander ' s coefficient ( n = 30 analyzed in 3 independent experiments ) . Data are presented as mean ± . * p < 0 . 05 , two - Student ' s t - .

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(B) After 48 h, the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander's coefficient (n= 30 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test.


I - K ) Mitochondrial Ca2 + was determined in WT Mfn2 KO MEFs loaded with Rhod2 . effect of ( I ) caffeine - induced ( 20 mM ) , ( J ) IP3R activation with ATP - induced ( 100 μM ) ( K ) histamine - induced ( 100 μM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 30 from 3 independent experiments ) . Data are presented as mean ± .

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I-K) Mitochondrial Ca2+ was determined in WT and Mfn2 KO MEFs loaded with Rhod2. The effect of (I) caffeine-induced (20 mM), (J) IP3R activation with ATP-induced (100 μM) and (K) histamine-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.


WT Mfn2 KO were transfected with plasmids encoding : GFP indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( R ) MMP was analyzed ( n = 90 analyzed in 3 independent experiments ) .

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WT and Mfn2 KO cells were transfected with plasmids encoding: GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (R) MMP was analyzed (n= 90 cells analyzed in 3 independent experiments).


WT Mfn2 KO were transfected with plasmids encoding GFP indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( S ) ATP of GFP + was analyzed after 24 h treatment with 2DG ( 10 mM ) ( n = 3 independent experiments ) .

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WT and Mfn2 KO cells were transfected with plasmids encoding GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (S) ATP levels of GFP+ cells was analyzed after 24 h treatment with 2DG (10 mM) (n= 3 independent experiments).


WT Mfn2 KO MEFs were co - transfected with plasmids encoding : mt - RFP ER - Mfn2 or control ( CT ) plasmid . After 48 h , were fixed . ( C ) Representative . Scale bar = 5 μm .

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WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: mt-RFP and ER-Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (C) Representative images. Scale bar= 5 μm.


WT Mfn2 KO MEFs were co - transfected with plasmids encoding : J - N ) GFP ER - Mfn2 or control ( CT ) plasmid . ( J ) MMP was determined . values were normalized to surrounding untransfected . ( n = 90 analyzed in 3 independent experiments ) . ( K ) After 24 h , transfected were sorted plated for another 24 h , after which were treated with 2 - DG ( 10 mM ) for 6 h , ATP were measured ( n = 3 independent experiments ) . ( L ) Oxygen consumption was measured ( M ) RCR ( N ) SRC was calculated ( n = 4 independent experiments ) . Data are presented as mean ± . Data information : * p < 0 . 05 , one - way ANOVA followed by Tukey ' s post hoc . Note that experiments were performed at same time , so values of WT Mfn2 KO transfected with control plasmid are same but figures have been split in two for sake of linearity .

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WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: J-N) GFP and ER-Mfn2 or control (CT) plasmid. (J) MMP was determined. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (K) After 24 h, the transfected cells were sorted and plated for another 24 h, after which cells were treated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). (L) Oxygen consumption was measured and (M) RCR and (N) SRC was calculated (n= 4 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT Mfn1 / 2 double KO were transfected with : A ) mt - RFP , ER - GFP plasmids expressing indicated . Mitochondria - ER colocalization was analyzed using Mander ' s coefficient ( n = 15 analyzed in 3 independent experiments ) . Data are presented as mean ± . B ) GFP ERMITO - RLuc indicated expression vectors . After 24 h , transfected were sorted plated for another 24 h , after which RLuc was analyzed ( n = 3 independent experiments ) . Data are presented as mean ± . C ) Data information : red dashed indicates WT results . * p < 0 . 05 versus Mfn1 / 2 DKO , one - way ANOVA followed by Tukey ' s post hoc . Note that experiments were performed at same time , so values of WT Mfn2 KO transfected with control plasmid are same but figures have been split in two for sake of linearity .

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WT and Mfn1/2 double KO were transfected with: A) mt-RFP, ER-GFP and plasmids expressing the indicated proteins. Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. B) GFP and ERMITO-RLuc and indicated expression vectors. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 3 independent experiments). Data are presented as mean ± SEM. C) Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT Mfn1 / 2 double KO were transfected with : C ) GFP indicated plasmids . After 48 h , mitochondrial Ca2 + was determined ( n = 30 from 3 independent experiments ) . Data are presented as mean ± . Data information : red dashed indicates WT results . * p < 0 . 05 versus Mfn1 / 2 DKO , one - way ANOVA followed by Tukey ' s post hoc . Note that experiments were performed at same time , so values of WT Mfn2 KO transfected with control plasmid are same but figures have been split in two for sake of linearity .

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WT and Mfn1/2 double KO were transfected with: C) GFP and the indicated plasmids. After 48 h, mitochondrial Ca2+ was determined (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT Mfn1 / 2 double KO were transfected with : D ) LAR - GECO1 indicated plasmids . After 48 h ER Ca2 + was released with caffeine ( 20 mM ) as indicated ( n = 25 - 27 from 3 independent experiments ) . Data are presented as mean ± . Data information : red dashed indicates WT results . * p < 0 . 05 versus Mfn1 / 2 DKO , one - way ANOVA followed by Tukey ' s post hoc . Note that experiments were performed at same time , so values of WT Mfn2 KO transfected with control plasmid are same but figures have been split in two for sake of linearity .

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WT and Mfn1/2 double KO were transfected with: D) LAR-GECO1 and the indicated plasmids. After 48 h ER Ca2+ was released with caffeine (20 mM) as indicated (n= 25-27 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT Mfn1 / 2 double KO were transfected with : E ) GFP indicated plasmid . After 48 h , effect of caffeine - induced ( 20 mM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 25 from 3 independent experiments ) . Data are presented as mean ± .

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WT and Mfn1/2 double KO were transfected with: E) GFP and the indicated plasmid. After 48 h, the effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 25 cells from 3 independent experiments). Data are presented as mean ± SEM.


B ) Representative Western blots of indicated during neuronal maturation in vitro ( n = 3 independent experiments ) . C ) Representative Western blots of indicated of WT Mfn2 KO neurons at DIV7 ( n = 3 independent experiments ) . D

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B) Representative Western blots of the indicated proteins during the neuronal maturation in vitro (n= 3 independent experiments). C) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 (n= 3 independent experiments). D


D ) neurons of tamoxifen - inducible Mfn2 KO mice were transfected at DIV3 with ER - GFP , mt - RFP plus indicated plasmids . Tamoxifen - treated ( for 72 h ) vehicle - treated neurons ( WT ; red dashed ) were fixed mitochondria - ER co - localization was analyzed using Mander ' s coefficient ( n = 15 neurons analyzed in 3 independent experiments ) . Data are presented as mean ± . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc . Scale bar : 500 μm .

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D) Primary neurons of tamoxifen-inducible Mfn2 KO mice were transfected at DIV3 with ER-GFP, mt-RFP plus the indicated plasmids. Tamoxifen-treated (for 72 h) and vehicle-treated neurons (WT; red dashed line) were fixed and mitochondria-ER co-localization was analyzed using Mander's coefficient (n= 15 neurons analyzed in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm.


( F ) neurite measurement representative tracings ( G ) of cortical neurons of WT Mfn2 KO mice co - transfected at DIV4 with GFP indicated plasmids . After 72 h , neurons were fixed , immunofluorescence with anti - GFP antibodies was performed neurite was measured ( n = 30 neurons from 3 independent experiments ) . Data are presented as mean ± . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc . Scale bar : 500 μm . E

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(F) neurite length measurement and representative tracings (G) of primary cortical neurons of WT and Mfn2 KO mice co-transfected at DIV4 with GFP and the indicated plasmids. After 72 h, the neurons were fixed, immunofluorescence with anti-GFP antibodies was performed and neurite length was measured (n= 30 neurons from 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm. E


( a ) Representative differential interference contrast microscopy of midgut from wild - type at early third instar larval ( Early 3rd ) , late third instar larval ( Late 3rd ) at puparium formation ( prepupal , WPP ) stages .

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(a) Representative differential interference contrast microscopy images of midgut cells from wild-type animals at the early third instar larval (Early 3rd), late third instar larval (Late 3rd) and at puparium formation (white prepupal, WPP) stages.


( b ) Autophagy detected by formation of mCherry - Atg8a punctate spots in midgut from wild - type at indicated stages . Representative are shown .

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(b) Autophagy detected by formation of mCherry-Atg8a punctate spots in midgut cells from wild-type animals at indicated stages. Representative images are shown.


( c ) Midguts from control Atg18KG03090 / wild - type ( Atg18 / + ) , n = 14 , Atg18KG03090 / Df ( 3L ) 6112 mutant ( Atg18 / Df ) , n = 11 , at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( d ) Wild - type , control ( Atg18KG03090 / + ) Atg18 mutant ( Atg18 / Df ) midgut ( μm2 ) at indicated stages ; n = 10 intestines per genotype with 5 measured per intestine per stage .

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(c) Midguts from control Atg18KG03090/wild-type (Atg18/+), n = 14, and Atg18KG03090/Df(3L)6112 mutant (Atg18/Df), n = 11, animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (d) Wild-type, control (Atg18KG03090/+) and Atg18 mutant (Atg18/Df) midgut cell size quantification (μm2) at indicated stages; n = 10 animal intestines per genotype with 5 cells measured per intestine per stage.


( e ) Differential interference contrast of midgut from Atg2EP3697 / wild - type control ( Atg2 / + ) Atg2EP3697 / Df ( 3L ) 6091 mutant ( Atg2 / Df ) at puparium formation . Representative are shown . ( f ) ( μm2 ) from e , n = 12 ( Atg2 / + ) n = 7 ( Atg2 / Df ) intestines per genotype with 5 measured per intestine .

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(e) Differential interference contrast images of midgut cells from Atg2EP3697/wild-type control (Atg2/+) and Atg2EP3697/Df(3L)6091 mutant (Atg2/Df) animals at puparium formation. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 12 (Atg2/+) and n = 7 (Atg2/Df) animal intestines per genotype with 5 cells measured per intestine.


( g - j ) Representative of intestine 2 h after puparium formation . ( g , i ) Control Atg18KG03090 / wild - type ( Atg18 / + , g ) control Atg2EP3697 / wild - type ( Atg2 / + , i ) with an enlarged image showing a double - structure ( arrowhead ) that surrounds a mitochondrion endoplasmic reticulum . Arrows indicate autolysosomes . ( h , j ) Atg18KG03090 / Df ( 3L ) 6112mutant ( Atg18 / Df , h ) Atg2EP3697 / Df ( Atg2 / Df , j ) mutant lacking autophagic structures . is shown as mean ± s . d . Scale bars , 20 μm ( a - c e ) 1 μm ( g - j ) .

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(g-j) Representative TEM images of intestine cells 2 h after puparium formation. (g,i) Control Atg18KG03090/wild-type (Atg18/+, g) and control Atg2EP3697/wild-type (Atg2/+, i) cells with an enlarged image showing a double-membrane structure (arrowhead) that surrounds a mitochondrion and endoplasmic reticulum. Arrows indicate autolysosomes. (h,j) Atg18KG03090/Df(3L)6112mutant (Atg18/Df, h) and Atg2EP3697/Df (Atg2/Df, j) mutant cells lacking autophagic structures. Quantification is shown as mean ± s.d. Scale bars, 20 μm (a-c and e) and 1 μm (g-j).


( a ) Midguts dissected from expressing Atg18IR specifically in DsRed - marked clones of at puparium formation analysed by differential interference contrast ( DIC ) microscopy . Representative are shown . ( b ) ( μm2 ) from a , n = 12 intestines per genotype with 1 - 5 measured per intestine .

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(a) Midguts dissected from animals expressing Atg18IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Quantification (μm2) from a, n = 12 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts expressing mCherry - Atg8a in , expressing Atg18IR specifically in GFP - marked clones dissected from at 2 h after puparium formation . Representative are shown .

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(c) Midguts expressing mCherry-Atg8a in all cells, and expressing Atg18IR specifically in GFP-marked cell clones dissected from animals at 2 h after puparium formation. Representative images are shown.


( d ) Midguts dissected from at puparium formation that contain an Atg1Δ3D loss - of - function mutant clone ( lacking GFP ) analysed by differential interference contrast microscopy . Wild - type ( + / + ) control possesses stronger GFP heterozygous Atg1Δ3D / wild - type ( Δ3D / + ) have weaker GFP . Representative are shown . ( e ) ( μm2 ) from d , n = 7 ( + / + ) , n = 8 ( Δ3D / + ) n = 8 ( Δ3D / Δ3D ) intestines per genotype with 1 - 5 measured per intestine .

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(d) Midguts dissected from animals at puparium formation that contain an Atg1Δ3D loss-of-function mutant cell clone (lacking GFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cell possesses stronger GFP and heterozygous Atg1Δ3D/wild-type (Δ3D/+) cells have weaker GFP. Representative images are shown. (e) Quantification (μm2) from d, n = 7 (+/+), n = 8 (Δ3D/+) and n = 8 (Δ3D/Δ3D) animal intestines per genotype with 1-5 cells measured per intestine.


( f ) Midguts dissected from expressing mCherry - Atg8a in , expressing Atg1IR specifically in GFP - marked clones of at 2 h after puparium formation . Representative are shown .

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(f) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing Atg1IR specifically in GFP-marked clones of cells at 2 h after puparium formation. Representative images are shown.


( g - j ) Representative cut views from three - dimensional reconstitution from wild - type ( g , h ) Atg1 knockdown ( i , j ) at early third instar larval stage ( g , i ) at puparium formation ( h , j ) . ( k ) of volume ( μm3 ) from g - j , n = 5 intestines per genotype with 1 - 5 measured per intestine per stage . Black columns : early third instar larval stage . columns : puparium formation stage . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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(g-j) Representative cut views from three-dimensional reconstitution images from wild-type (g,h) and Atg1 knockdown (i,j) cells at early third instar larval stage (g,i) and at puparium formation (h,j). (k) Quantification of cell volume (μm3) from g-j, n = 5 animal intestines per genotype with 1-5 cells measured per intestine per stage. Black columns: early third instar larval stage. White columns: puparium formation stage. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


( a ) Midguts dissected from at puparium formation that contain Vps34Δm22 loss - of - function mutant clones ( lacking GFP ) analysed by differential interference contrast ( DIC ) microscopy . Wild - type ( + / + ) control possess stronger GFP heterozygous Vps34Δm22 / wild - type ( Δm22 / + ) have weaker GFP . Representative are shown . ( b ) ( μm2 ) from a , n = 14 intestines per genotype with 1 - 5 measured per intestine .

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(a) Midguts dissected from animals at puparium formation that contain Vps34Δm22 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Vps34Δm22/wild-type (Δm22/+) cells have weaker GFP. Representative images are shown. (b) Quantification (μm2) from a, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts dissected from expressing Atg8aIR specifically in GFP - marked clones of at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( d ) ( μm2 ) from c , n = 10 intestines per genotype with 1 - 5 measured per intestine .

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(c) Midguts dissected from animals expressing Atg8aIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (d) Cell size quantification (μm2) from c, n = 10 animal intestines per genotype with 1-5 cells measured per intestine.


( e ) Midguts dissected from expressing Atg12IR specifically in DsRed - marked clones of at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( f ) ( μm2 ) from e , n = 14 intestines per genotype with 1 - 5 measured per intestine .

(e) Midguts dissected from animals expressing Atg12IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.


( g - i ) Midguts dissected from early third instar larvae that mis - express Atg1GS10797 ( Atg1 ; g , GFP in nucleus cytoplasm ) , both Atg1 + Atg8aIR ( h ) , or both Atg1 + Atg7IR ( i ) stained to detect Discs large ( green ) on cortex of . Representative are shown . ( g ' i ' ) DIC of midguts expressing mCherry - Atg8a in , Atg1 ( g ' ) , Atg1 + Atg7IR ( i ' ) in GFP - marked clones of . mCherry - Atg8a are shown in insets . Representative are shown . is shown as mean ± s . d . Scale bars , 20 μm .

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(g-i) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1; g, GFP in nucleus and cytoplasm), both Atg1 +Atg8aIR (h), or both Atg1 +Atg7IR (i) and stained to detect Discs large (green) on the cortex of all cells. Representative images are shown. (g' and i') DIC images of midguts expressing mCherry-Atg8a in all cells, Atg1 (g'), and Atg1+Atg7IR (i') in GFP-marked clones of cells. mCherry-Atg8a puncta are shown in insets. Representative images are shown. Quantification is shown as mean ± s.d. Scale bars, 20 μm.


( a ) Midguts dissected from at puparium formation that contain Atg7d4 loss - of - function mutant clones ( lacking GFP ) analysed by differential interference contrast ( DIC ) microscopy . Wild - type ( + / + ) control possess stronger GFP heterozygous Atg7d4 / wild - type ( d4 / + ) have weaker GFP . Representative are shown . ( b ) ( μm2 ) from a , n = 8 intestines per genotype with 1 - 5 measured per intestine .

(a) Midguts dissected from animals at puparium formation that contain Atg7d4 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Atg7d4/wild-type (d4/+) cells have weaker GFP. Representative images are shown. (b) Cell size quantification (μm2) from a, n = 8 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts expressing mCherry - Atg8a in , with Atg7d4 loss - of - function clone ( lacking GFP ) at 2 h after puparium formation . Representative are shown .

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(c) Midguts expressing mCherry-Atg8a in all cells, and with Atg7d4 loss-of-function clone cells (lacking GFP) at 2 h after puparium formation. Representative images are shown.


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( f ) Midguts expressing GFP - Atg5 in enterocytes ( larger nuclei ) with an Atg7d4 loss - of - function clone ( lacking RFP ) at puparium formation . Representative are shown .

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(f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei) with an Atg7d4 loss-of-function clone cell (lacking RFP) at puparium formation. Representative images are shown.


( g , h ) Representative of intestine 2 h after puparium formation . Arrows indicate autolysosomes . ( g , h ) Control ( Atg7d30 / d14 ; g ) Atg7 mutant ( Atg7d77 / d14 ; h ) both contain autophagic structures . ( h , right ) Enlarged Atg7 mutant image of a double - autophagosome ( arrowhead ) surrounding a mitochondrion . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm ( a , c , d f ) 1 μm ( g , h ) .

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(g,h) Representative TEM images of intestine cells 2 h after puparium formation. Arrows indicate autolysosomes. (g,h) Control (Atg7d30/d14; g) and Atg7 mutant (Atg7d77/d14; h) cells both contain autophagic structures. (h, right) Enlarged Atg7 mutant cell image of a double-membrane autophagosome (arrowhead) surrounding a mitochondrion. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm (a,c,d and f) and 1 μm (g,h).


( a ) of mitochondria numbers from Atg18 control ( Atg18KG03090 / wild type ) mutant ( Atg18KG03090 / Df ( 3L ) 6112 ) or Atg7 control ( Atg7d30 / d14 ) mutant ( Atg7d77 / d14 ) . Mitochondria were quantified from 2 distinct 25 μm2 regions per from 2 per from at least 3 different per genotype .

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(a) Quantification of mitochondria numbers from Atg18 control (Atg18KG03090/wild type) and mutant (Atg18KG03090/Df(3L)6112) or Atg7 control (Atg7d30/d14) and mutant (Atg7d77/d14) TEM images. Mitochondria were quantified from 2 distinct 25 μm2 regions per cell from 2 cells per animal from at least 3 different animals per genotype.


( b , c ) Midguts dissected from either early third instar larvae ( b ) or at puparium formation ( c ) that express GFP - labelled mitochondria in contain an Atg1Δ3D loss - of - function mutant clone ( lacking RFP ) . Wild - type control possess stronger RFP heterozygous have weaker RFP . Representative are shown . is shown as mean ± s . d . NS , not significant . Scale bars , 0 μm .

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(b,c) Midguts dissected from either early third instar larvae (b) or at puparium formation (c) that express GFP-labelled mitochondria in all cells and contain an Atg1Δ3D loss-of-function mutant cell clone (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 0 μm.


( a ) Midguts dissected from expressing Uba1IR specifically in GFP - marked clones of at puparium formation analysed by differential interference contrast ( DIC ) microscopy . Representative are shown . ( b ) ( μm2 ) from a ; n = 16 intestines per genotype with 1 - 5 measured per intestine .

(a) Midguts dissected from animals expressing Uba1IR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Cell size quantification (μm2) from a; n = 16 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function mutant clones ( lacking RFP ) analysed by differential interference contrast microscopy . Wild - type ( + / + ) control possess stronger RFP heterozygous Uba1H33 / wild - type ( H33 / + ) have weaker RFP . Representative are shown . ( d ) ( μm2 ) from c , n = 8 ( + / + ) , n = 11 ( H33 / + ) n = 11 ( H33 / H33 ) intestines per genotype with 1 - 5 measured per intestine .

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(c) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function mutant cell clones (lacking RFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cells possess stronger RFP and heterozygous Uba1H33/wild-type (H33/+) cells have weaker RFP. Representative images are shown. (d) Quantification (μm2) from c, n = 8 (+/+), n = 11 (H33/+) and n = 11 (H33/H33) animal intestines per genotype with 1-5 cells measured per intestine.


( e ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function MARCM mutant clones ( GFP - positive ) that also have mCherry - Atg8a expressed in analysed by microscopy . Control wild - type heterozygous have no GFP . Representative are shown .

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(e) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that also have mCherry-Atg8a expressed in all cells and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown.


( f ) Midguts expressing GFP - Atg5 in enterocytes ( larger nuclei ) , with an Uba1H33 loss - of - function clone ( lacking RFP ) at puparium formation analysed by microscopy . Representative are shown .

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(f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei), and with an Uba1H33 loss-of-function clone cell (lacking RFP) at puparium formation analysed by fluorescence microscopy. Representative images are shown.


( g ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function MARCM mutant clones ( GFP - positive ) that are stained with p62 antibody analysed by microscopy . Control wild - type heterozygous have no GFP . Representative are shown .

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(g) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that are stained with p62 antibody and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown.


( h ) Midguts dissected from expressing Dts7 , a dominant - sensitive mutant of β2 subunit of proteasome , specifically in GFP - marked at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( i ) ( μm2 ) from h , n = 6 intestines per genotype with 1 - 5 measured per intestine . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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(h) Midguts dissected from animals expressing Dts7, a dominant temperature-sensitive mutant of the β2 subunit of the proteasome, specifically in GFP-marked cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (i) Cell size quantification (μm2) from h, n = 6 animal intestines per genotype with 1-5 cells measured per intestine. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


( a , b ) E1 - charging for Uba1 Atg7 with either Atg8a ( a ) or ubiquitin ( Ub ; b ) , n = 2 experiments with independently isolated analyses . Flag - tagged baculoviral - expressed E1s were incubated with either His - tagged Atg8a or Ub in presence or absence of β - mercaptoethanol , separated by electrophoresis blotted . Band shift was detected with anti - Flag antibody . Relative molecular mass ( Mr K ) ladders are indicated . Representative are shown . Relative molecular masses : Flag - Uba1 , 135K ; Flag - Atg7 , 81K ; His - Atg8a , 29K ; Ub , 9K .

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(a,b) E1-charging assay for Uba1 and Atg7 with either Atg8a (a) or ubiquitin (Ub; b), n = 2 experiments with independently isolated proteins and analyses. Flag-tagged baculoviral-expressed E1s were incubated with either His-tagged Atg8a or Ub in the presence or absence of β-mercaptoethanol, separated by electrophoresis and blotted. Band shift was detected with anti-Flag antibody. Relative molecular mass (Mr K) ladders are indicated. Representative images are shown. Relative molecular masses: Flag-Uba1, 135K; Flag-Atg7, 81K; His-Atg8a, 29K; Ub, 9K.


( c ) Midgut extracts at puparium formation from wild - type , Atg7 mutant ( Atg7d77 / d14 ) Atg8 ( Atg8KG07569 ) mutant blotted with anti - Atg8 anti - Tubulin , n = 3 independent biological experiments . Representative are shown .

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(c) Midgut protein extracts at puparium formation from wild-type, Atg7 mutant (Atg7d77/d14) and Atg8 (Atg8KG07569) mutant animals blotted with anti-Atg8 and anti-Tubulin, n = 3 independent biological experiments. Representative images are shown.


( d ) Midguts from control Atg310 / wild - type ( Atg3 / + ) Atg310 / Df ( 3L ) cat mutant ( Atg3 / Df ) at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( e ) ( μm2 ) from d , n = 11 ( Atg3 / + ) n = 9 ( Atg3 / Df ) intestines per genotype with 5 measured per intestine .

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(d) Midguts from control Atg310/wild-type (Atg3/+) and Atg310/Df(3L)cat mutant (Atg3/Df) animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (e) Cell size quantification (μm2) from d, n = 11 (Atg3/+) and n = 9 (Atg3/Df) animal intestines per genotype with 5 cells measured per intestine.


( f ) Midguts dissected from early third instar larvae that mis - express Atg1GS10797 ( Atg1 ) Atg3IR only in DsRed - marked clone analysed by differential interference contrast ( DIC ) microscopy . Representative are shown .

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(f) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1) and Atg3IR only in the DsRed-marked cell clone and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown.


( g ) Midguts dissected from expressing Atg4DN specifically in DsRed - marked clones of at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( h ) ( μm2 ) from g , n = 10 intestines per genotype with 1 - 5 measured per intestine .

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(g) Midguts dissected from animals expressing Atg4DN specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (h) Cell size quantification (μm2) from g, n = 10 animal intestines per genotype with 1-5 cells measured per intestine.


( i ) Midguts dissected from expressing mCherry - Atg8a in , expressing UbIR specifically in GFP - marked clones of at puparium formation . Representative are shown .

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(i) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing UbIR specifically in GFP-marked clones of cells at puparium formation. Representative images are shown.


( j ) Midguts dissected from expressing UbIR specifically in GFP - marked clones of at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( k ) ( μm2 ) from ( j ) , n = 12 intestines per genotype with 1 - 5 measured per intestine .

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(j) Midguts dissected from animals expressing UbIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (k) Cell size quantification (μm2) from (j), n = 12 animal intestines per genotype with 1-5 cells measured per intestine.


( l , m ) Midguts dissected from early third instar larvae that exhibit Atg1GS10797 ( Atg1 ) mis - expression in either a wild - type GFP - positive clone ( l ) or a Uba1H33 loss - of - function MARCM mutant clone ( GFP - positive ; m ) analysed by differential interference contrast microscopy . Control wild - type heterozygous Uba1H33 / wild - type have no GFP . Representative are shown . ( n ) ( μm2 ) from l m , n = 22 ( control ) , n = 16 ( Atg1 ) n = 6 ( Atg1 + Uba1H33 ) intestines per genotype with 1 - 5 measured per intestine . data for control ( l , m ) were pooled because they are genetically identical . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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(l,m) Midguts dissected from early third instar larvae that exhibit Atg1GS10797 (Atg1) mis-expression in either a wild-type GFP-positive cell clone (l) or a Uba1H33 loss-of-function MARCM mutant cell clone (GFP-positive; m) and analysed by fluorescence and differential interference contrast microscopy. Control wild-type and heterozygous Uba1H33/wild-type cells have no GFP. Representative images are shown. (n) Quantification (μm2) from l and m, n = 22 (control), n = 16 (Atg1) and n = 6 (Atg1 +Uba1H33) animal intestines per genotype with 1-5 cells measured per intestine. Quantification data for control cells (l,m) were pooled because they are genetically identical. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


( a , b ) Midguts dissected from either early third instar larvae ( a ) or at puparium formation ( b ) that express GFP - labelled mitochondria in enterocytes ( larger nuclei ) contain Uba1H33 loss - of - function mutant clones ( lacking RFP ) . Wild - type control possess stronger RFP heterozygous have weaker RFP . Representative are shown .

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(a,b) Midguts dissected from either early third instar larvae (a) or at puparium formation (b) that express GFP-labelled mitochondria in enterocytes (larger nuclei) and contain Uba1H33 loss-of-function mutant cell clones (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown.


( c ) Midguts dissected at puparium formation that express Uba1IR ( GFP in nucleus cytoplasm ) stained with ATP synthase V ( ATPV ) to detect mitochondria in . Representative are shown .

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(c) Midguts dissected at puparium formation that express Uba1IR (GFP in nucleus and cytoplasm) and stained with ATP synthase complex V (ATPV) to detect mitochondria in all cells. Representative images are shown.


( d - f ) Representative immuno - of Uba1IRclone at puparium formation . Uba1IR - expressing possess gold particles ; control lack gold particles ( d ) . Control possess numerous autolysosomes in cytoplasm ( arrowheads ) few mitochondria ( asterisks ) ; Uba1IR - expressing possess numerous mitochondria few autophagic structures . Scale bars , 20 μm ( a - c ) 1 μm ( d - f ) .

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(d-f) Representative immuno-TEM images of Uba1IRclone cells at puparium formation. Uba1IR-expressing cells possess gold particles; control cells lack gold particles (d). Control cells possess numerous autolysosomes in the cytoplasm (arrowheads) and few mitochondria (asterisks); Uba1IR-expressing cells possess numerous mitochondria and few autophagic structures. Scale bars, 20 μm (a-c) and 1 μm (d-f).


( C - I ) BMDMs were primed with LPS for 4 h then treated with LicoB for 1 h , prior to stimulation with nigericin for 45 min or ATP for 1 h . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , ASC ( arrow indicates ASC in whole lysate ( WCL ) ; activated caspase - 1 ( p20 ) IL - 1β ( p17 ) in culture supernatants ( SN ) of BMDMs ( C , G ) . Caspase - 1 activity ( D , H ) , IL - 1β secretion ( E , I ) , LDH release ( F , J ) in SN were measured . Coomassie Blue was used as supernatant loading control , while lamin B was used as lysate loading control .

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(C-I) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC (the arrow indicates ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (C, G). Caspase-1 activity (D, H), IL-1β secretion (E, I), and LDH release (F, J) in the SN were measured. Coomassie Blue staining was used as the supernatant loading control, while lamin B was used as the lysate loading control.


( A - C ) BMDMs were primed with LPS then treated with LicoB ( 20 μΜ ) for 1 h , followed by stimulation with nigericin , ATP , poly ( I : C ) , or MSU . Pam3CSK4 - primed BMDMs were treated with LicoB ( 20 μM ) then transfected with LPS . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , ASC in whole lysate ( WCL ) ; activated caspase - 1 ( p20 ) IL - 1β ( p17 ) in culture supernatants ( SN ) of BMDMs . ( A ) . Caspase - 1 activity ( B ) IL - 1β secretion ( C ) in SN were measured .

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(A-C) BMDMs were primed with LPS and then treated with LicoB (20 μΜ) for 1 h, followed by stimulation with nigericin, ATP, poly (I:C), or MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then transfected with LPS. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs. (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured.


( D - F ) LPS - primed BMDMs were treated with LicoB ( 20 μM ) for 1 h then stimulated with nigericin for 45 min , or poly ( dA : dT ) / Salmonella for 6 h . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , ASC in whole lysate ( WCL ) ; activated caspase - 1 ( p20 ) IL - 1β ( p17 ) in culture supernatants ( SN ) of BMDMs ( D ) . Caspase - 1 activity ( E ) IL - 1β secretion ( F ) in SN were measured .

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(D-F) LPS-primed BMDMs were treated with LicoB (20 μM) for 1 h and then stimulated with nigericin for 45 min, or poly(dA:dT)/Salmonella for 6 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (D). Caspase-1 activity (E) and IL-1β secretion (F) in the SN were measured.


( A - C ) LPS - primed BMDMs were pre - treated with indicated dose of LicoB for 1 h then stimulated with ATP for 1 h . Western blot was used to detect cross - linked ASC in Triton X - insoluble pellet , arrow indicates ASC ( A ) . Caspase - 1 activity ( B ) IL - 1β secretion ( C ) in SN were measured again to verify NLRP3 activation .

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(A-C) LPS-primed BMDMs were pre-treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h. Western blot analysis was used to detect cross-linked ASC in the Triton X-insoluble pellet, the arrow indicates ASC (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured again to verify NLRP3 activation.


( E ) Western blot of cross - linked ASC in Triton X - insoluble pellet from LPS - primed BMDMs pre - treated with LicoB ( 20 μM ) or vehicle then stimulated with nigericin , poly ( dA : dT ) , or Salmonella .

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(E) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, poly (dA:dT), or Salmonella.


( A ) Western blot of in whole lysates ( WCL ) from BMDMs stimulated with LPS for 3 h then treated with LicoB for 1 h ( LicoB after LPS ) , or BMDMs first treated with LicoB for 1 h then stimulated with LPS for 3 h ( LicoB before LPS ) .

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(A) Western blot analysis of protein in whole cell lysates (WCL) from BMDMs stimulated with LPS for 3 h and then treated with LicoB for 1 h (LicoB after LPS), or BMDMs first treated with LicoB for 1 h and then stimulated with LPS for 3 h (LicoB before LPS).


( I ) lysates of LPS - primed BMDMs treated with / without nigericin were incubated with sepharose or LicoB - sepharose . pull - down samples input were analysed using Western blot . ( J ) lysates of LPS - primed BMDMs were incubated with sepharose or LicoB - sepharose in presence of different concentrations of free LicoB ( 0 . 5 mM 1 mM ) . pull - down samples input were analysed using Western blot .

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(I) Cell lysates of LPS-primed BMDMs treated with/without nigericin were incubated with sepharose or LicoB-sepharose. The pull-down samples and input were analysed using Western blot analysis. (J) Cell lysates of LPS-primed BMDMs were incubated with sepharose or LicoB-sepharose in the presence of different concentrations of free LicoB (0.5 mM and 1 mM). The pull-down samples and input were analysed using Western blot.


( K ) HEK - 293T were transfected with Flag - NLRP3 or Flag - vector then treated with LicoB ( 40 μM ) . Immunoprecipitation was performed with anti - DYKDDDDK ( Flag ) affinity gel agarose beads ; Western blot has been shown .

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(K) HEK-293T cells were transfected with Flag-NLRP3 or Flag-vector and then treated with LicoB (40 μM). Immunoprecipitation was performed with anti-DYKDDDDK (Flag) affinity gel agarose beads; Western blot analysis has been shown.


( A ) Survival of WT mice pre - treated with vehicle , LicoB ( 20 mg / kg ) , LicoB ( 40 mg / kg ) , MCC950 ( 40 mg / kg ) , or LicoB ( 40 mg / kg ) + MCC950 ( 40 mg / kg ) , followed by i . p . injection with LPS ( 20 mg / kg ) ; mice survival was monitored for 72 h ( n = 10 mice per group ) .

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(A) Survival of WT mice pre-treated with vehicle, LicoB (20 mg/kg), LicoB (40 mg/kg), MCC950 (40 mg/kg), or LicoB (40 mg/kg) + MCC950 (40 mg/kg), followed by i.p. injection with LPS (20 mg/kg); mice survival was monitored for 72 h (n=10 mice per group).


C57BL / 6 mice received vehicle or LicoB ( 40 mg / kg ) for 17 consecutive doses , once every two days for 34 days . Following that treatment , serum of ALT ( H ) ( n = 8 mice per group ) .

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C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of ALT (H) (n=8 mice per group).


C57BL / 6 mice received vehicle or LicoB ( 40 mg / kg ) for 17 consecutive doses , once every two days for 34 days . Following that treatment , serum of AST ( I ) , CRE ( J ) were measured , ( n = 8 mice per group ) .

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C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of AST (I), and CRE (J) were measured, (n=8 mice per group).


( A - D ) Mice were pre - treated with LicoB or MCC950 for 1 h , then i . p . injected with MSU ( 50 mg / kg ) treated for 6 h ( n = 6 mice per group ) . of IL - 1β in peritoneal lavage fluid ( A ) serum ( B ) were measured using ELISA . of peritoneal exudate ( PECs ) ( C ) neutrophils ( Ly6G CD11b ) ( D ) using flow cytometry .

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(A-D) Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with MSU (50 mg/kg) and treated for 6 h (n=6 mice per group). The levels of IL-1β in the peritoneal lavage fluid (A) and serum (B) were measured using ELISA. Quantification of peritoneal exudate cells (PECs) (C) and neutrophils (Ly6G and CD11b) (D) using flow cytometry.


Under same growth conditions ( permitted water , free food , a 12 h / 12 h / cycle at 20 ± 2 ° C ) , eight - week - old C57BL / 6 mice were continuously fed with methionine - choline - supplemented ( MCS ) or methionine - choline - deficient ( MCD ) diets for 6 , at same time , gavaged with LicoB , MCC950 , or a combination of LicoB MCC950 ( n = 8 mice per group ( B , C ( A ) Representative liver section stained with H & E , Sirius Red , Masson ' s trichrome stains . Scale bar : 0 . 5 cm ( top row ) 200 μm ( 3 bottom ows ) . ( B , C ) serum of ALT AST ( n = 8 mice per group ) were measured using ELISA .

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

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950, or a combination of LicoB and MCC950 (n=8 mice per group (B, C (A) Representative liver section images stained with H&E, Sirius Red, and Masson's trichrome stains. Scale bar: 0.5 cm (top row) and 200 μm (3 bottom ows). (B, C) The serum levels of ALT and AST (n=8 mice per group) were measured using ELISA.


Under same growth conditions ( permitted water , free food , a 12 h / 12 h / cycle at 20 ± 2 ° C ) , eight - week - old C57BL / 6 mice were continuously fed with methionine - choline - supplemented ( MCS ) or methionine - choline - deficient ( MCD ) diets for 6 , at same time , gavaged with LicoB , MCC950 n = 6 mice per group Real - time quantitative PCR was used to detect mRNA of Col1a1 ( E ) , TNF - α ( F ) in mice livers , as described in ( A ) ( n = 6 mice per group ) .

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

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of Col1a1 (E), TNF-α (F) in the mice livers, as described in (A) (n=6 mice per group).


Under same growth conditions ( permitted water , free food , a 12 h / 12 h / cycle at 20 ± 2 ° C ) , eight - week - old C57BL / 6 mice were continuously fed with methionine - choline - supplemented ( MCS ) or methionine - choline - deficient ( MCD ) diets for 6 , at same time , gavaged with LicoB , MCC950 n = 6 mice per group Real - time quantitative PCR was used to detect mRNA of IL - 1β ( G ) , IL - 18 ( H ) in mice livers , as described in ( A ) ( n = 6 mice per group ) .

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

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of IL-1β (G), and IL-18 (H) in the mice livers, as described in (A) (n=6 mice per group).


Release of IL - 6 / or TNF release ( ELISA ) from ( C ) human MoMacs ( n = 6 - 8 ) Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicates ( human donor * p < 0 . 05 according to Wilcoxon signed rank sum (

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Release of IL-6 and/or TNF release (ELISA) from (C) human MoMacs (n=6-8) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicates (human donor * p<0.05 according to Wilcoxon signed rank sum (


Release of IL - 6 / or TNF release ( ELISA ) fro ( D ) murine BMDMs ( n = 6 Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate mic * p < 0 . 05 according to Wilcoxon signed rank sum ( C ,

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Release of IL-6 and/or TNF release (ELISA) fro (D) murine BMDMs (n=6 Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic * p<0.05 according to Wilcoxon signed rank sum (C, D


Release of IL - ( ELISA ) fro THP - 1 ( n = 3 ) Data information I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD ) Student ' s t -

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Release of IL- (ELISA) fro THP-1 cells (n=3) Data information I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD) Student's t-tes


( f ) GFP in vivo post intradermal injection of chitin oligomers into LysMEGFP / + C57BL / 6 mice ( n = 3 / group ) Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate

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(f) GFP in vivo fluorescence post intradermal injection of chitin oligomers into LysMEGFP/+ C57BL/6 mice (n=3/group) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic


( G ) Representative in vivo imaging results I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD )

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(G) Representative in vivo imaging results I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD)


Leukocyte infiltration in vivo after intratracheal administration assessed by ( H ) BALF flow cytometry ( n = 3 - 4 / group Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate mic ) , one - way ANOVA (

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Leukocyte infiltration in vivo after intratracheal administration assessed by (H) BALF flow cytometry (n=3-4/group Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic ), one-way ANOVA (H


Leukocyte infiltration in vivo after intratracheal administration assessed b ( I ) histology ( n = 2 ) , scale bar = 200 μm , arrows show infiltrate I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD )

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Leukocyte infiltration in vivo after intratracheal administration assessed b (I) histology analysis (n=2), scale bar = 200 μm, arrows show cellular infiltrate I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD)


A . thaliana seedling ( J ) target gene mRNA induction ( n = 3 I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD ) Student ' s t -

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A. thaliana seedling (J) target gene mRNA induction (n=3 I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD) Student's t-tes


A . thalian leaf piece ( K ) ROS production ( 0 - 45 min , n = 6 ) Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate

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A. thalian leaf piece (K) ROS production (0-45 min, n=6) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate plant leave


TNF released from WT or Myd88 ( A KO BMDM ( total n = 6 - 9 / group combined from 3 experiments ) Data information : I represent data ( mean + ) combined from ' n ' technical or biological replicate mic Student ' t -

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TNF released from WT or Myd88 (A KO BMDM (total n=6-9/group combined from 3 experiments) Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate mic Student' t-test


TNF released from W or Tlr KO BMDM ( total n = 6 - 9 / group combined from 3 experiments ) Student ' s t -

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TNF released from W or Tlr KO BMDM (total n=6-9/group combined from 3 experiments) Student's t-tes


( C ) IL - 6 released from MoMacs ( n = 5 ) treated with non - targeting ( NT ) , TLR2 - or MyD88 - specific siRNA relative to NT condition one - sample t -

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(C) IL-6 released from primary MoMacs (n=5) treated with non-targeting (NT), TLR2- or MyD88-specific siRNA relative to NT condition one-sample t-tes


( D ) CD62L shedding from human PMNs ( n = 4 - 8 ) with or without isotype control or anti - TLR2 blocking antibody Data information : I represent data ( mean + ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann -

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(D) CD62L shedding from human PMNs (n=4-8) with or without isotype control or anti-TLR2 blocking antibody Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney


( E ) IL - 6 released from mock or TLR2 Cas9 - CRISPR - edited THP - 1 ( n = 3 ) I one representative of ' n ' independent experiments is shown ( mean + SD ) ) , Student ' s t -

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(E) IL-6 protein released from mock or TLR2 Cas9-CRISPR-edited THP-1 cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD) ), Student's t-tes


( F ) NF - κB activity in empty vector ( EV ) or TLR2 - transfected HEK293T ( n = 3 ) I one representative of ' n ' independent experiments is shown ( mean + SD )

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(F) NF-κB activity in empty vector (EV) or TLR2-transfected HEK293T cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD)


( G ) MPO release from intradermally injected WT or Tlr2 KO mice ( n = 5 - 15 biopsies each ) Data information I represent data ( mean + ) combined from ' n ' technical or biological replicate mic ) , Student ' s t -

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(G) MPO release from intradermally injected WT or Tlr2 KO mice (n=5-15 biopsies each) Data information I represent data (mean+SEM) combined from 'n' technical or biological replicate mic ), Student's t-tes