Figure 2 ( A ) Respiratory chain super - complex levels are slightly restored by over - expression of Mcp3 . Mitochondria isolated from the indicated strains were lysed in 1 % digitonin and subjected to a 4 - 8 % BN - PAGE . Proteins were analysed by immunodecoration with antibodies against either Cor1 of complex III or Cox2 of complex IV . ( B ) Assembly defects in TOM and TOB complexes in mdm10 cells are partially restored by Mcp3 over - expression . Mitochondria of the indicated cells were lysed in 1 % digitonin and subjected to a 6 - 13 % BN - PAGE and immunoblotting with antibodies against Tom40 ( left ) or Tob55 ( right ) . The assembled TOM complex and an unassembled Tom40 species at ca . 100 kDa ( arrowhead ) are indicated as well as the TOB complex and an additional higher molecular weight species of the TOB complex ( asterisk ) . ( C ) Over - expression of Mcp3 partially rescues the alterations in lipid composition of cells lacking Mdm10 . Lipids were extracted from highly pure mitochondria isolated from the indicated cells and then analysed by mass spectrometry analysis . The level of each phospholipid species in wild - type mitochondria was set to 100 % and the ERG / PL ratio to 1 . Relative changes in mitochondria from other cells are presented . The mean with standard deviations of three biological repeats with two technical repeats for each ( n = 6 ; SD ; * , p < 0 . 05 ) is given . ( D ) Mcp3 rescues loss of Mdm12 . Wild - type ( WT ) or mdm12Δ cells transformed with the empty plasmid pYX142 ( ) or mdm12Δ cells over - expressing either Mcp3 or Mdm12 ( as a control ) were analysed at 30 ° C or 37 ° C by drop dilution assay on rich medium containing glycerol ( YPG ) . ( E ) Mcp3 rescues loss of Mmm1 . WT and mmm1Δ cells were transformed and analysed as described in ( D ) . ( F ) Mcp3 rescues loss of Mdm34 . WT and mdm34Δ cells were transformed and analysed as described in ( D ) .

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Figure 2(A) Respiratory chain super-complex levels are slightly restored by over-expression of Mcp3. Mitochondria isolated from the indicated strains were lysed in 1% digitonin and subjected to a 4-8% BN-PAGE. Proteins were analysed by immunodecoration with antibodies against either Cor1 of complex III or Cox2 of complex IV.(B) Assembly defects in TOM and TOB complexes in mdm10 cells are partially restored by Mcp3 over-expression. Mitochondria of the indicated cells were lysed in 1% digitonin and subjected to a 6-13% BN-PAGE and immunoblotting with antibodies against Tom40 (left) or Tob55 (right). The assembled TOM complex and an unassembled Tom40 species at ca. 100 kDa (arrowhead) are indicated as well as the TOB complex and an additional higher molecular weight species of the TOB complex (asterisk).(C) Over-expression of Mcp3 partially rescues the alterations in lipid composition of cells lacking Mdm10. Lipids were extracted from highly pure mitochondria isolated from the indicated cells and then analysed by mass spectrometry analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from other cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD; *, p < 0.05) is given.(D) Mcp3 rescues loss of Mdm12. Wild-type (WT) or mdm12Δ cells transformed with the empty plasmid pYX142 () or mdm12Δ cells over-expressing either Mcp3 or Mdm12 (as a control) were analysed at 30°C or 37°C by drop dilution assay on rich medium containing glycerol (YPG).(E) Mcp3 rescues loss of Mmm1. WT and mmm1Δ cells were transformed and analysed as described in (D).(F) Mcp3 rescues loss of Mdm34. WT and mdm34Δ cells were transformed and analysed as described in (D).


Figure 3 ( A ) Growth phenotype of mcp3 cells . WT or mcp3 ∆ cells were grown to an OD600 of 1 . 0 in YPD medium and spotted on YPD or YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at the indicated temperatures . ( B ) mcp3 cells have normal mitochondrial morphology . WT and mcp3 ∆ cells expressing mitochondrially targeted GFP ( mtGFP ) were grown to mid - logarithmic phase then analysed by fluorescence microscopy . Typical images of the two different strains are shown ( scale bar = 5 µm ) . The quantification of the depicted strains shows the average percentage with standard deviation bars of three independent experiments with at least 100 cells per experiment ( n = 3 ; SD ; p as indicated ) . ( C , D ) Loss of Mcp3 has no effect on the phospholipid composition of mitochondria . Highly - pure mitochondria were isolated from the indicated yeast cells . Lipids were extracted and then analysed by mass spectrometry analysis . The level of each phospholipid species in wild - type mitochondria was set to 100 % and the ERG / PL ratio to 1 . Relative changes in mitochondria from mcp3 cells are presented . The mean with standard deviations of three biological repeats with two technical repeats for each ( n = 6 ; SD ) is given . ( E ) Over - expression of Mcp3 leads to alterations of the tubular mitochondrial network . Wild - type cells expressing mitochondrially targeted GFP ( mtGFP ) were transformed with a plasmid over - expressing Mcp3 ( MCP3 ) or an empty plasmid as control ( ) . Cells were grown to mid - logarithmic phase and then analysed by fluorescence microscopy . Typical images of the two different strains are shown ( scale bar = 5 µm ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Growth phenotype of mcp3 cells. WT or mcp3∆ cells were grown to an OD600 of 1.0 in YPD medium and spotted on YPD or YPG plates in a 1:5 dilution series. Plates were incubated for growth at the indicated temperatures.(B) mcp3 cells have normal mitochondrial morphology. WT and mcp3∆ cells expressing mitochondrially targeted GFP (mtGFP) were grown to mid-logarithmic phase then analysed by fluorescence microscopy. Typical images of the two different strains are shown (scale bar = 5 µm). The quantification of the depicted strains shows the average percentage with standard deviation bars of three independent experiments with at least 100 cells per experiment (n=3; SD; p as indicated).(C, D) Loss of Mcp3 has no effect on the phospholipid composition of mitochondria. Highly-pure mitochondria were isolated from the indicated yeast cells. Lipids were extracted and then analysed by mass spectrometry analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from mcp3 cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD) is given.(E) Over-expression of Mcp3 leads to alterations of the tubular mitochondrial network. Wild-type cells expressing mitochondrially targeted GFP (mtGFP) were transformed with a plasmid over-expressing Mcp3 (MCP3) or an empty plasmid as control (). Cells were grown to mid-logarithmic phase and then analysed by fluorescence microscopy. Typical images of the two different strains are shown (scale bar = 5 µm).


Figure 4 ( B ) Internally tagged HA - Mcp3 is functional . mdm10 ∆ cells were transformed with the empty plasmid pYX142 ( ) or pYX142 encoding Mdm10 , Mcp3 , or an internally HA - tagged Mcp3 ( MDM10 , MCP3 , HA - MCP3 ) . Cells were grown to an OD600 of 1 . 0 and spotted on YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at 37 ° C for 4 days . ( C ) HA - Mcp3 is processed in vivo . Mitochondria isolated from mdm10 ∆ ( ∆ ) cells containing the empty plasmid or expressing HA - Mcp3 were analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins . Two different bands are detectable in case of HA - Mcp3 ( p , precursor ; m , mature protein ) . The MOM protein Tom40 serves as loading control . ( D ) Mcp3 is a mitochondrial protein . Whole cell lysate ( WCL ) and fractions corresponding to cytosol ( Cyt . ) , light microsomal fraction ( ER ) and mitochondria ( Mito . ) of mcp3 ∆ cells either containing the empty plasmid ( ) or expressing HA - Mcp3 were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , the mitochondrial protein Tom70 , a marker protein for the cytosol ( Bmh1 ) and an ER marker protein ( Erv2 ) . m , mature protein . ( E ) Mcp3 co - localizes with the mitochondrial marker protein AAC . Cells over - expressing HA - Mcp3 were fixed and analysed by fluorescence microscopy using antibodies against the HA - epitope ( αHA ) and the mitochondrial ADP / ATP carrier ( αAAC ) . ( scale bar = 5 µm ) . ( F ) C - terminally tagged Mcp3 - HA is located to mitochondria . A mitochondria enriched fraction ( Mito . ) and the post mitochondrial supernatant ( PMS ) of wild - type cells carrying the empty plasmid ( ) or over - expressing the C - terminally HA - tagged Mcp3 ( Mcp3 - HA ) were analysed by SDS - PAGE and immunodecortion with antibodies against the HA - Tag , the mitochondrial protein Tom40 and a marker protein for the cytosol ( Hxk1 ) . m , mature protein . ( G ) Mcp3 is membrane - embedded . Mitochondria isolated from mcp3Δ cells expressing HA - Mcp3 were left untreated ( I , input ) or subjected to alkaline extraction . The supernatant ( SN ) and pellet ( P ) fractions were analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins . Tom20 an integral MOM proteins ; Aco1 , soluble matrix protein . m , mature protein . ( H ) Mcp3 is a MOM protein . Mitochondria isolated from cells expressing HA - Mcp3 were treated with proteinase K ( PK , 100 µg / ml ) in the absence or presence of the detergent Triton X - 100 ( TX ) . Samples were precipitated with TCA and analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag or the indicated mitochondrial proteins . Tom20 , a MOM protein exposed to the cytosol ; Dld1 , a protein located in the IMS . ( I ) Mcp3 behaves like an outer membrane protein in density gradient centrifugation . Mitochondrial vesicles isolated from cells over - expressing HA - Mcp3 were subjected to sucrose density gradient centrifugation . Fractions of the gradient were collected and analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins . The processed form of HA - Mcp3 is shown . Right panel : The intensities of the various bands were quantified and depicted . The sum of all intensities for each protein was set to 100 % . ( J ) Mcp3 lacking either transmembrane domain fails to rescue loss of Mdm10 . Wild - type cells carrying the empty plasmid ( wt + ) or mdm10 ∆ cells transformed with the empty plasmid ( ) or plasmid encoding Mdm10 , Mcp3 , or Mcp3 lacking TMD1 or TMD2 ( MDM10 , MCP3 , ΔTMD1 , ΔTMD2 ) were grown to an OD600 of 1 . 0 and spotted on YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at 37 ° C for 4 days . ( K ) Mcp3 lacking either of the two transmembrane domains is unstable . Crude mitochondria were obtained from wild - type cells containing an empty plasmid ( Ø ) or mcp3Δ cells ( Δ ) transformed with plasmid encoding Mcp3 or Mcp3 lacking TMD1 or TMD2 ( MCP3 , ΔTMD1 , ΔTMD2 ) . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against Mcp3 and Tom20 as loading control .

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Figure 4(B) Internally tagged HA-Mcp3 is functional. mdm10∆ cells were transformed with the empty plasmid pYX142 () or pYX142 encoding Mdm10, Mcp3, or an internally HA-tagged Mcp3 (MDM10, MCP3, HA-MCP3). Cells were grown to an OD600 of 1.0 and spotted on YPG plates in a 1:5 dilution series. Plates were incubated for growth at 37°C for 4 days.(C) HA-Mcp3 is processed in vivo. Mitochondria isolated from mdm10∆ (∆) cells containing the empty plasmid or expressing HA-Mcp3 were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. Two different bands are detectable in case of HA-Mcp3 (p, precursor; m, mature protein). The MOM protein Tom40 serves as loading control.(D) Mcp3 is a mitochondrial protein. Whole cell lysate (WCL) and fractions corresponding to cytosol (Cyt.), light microsomal fraction (ER) and mitochondria (Mito.) of mcp3∆ cells either containing the empty plasmid () or expressing HA-Mcp3 were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, the mitochondrial protein Tom70, a marker protein for the cytosol (Bmh1) and an ER marker protein (Erv2). m, mature protein.(E) Mcp3 co-localizes with the mitochondrial marker protein AAC. Cells over-expressing HA-Mcp3 were fixed and analysed by fluorescence microscopy using antibodies against the HA-epitope (αHA) and the mitochondrial ADP/ATP carrier (αAAC). (scale bar = 5 µm).(F) C-terminally tagged Mcp3-HA is located to mitochondria. A mitochondria enriched fraction (Mito.) and the post mitochondrial supernatant (PMS) of wild-type cells carrying the empty plasmid () or over-expressing the C-terminally HA-tagged Mcp3 (Mcp3-HA) were analysed by SDS-PAGE and immunodecortion with antibodies against the HA-Tag, the mitochondrial protein Tom40 and a marker protein for the cytosol (Hxk1). m, mature protein.(G) Mcp3 is membrane-embedded. Mitochondria isolated from mcp3Δ cells expressing HA-Mcp3 were left untreated (I, input) or subjected to alkaline extraction. The supernatant (SN) and pellet (P) fractions were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. Tom20 an integral MOM proteins; Aco1, soluble matrix protein. m, mature protein.(H) Mcp3 is a MOM protein. Mitochondria isolated from cells expressing HA-Mcp3 were treated with proteinase K (PK, 100 µg/ml) in the absence or presence of the detergent Triton X-100 (TX). Samples were precipitated with TCA and analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag or the indicated mitochondrial proteins. Tom20, a MOM protein exposed to the cytosol; Dld1, a protein located in the IMS.(I) Mcp3 behaves like an outer membrane protein in density gradient centrifugation. Mitochondrial vesicles isolated from cells over-expressing HA-Mcp3 were subjected to sucrose density gradient centrifugation. Fractions of the gradient were collected and analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. The processed form of HA-Mcp3 is shown. Right panel: The intensities of the various bands were quantified and depicted. The sum of all intensities for each protein was set to 100%.(J) Mcp3 lacking either transmembrane domain fails to rescue loss of Mdm10. Wild-type cells carrying the empty plasmid (wt+) or mdm10∆ cells transformed with the empty plasmid () or plasmid encoding Mdm10, Mcp3, or Mcp3 lacking TMD1 or TMD2 (MDM10, MCP3, ΔTMD1, ΔTMD2) were grown to an OD600 of 1.0 and spotted on YPG plates in a 1:5 dilution series. Plates were incubated for growth at 37°C for 4 days.(K) Mcp3 lacking either of the two transmembrane domains is unstable. Crude mitochondria were obtained from wild-type cells containing an empty plasmid (Ø) or mcp3Δ cells (Δ) transformed with plasmid encoding Mcp3 or Mcp3 lacking TMD1 or TMD2 (MCP3, ΔTMD1, ΔTMD2). Samples were analysed by SDS-PAGE and immunodecoration with antibodies against Mcp3 and Tom20 as loading control.


Figure 5 ( A ) Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . The arrowhead marks an additional band of the size of the cleaved N - terminus of Mcp3 . p and m , precursor and mature forms , respectively . ( B ) Import of Mcp3 is dependent on the receptor Tom70 . Mitochondria isolated from wild - type or tom70 / 71Δ cells were incubated with radiolabelled Mcp3 or pSu9 - DHFR ( as control ) for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . p and m , precursor and mature forms , respectively . I , 20 % of radiolabelled precursor protein used in each import reaction . Bands corresponding to the mature ( m ) form were quantified . Import into wild - type mitochondria after 15 min was set to 100 % . The mean with standard deviations is depicted ( n = 3 ; SD ) . ( C ) Reduced steady state levels of HA - Mcp3 in cells lacking Tom70 . Crude mitochondria isolated from the indicated cells were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , AAC as control substrate of Tom70 , and Fis1 as a loading control . HA - Mcp3 levels were quantified in relation to Fis1 levels and the levels in WT cells were set to 100 % . The bar diagram shows the mean with standard deviation ( n = 3 ; SD ; * , p < 0 . 05 ) . ( D ) Import of Mcp3 is dependent on Tom40 . Mitochondria isolated from wild - type and tom4025 cells were incubated with radiolabelled Mcp3 for the indicated time periods . Next , mitochondria were reisolated and analysed by SDS - PAGE and autoradiography as described in ( B ) . As a control Ugo1 was imported into the same mitochondria . Organelles were reisolated and analysed by BN - PAGE and autoradiography . The band corresponding to the Ugo1 dimer at around 150 kDa was quantified . The mean with standard deviation is shown ( n = 3 ; SD ) . ( E ) Steady state levels of HA - Mcp3 are lower in cells harbouring a mutant TOM40 allele . Crude mitochondria from wild - type and tom40 - 25 cells were obtained and analysed as described in ( C ) . Immunodecoration was performed with antibodies against the HA - tag , Tom40 , and Fis1 as loading control .

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Figure 5(A) Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3. p and m, precursor and mature forms, respectively.(B) Import of Mcp3 is dependent on the receptor Tom70. Mitochondria isolated from wild-type or tom70/71Δ cells were incubated with radiolabelled Mcp3 or pSu9-DHFR (as control) for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. p and m, precursor and mature forms, respectively. I, 20% of radiolabelled precursor protein used in each import reaction. Bands corresponding to the mature (m) form were quantified. Import into wild-type mitochondria after 15 min was set to 100%. The mean with standard deviations is depicted (n=3; SD).(C) Reduced steady state levels of HA-Mcp3 in cells lacking Tom70. Crude mitochondria isolated from the indicated cells were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, AAC as control substrate of Tom70, and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels and the levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05).(D) Import of Mcp3 is dependent on Tom40. Mitochondria isolated from wild-type and tom4025 cells were incubated with radiolabelled Mcp3 for the indicated time periods. Next, mitochondria were reisolated and analysed by SDS-PAGE and autoradiography as described in (B). As a control Ugo1 was imported into the same mitochondria. Organelles were reisolated and analysed by BN-PAGE and autoradiography. The band corresponding to the Ugo1 dimer at around 150 kDa was quantified. The mean with standard deviation is shown (n=3; SD).(E) Steady state levels of HA-Mcp3 are lower in cells harbouring a mutant TOM40 allele. Crude mitochondria from wild-type and tom40-25 cells were obtained and analysed as described in (C). Immunodecoration was performed with antibodies against the HA-tag, Tom40, and Fis1 as loading control.


Figure 6 ( A ) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors . Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70 , Tom20 or Tom22 prebound to glutathione beads or with unloaded beads . After washing bound material was eluted with sample buffer . Proteins were analysed by SDS - PAGE , blotting onto a membrane and autoradiography . To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S . I , input 5 % of precursor used . ( B ) Mcp3 precursor binds to Tom22 in organello . Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His - Tom22 containing cells . Mitochondria were pre - incubated with CCCP ( 40 µM ) to halt import . After lysis with β - dodecyl maltoside samples were incubated with Ni - NTA beads . After washing bound proteins were eluted with sample buffer and analysed by SDS - PAGE and autoradiography . I , 5 % of 35S - Mcp3 used ; pel . , 2 % insoluble material after clarifying spin ; sup . , 2 % of material incubated with Ni - NTA ; n . b . 2 % of non - bound material after binding to Ni - NTA ; eluate , 100 % of bound material . ( C ) Radiolabelled Mcp3 was incubated with organelles described in ( B ) . After import mitochondria were solubilized in digitonin , the lysate was cleared by centrifugation and incubated with or without an antibody against the His - tag ( α - His ) . Samples were analysed by BN - PAGE , autoradiography and afterwards immunodecoration with an antibody against Tom40 . Bands shifted by the anti - His antibody are indicated by arrowhead .

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Figure 6(A) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors. Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70, Tom20 or Tom22 prebound to glutathione beads or with unloaded beads. After washing bound material was eluted with sample buffer. Proteins were analysed by SDS-PAGE, blotting onto a membrane and autoradiography. To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used.(B) Mcp3 precursor binds to Tom22 in organello. Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His-Tom22 containing cells. Mitochondria were pre-incubated with CCCP (40 µM) to halt import. After lysis with β-dodecyl maltoside samples were incubated with Ni-NTA beads. After washing bound proteins were eluted with sample buffer and analysed by SDS-PAGE and autoradiography. I, 5% of 35S-Mcp3 used; pel., 2% insoluble material after clarifying spin; sup., 2% of material incubated with Ni-NTA; n.b. 2% of non-bound material after binding to Ni-NTA; eluate, 100% of bound material.(C) Radiolabelled Mcp3 was incubated with organelles described in (B). After import mitochondria were solubilized in digitonin, the lysate was cleared by centrifugation and incubated with or without an antibody against the His-tag (α-His). Samples were analysed by BN-PAGE, autoradiography and afterwards immunodecoration with an antibody against Tom40. Bands shifted by the anti-His antibody are indicated by arrowhead.


Figure 7 ( A ) Import of Mcp3 is dependent on the TIM23 complex . Mitochondria isolated from wild - type and tim23ts cells were incubated with radiolabelled Mcp3 for the indicated time periods . Further treatment and analysis ( n = 3 ; SD ) was as described for Fig . 5D . ( B ) Steady state levels of HA - Mcp3 are lower in cells harbouring a temperature sensitive TIM23 allele . Crude mitochondria were obtained at the non - permissive temperature from WT or tim23ts cells containing a plasmid expressing HA - Mcp3 . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , the matrix proteins Mge1 and Yah1 as typical TIM23 substrates , Ugo1 and Fis1 as TIM23 - independent substrates . HA - Mcp3 levels were quantified in relation to Fis1 levels . Levels in WT cells were set to 100 % . The bar diagram shows the mean with standard deviation of six independent experiments ( n = 6 ; SD ; * * , p < 0 . 01 ) . ( C ) Import and processing of Mcp3 is dependent on the mitochondrialmembrane potential . Isolated mitochondria were incubated with radiolabelled Mcp3 or pSu9 - DHFR as control for the indicated time periods or for 15 minutes in the presence of either CCCP or o - phenantroline and EDTA ( o - Phen . / EDTA ) . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . L + MPP : radiolabelled proteins were incubated with recombinant MPP . ( D ) Mcp3 is processed by Imp1 / 2 . Single deletion strains of known mitochondrial proteases and peptidases were transformed with a plasmid expressing HA - Mcp3 . Crude mitochondria were isolated and analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins and the HA - epitope . Cox2 , a known substrate of IMP ; Tom70 , a mitochondrial outer membrane protein . ( E ) C - terminally tagged Mcp3 - HA is processed by Imp1 . WT and imp1Δ cells were transformed with a plasmid expressing Mcp3 - HA and further analysis was performed as described in ( D ) . ( F ) Mcp3 is processed by Imp1 / 2 after in vitro import into mitochondria . Mitochondria isolated from WT , imp1Δ or imp2Δ strains were incubated with radiolabelled precursor of Mcp3 or pSu9 - DHFR as control . Further treatment and analysis was as described in Fig . 5A . ( G ) Full - length Mcp3 precursor accumulates during import if IMP processing is abolished . Radiolabelled internally HA - tagged Mcp3 without ( HA - Mcp3 ) or with mutation in the Imp1 cleavage site ( D70G + HA ) were incubated with mitochondria as in ( F ) . After import samples were incubated with or without PK ( 20 µg / ml ) . Samples were analysed by SDS - PAGE and autoradiography . ( H ) Mitochondrial membrane potential is required for efficient import of Mcp3 precursor into mitochondria . Isolated mitochondria were incubated with radiolabelled Mcp3 in the presence or absence of CCCP . After import mitochondria were incubated with PK as in ( G ) and analysed by SDS - PAGE and autoradiography . I , 20 % of radiolabelled precursor protein used in each import reaction ; The arrowhead marks an additional band of the size of the cleaved N - terminus of Mcp3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Import of Mcp3 is dependent on the TIM23 complex. Mitochondria isolated from wild-type and tim23ts cells were incubated with radiolabelled Mcp3 for the indicated time periods. Further treatment and analysis (n=3; SD) was as described for Fig. 5D.(B) Steady state levels of HA-Mcp3 are lower in cells harbouring a temperature sensitive TIM23 allele. Crude mitochondria were obtained at the non-permissive temperature from WT or tim23ts cells containing a plasmid expressing HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, the matrix proteins Mge1 and Yah1 as typical TIM23 substrates, Ugo1 and Fis1 as TIM23-independent substrates. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation of six independent experiments (n=6; SD; **, p < 0.01).(C) Import and processing of Mcp3 is dependent on the mitochondrialmembrane potential. Isolated mitochondria were incubated with radiolabelled Mcp3 or pSu9-DHFR as control for the indicated time periods or for 15 minutes in the presence of either CCCP or o-phenantroline and EDTA (o-Phen./EDTA). After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. L+MPP: radiolabelled proteins were incubated with recombinant MPP.(D) Mcp3 is processed by Imp1/2. Single deletion strains of known mitochondrial proteases and peptidases were transformed with a plasmid expressing HA-Mcp3. Crude mitochondria were isolated and analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins and the HA-epitope. Cox2, a known substrate of IMP; Tom70, a mitochondrial outer membrane protein.(E) C-terminally tagged Mcp3-HA is processed by Imp1. WT and imp1Δ cells were transformed with a plasmid expressing Mcp3-HA and further analysis was performed as described in (D).(F) Mcp3 is processed by Imp1/2 after in vitro import into mitochondria. Mitochondria isolated from WT, imp1Δ or imp2Δ strains were incubated with radiolabelled precursor of Mcp3 or pSu9-DHFR as control. Further treatment and analysis was as described in Fig. 5A.(G) Full-length Mcp3 precursor accumulates during import if IMP processing is abolished. Radiolabelled internally HA-tagged Mcp3 without (HA-Mcp3) or with mutation in the Imp1 cleavage site (D70G+HA) were incubated with mitochondria as in (F). After import samples were incubated with or without PK (20 µg/ml). Samples were analysed by SDS-PAGE and autoradiography.(H) Mitochondrial membrane potential is required for efficient import of Mcp3 precursor into mitochondria. Isolated mitochondria were incubated with radiolabelled Mcp3 in the presence or absence of CCCP. After import mitochondria were incubated with PK as in (G) and analysed by SDS-PAGE and autoradiography. I, 20% of radiolabelled precursor protein used in each import reaction; The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3.


Figure 8 ( A ) Import of Mcp3 is mediated by the MIM complex . Mitochondria isolated from WT , mim1Δ , or mim2Δ cells were incubated with radiolabelled Mcp3 for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . Bands corresponding to the mature ( m ) form were quantified . Import after 15 min into wild - type mitochondria was set to 100 % . The mean with standard deviations is depicted ( n = 3 ; SD ) . I , 20 % of radiolabelled precursor protein used in each import reactionB ) Steady state levels of over - expressed HA - Mcp3 are reduced in cells lacking MIM subunits . Crude mitochondria were obtained from WT cells containing an empty plasmid ( Ø ) and from wild - type , mim1Δ or mim2Δ cells containing a plasmid encoded HA - Mcp3 . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , Tom20 as MIM substrate and Fis1 as a loading control . HA - Mcp3 levels were quantified in relation to Fis1 levels . Levels in wild - type cells were set to 100 % . The bar diagram shows the mean with standard deviation ( n = 3 ; SD ; * , p < 0 . 05 ; * * , p < 0 . 01 ) . ( C ) Mcp3 precursor binds in vitro to Mim1 . Radiolabelled Mcp3 was incubated with amylose beads harbouring recombinant MBP or MBP fused to Mim1 . After washing proteins were eluted with sample buffer . Analysis was performed by SDS - PAGE , blotting and autoradiography . To demonstrate equal amounts of MBP fusion proteins the membrane was stained with Ponceau S . I , input 5 % of precursor used ; n . b . , 5 % of unbound material ; . b , 100 % of bound material .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Import of Mcp3 is mediated by the MIM complex. Mitochondria isolated from WT, mim1Δ, or mim2Δ cells were incubated with radiolabelled Mcp3 for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. Bands corresponding to the mature (m) form were quantified. Import after 15 min into wild-type mitochondria was set to 100%. The mean with standard deviations is depicted (n=3; SD). I, 20% of radiolabelled precursor protein used in each import reactionB) Steady state levels of over-expressed HA-Mcp3 are reduced in cells lacking MIM subunits. Crude mitochondria were obtained from WT cells containing an empty plasmid (Ø) and from wild-type, mim1Δ or mim2Δ cells containing a plasmid encoded HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, Tom20 as MIM substrate and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in wild-type cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05; **, p < 0.01).(C) Mcp3 precursor binds in vitro to Mim1. Radiolabelled Mcp3 was incubated with amylose beads harbouring recombinant MBP or MBP fused to Mim1. After washing proteins were eluted with sample buffer. Analysis was performed by SDS-PAGE, blotting and autoradiography. To demonstrate equal amounts of MBP fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used; n.b., 5% of unbound material;.b, 100% of bound material.


Figure 1Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and BMI Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression , plasma alanine aminotransferase ( ALAT ) ( B ) Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and liver fat content ( C ) . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . ASK1 mRNA expression in patients with different NASH scores Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * * Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG5 ( E ) mRNA expression . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * * Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG7 Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG12 mRNA expression . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * *

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and BMI Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).Scatter plot and correlation coefficient (r) of ASK1 mRNA expression , plasma alanine aminotransferase (ALAT) (B) Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and liver fat content (C). Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).ASK1 mRNA expression in patients with different NASH scores Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG5 (E) mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG7 Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG12 mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***


Figure 2Hepatocytes were treated with BSA , Palm or Palm + Rap for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) . Scale bar represents 100 µm . Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ) or non - targeting siRNA control ( siCtrl ) . Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm , Palm + Rap and stained as mentioned above ( n = 4 biological replicates ) . Shown is one representative Western blot from two independent experiments and quantification of LC3 - II / I and p62 protein levels in siCtrl ( n = 4 biological replicates ) or siASK1 ( n = 4 biological replicates ) cells treated with Palm . Colocalization of LC3 - II punctate ( red ; arrow ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in siCtrl or siASK1 cells treated with BSA , Palm or Palm + Rap . Scale bar represents 100 µm . Colocalization of LC3 - II punctate with lipid droplets ( BODIPY 493 / 503 , green ) was quantified in hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ; grey bars ) or non - targeting siRNA control ( siCtrl ; black bars ) and treated with BSA or BSA + Baf for 24 hours ( n = 4 biological replicates ) . Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm or Palm + Baf and stained as mentioned above ( n = 4 biological replicates ) . Values are expressed as mean ± SEM . *

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Hepatocytes were treated with BSA, Palm or Palm+Rap for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates). Scale bar represents 100 µm.Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 (siASK1) or non-targeting siRNA control (siCtrl).Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm, Palm+Rap and stained as mentioned above (n=4 biological replicates).Shown is one representative Western blot from two independent experiments and quantification of LC3-II/I and p62 protein levels in siCtrl (n=4 biological replicates) or siASK1 (n=4 biological replicates) cells treated with Palm.Colocalization of LC3-II punctate (red; arrow) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in siCtrl or siASK1 cells treated with BSA, Palm or Palm+Rap. Scale bar represents 100 µm.Colocalization of LC3-II punctate with lipid droplets (BODIPY 493/503, green) was quantified in hepatocytes transfected with siRNA targeting ASK1 (siASK1; grey bars) or non-targeting siRNA control (siCtrl; black bars) and treated with BSA or BSA+Baf for 24 hours (n=4 biological replicates).Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm or Palm+Baf and stained as mentioned above (n=4 biological replicates). Values are expressed as mean ± SEM. *


Figure 3Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm . Liver triglyceride ( TG ) content of mice fed a chow ( ASK1F / F n = 6 mice ; ASK1Δhep n = 6 mice ) or HFD ( ASK1F / F n = 14 mice ; ASK1Δhep n = 15 mice ) . Relative mRNA expression of perilipin ( Plin ) and very low - density lipoprotein receptor ( Vldlr ) in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( Vldlr ) or n = 8 mice ( Plin ) ) . Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( F4 / 80 , Mcp1 ) or ASK1Δhep n = 8 mice ( Il - 6 ) ) . Relative mRNA expression of collagen , type I , alpha 1 ( Col1a1 ) ( ASK1F / F n = 11 mice ; ASK1Δhep n = 15 mice ) and transforming growth factor beta 1 ( Tgfβ1 ) ( ASK1F / F n = 6 mice ; ASK1Δhep n = 8 mice ) in liver of HFD - fed mice . Representative images of liver sections stained with Sirius Red ( scale bar represents 100 µm ) quantification of Sirius Red positive area in liver of mice fed a HFD ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) . Representative images of liver sections stained with α - SMA from mice fed a HFD for 20 weeks . Scale bar represents 100 µm . Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) . Liver triglyceride ( TG ) content ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) and quantification of Sirius Red positive area in livers ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) of 15 months old chow - fed mice . Values are expressed as mean ± SEM . #

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.Liver triglyceride (TG) content of mice fed a chow (ASK1F/F n=6 mice; ASK1Δhep n=6 mice) or HFD (ASK1F/F n=14 mice; ASK1Δhep n=15 mice).Relative mRNA expression of perilipin (Plin) and very low-density lipoprotein receptor (Vldlr) in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (Vldlr) or n=8 mice (Plin)).Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (F4/80, Mcp1) or ASK1Δhep n=8 mice (Il-6)).Relative mRNA expression of collagen, type I, alpha 1 (Col1a1) (ASK1F/F n=11 mice; ASK1Δhep n=15 mice) and transforming growth factor beta 1 (Tgfβ1) (ASK1F/F n=6 mice; ASK1Δhep n=8 mice) in liver of HFD-fed mice.Representative images of liver sections stained with Sirius Red (scale bar represents 100 µm)quantification of Sirius Red positive area in liver of mice fed a HFD (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).Representative images of liver sections stained with α-SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).Liver triglyceride (TG) content (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) and quantification of Sirius Red positive area in livers (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) of 15 months old chow-fed mice. Values are expressed as mean ± SEM. #


Figure 4Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) . Representative immunofluorescence images of liver sections stained for LC3 - II punctate ( red ) and nuclei ( DAPI , blue ) in mice . Scale bar represents 100 µm .

O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).Representative immunofluorescence images of liver sections stained for LC3-II punctate (red) and nuclei (DAPI, blue) in mice. Scale bar represents 100 µm.


Figure 5Primary hepatocytes isolated from chow - fed ASK1F / F ( ) and ASK1Δhep ( ) mice were treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) . Colocalization of LC3 - II punctate ( red ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in primary hepatocytes treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf . Scale bar represents 100 µm . Values are expressed as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Primary hepatocytes isolated from chow-fed ASK1F/F () and ASK1Δhep () mice were treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates).Colocalization of LC3-II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in primary hepatocytes treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf. Scale bar represents 100 µm. Values are expressed as mean ± SEM.


Figure 6Western blots and quantification of respective protein levels in liver of HFD - fed mice treated with or without ASK1 inhibitor ( n = 4 mice per treatment ) . Representative images of liver sections stained with H & E ( scale bar represents 100 µm ) liver triglyceride ( TG ) content of HFD - fed mice treated with or without ASK1 inhibitor ( n = 8 mice per treatment ) .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Western blots and quantification of respective protein levels in liver of HFD-fed mice treated with or without ASK1 inhibitor (n=4 mice per treatment).Representative images of liver sections stained with H&E (scale bar represents 100 µm)liver triglyceride (TG) content of HFD-fed mice treated with or without ASK1 inhibitor (n=8 mice per treatment).


Figure 7Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm . Liver triglyceride ( TG ) content ( ASK1F / F n = 11 mice ; ASK1 + hep n = 10 mice ) in livers of mice fed a HFD for 20 weeks . Western blots and quantification of protein levels of respective targets in liver of HFD - fed mice ( n = 8 mice per genotype for pJNK and ATG12 , n = 4 mice per genotype for pBCN1 ) . Quantification of Sirius Red positive area ( ASK1F / F n = 11 mice ; ASK1 + hep n = 9 mice ) in livers of CCl4 - injected mice . Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM . # Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.Liver triglyceride (TG) content (ASK1F/F n=11 mice; ASK1+hep n=10 mice) in livers of mice fed a HFD for 20 weeks.Western blots and quantification of protein levels of respective targets in liver of HFD-fed mice (n=8 mice per genotype for pJNK and ATG12, n=4 mice per genotype for pBCN1).Quantification of Sirius Red positive area (ASK1F/F n=11 mice; ASK1+hep n=9 mice) in livers of CCl4-injected mice.Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM. #Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM.


Figure 1 A Whole mount FISH for piwi - 1 and immunostaining for BrdU at 1 - day , 1 - week and 5 - weeks post - BrdU administration . White boxes denote the magnified regions shown in the bottom panels . Scale bars , 30 µm . B Quantification of the percentage of BrdU label - retaining neoblasts . Data are presented as mean ± s . d . ( n = 4 - 6 animals per time point ) . C Double pulse experiment showing colocalization of BrdU after a 2 - week chase period with EdU after a 1 - day chase period . White boxes denote the magnified region shown on the left . Dashed lines mark the edges of the animal and white arrows mark colocalization . Scale bar , 50 µm .

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Figure 1 A Whole mount FISH for piwi-1 and immunostaining for BrdU at 1-day, 1-week and 5-weeks post-BrdU administration. White boxes denote the magnified regions shown in the bottom panels. Scale bars, 30 µm.  B Quantification of the percentage of BrdU label-retaining neoblasts. Data are presented as mean ± s.d. (n = 4-6 animals per time point).  C Double pulse experiment showing colocalization of BrdU after a 2-week chase period with EdU after a 1-day chase period. White boxes denote the magnified region shown on the left. Dashed lines mark the edges of the animal and white arrows mark colocalization. Scale bar, 50 µm.


Figure 2 A Dissociated cells from unirradiated , 1 dpi or 7 dpi animals ( 60 Gy of irradiation ) were stained with Hoechst ( DNA ) and Pyronin Y ( RNA ) and sorted by FACS to reveal an RNAlow population . Percentages indicate the proportion of events within each gate . B Cell size and nucleus : cytoplasm ratio measured by CellMask ( cytoplasm ) and DAPI ( nucleus ) staining of FACS sorted cells at homeostasis ( n ≥ 47 per sample ) . Representative images are shown , with dashed lines to indicate the area measured . Scale bars , 5 µm . C EdU pulse - chase time course of label - retention in sorted RNAlow and S / G2 / M neoblasts ( n ≥ 3 individual experiments per time point ) . Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S / G2 / M or RNAlow neoblasts , respectively . Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S / G2 / M neoblasts . D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C . Red boxes denote examples of EdU - cells . Scale bars , 5 µm .

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

Figure 2 A Dissociated cells from unirradiated, 1 dpi or 7 dpi animals (60 Gy of irradiation) were stained with Hoechst (DNA) and Pyronin Y (RNA) and sorted by FACS to reveal an RNAlow population. Percentages indicate the proportion of events within each gate.  B Cell size and nucleus:cytoplasm ratio measured by CellMask (cytoplasm) and DAPI (nucleus) staining of FACS sorted cells at homeostasis (n ≥ 47 per sample). Representative images are shown, with dashed lines to indicate the area measured. Scale bars, 5 µm.  C EdU pulse-chase time course of label-retention in sorted RNAlow and S/G2/M neoblasts (n ≥ 3 individual experiments per time point). Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S/G2/M or RNAlow neoblasts, respectively. Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S/G2/M neoblasts.  D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C. Red boxes denote examples of EdU- cells. Scale bars, 5 µm.


Figure 3 A Area measurements of cells from the RNAlow , G1 and S / G2 / M gates in intact animals and at 6 and 48 hpa ( n ≥ 28 for each sample in the RNAlow plot , n ≥ 24 in the G1 plot , n ≥ 42 in the S / G2 / M plot ) . B RNAlow neoblast size in intact animals and at 48 , 72 and 96 hpa ( n ≥ 54 ) . C RNAlow neoblast size in intact animals and at 48 hours following amputation , poke or incision injuries ( n ≥ 61 ) . D RNAlow neoblast size in unirradiated animals and at 1 , 2 and 3 dpi ( 12 . 5 Gy . of irradiation , n ≥ 52 ) .

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Figure 3 A Area measurements of cells from the RNAlow, G1 and S/G2/M gates in intact animals and at 6 and 48 hpa (n ≥ 28 for each sample in the RNAlow plot, n ≥ 24 in the G1 plot, n ≥ 42 in the S/G2/M plot).  B RNAlow neoblast size in intact animals and at 48, 72 and 96 hpa (n ≥ 54).  C RNAlow neoblast size in intact animals and at 48 hours following amputation, poke or incision injuries (n ≥ 61).  D RNAlow neoblast size in unirradiated animals and at 1, 2 and 3 dpi (12.5 Gy. of irradiation, n ≥ 52).


Figure 4 A Representative images of RNAlow , G1 and S / G2 / M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa . All images shown are from samples processed in the same experiment . Intact images are duplicated from Figure 2B . White arrows mark projections . Percentage of cells displaying projections in each condition is noted ( n > 100 cells counted ) . Scale bars , 5 µm . B Average projection length in RNAlow , G1 and S / G2 / M cells from intact animals and at 6 and 48 hpa ( n ≥ 13 ) . C - E Quantification of the number of projections per cell ( blue data ) and the sum total projection length per cell ( orange data ) in RNAlow ( C ) , G1 ( D ) or S / G2 / M ( E ) cells isolated from intact animals and at 6 and 48 hpa ( n ≥ 23 for projections / cell , n ≥ 10 for Σ projection length / cell ) .

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Figure 4 A Representative images of RNAlow, G1 and S/G2/M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa. All images shown are from samples processed in the same experiment. Intact images are duplicated from Figure 2B. White arrows mark projections. Percentage of cells displaying projections in each condition is noted (n > 100 cells counted). Scale bars, 5 µm. B Average projection length in RNAlow, G1 and S/G2/M cells from intact animals and at 6 and 48 hpa (n ≥ 13). C-E Quantification of the number of projections per cell (blue data) and the sum total projection length per cell (orange data) in RNAlow (C), G1 (D) or S/G2/M (E) cells isolated from intact animals and at 6 and 48 hpa (n ≥ 23 for projections/cell, n ≥ 10 for Σ projection length/cell).


Figure 5 B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment . C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals ( n ≥ 55 ) . D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals ( n = 5 - 10 ) . E RNAlow neoblast size in intact animals and at 48 hpa in control , raptor or rictor KD animals ( n ≥ 54 ) . F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals . Percentages indicate the proportion of events within each gate .

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

Figure 5 B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment.  C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals (n ≥ 55).  D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals (n = 5-10).  E RNAlow neoblast size in intact animals and at 48 hpa in control, raptor or rictor KD animals (n ≥ 54).  F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals. Percentages indicate the proportion of events within each gate.


Figure 6A Representative WISH images for Lrig - 1 . B RNAlow neoblast size in intact animals following control or Lrig - 1 knockdown ( n ≥ 53 technical replicates ) . C Quantification of H3P immunostaining in intact animals following control or Lrig - 1 knockdown ( n = 9 - 10 technical replicates ) . D Quantification of H3P immunostaining at homeostasis ( fixed at 7 days post - RNAi , white region ) and at 48 hpa ( amputated at 5 days post - RNAi and fixed at 7 days post - RNAi , yellow region ) during the first round of regeneration in control or Lrig - 1 KD animals ( n = 10 - 17 ) . E Quantification of unpigmented blastema area ( as % total tissue area ) in regenerating control or Lrig - 1 KD animals from 2 dpa to 7 dpa during the first round of regeneration ( n = 10 animals per condition per time point ) . The same animals were used for all timepoints . Representative images from 7 dpa are shown . Scale bars , 500 µm . F Quantification of H3P immunostaining at homeostasis ( fixed at 14 days post - RNAi , white region ) and at 48 hpa ( amputated at 12 days post - RNAi and fixed at 14 days post - RNAi , yellow region ) during a second round of regeneration in control or Lrig - 1 KD animals ( n = 10 - 18 ) . G Quantification of unpigmented blastema area ( as % total tissue area ) in regenerating control or Lrig - 1 KD animals from 2 dpa to 7 dpa during the second round of regeneration ( n = 9 - 10 animals per condition per time point ) . The same animals were used for all timepoints . Representative images from 7 dpa are shown . Scale bars , 500 µm .

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Figure 6A Representative WISH images for Lrig-1. B RNAlow neoblast size in intact animals following control or Lrig-1 knockdown (n ≥ 53 technical replicates).  C Quantification of H3P immunostaining in intact animals following control or Lrig-1 knockdown (n = 9-10 technical replicates).  D Quantification of H3P immunostaining at homeostasis (fixed at 7 days post-RNAi, white region) and at 48 hpa (amputated at 5 days post-RNAi and fixed at 7 days post-RNAi, yellow region) during the first round of regeneration in control or Lrig-1 KD animals (n = 10-17).  E Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the first round of regeneration (n = 10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.  F Quantification of H3P immunostaining at homeostasis (fixed at 14 days post-RNAi, white region) and at 48 hpa (amputated at 12 days post-RNAi and fixed at 14 days post-RNAi, yellow region) during a second round of regeneration in control or Lrig-1 KD animals (n = 10-18).  G Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the second round of regeneration (n = 9-10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.


Figure 1 A ) Transcriptome comparison of hemocytes from stage 16 embryos ( E16 ) and wandering 3rd instar larvae ( WL ) . The x - axis is the average genes expression levels ( n = 3 ) and the y - axis the Log2 Fold Change WL / E16 . P - values are indicated with the color code . Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in hemocytes Scatter plots as in ( A ) highlighting in black subsets of known genes associated with the GO terms extracellular matrix Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in phagocytosis F - G ) Phagocytosis assay on E16 ( F ) and WL hemocytes ( G ) srp ( hemo ) - moesin - RFP . The beads ( in green ) are phagocytosed by the hemocytes ( in red ) . The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure . Full stacks are displayed and the scale bars represent 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 A) Transcriptome comparison of hemocytes from stage 16 embryos (E16) and wandering 3rd instar larvae (WL). The x-axis is the average genes expression levels (n=3) and the y-axis the Log2 Fold Change WL/E16. P-values are indicated with the color code.  Scatter plots as in (A) highlighting in black subsets of known genes expressed in hemocytes  Scatter plots as in (A) highlighting in black subsets of known genes associated with the GO terms extracellular matrix  Scatter plots as in (A) highlighting in black subsets of known genes expressed in phagocytosis  F-G) Phagocytosis assay on E16 (F) and WL hemocytes (G) srp(hemo)-moesin-RFP. The beads (in green) are phagocytosed by the hemocytes (in red). The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure. Full stacks are displayed and the scale bars represent 20 µm.


Figure 2 A ) UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL ( NI dataset ) . D ) Top 5 markers of each cluster . The expression levels is represented by the gradient of purple levels ) and the percentage of cells with the size of the dots .

O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A) UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL (NI dataset). D) Top 5 markers of each cluster. The expression levels is represented by the gradient of purple levels) and the percentage of cells with the size of the dots.


Figure 3 A ) Identification of the position ( circulating / resident ) of the clusters within the larva by qPCR . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment . Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment ( n = 5 , mean + / - SD are represented on the graph ) . The p - values are estimated by bilateral paired student test B ) Circulating and resident hemocytes ( top and bottom panels , respectively ) from Mimic - Lsp1beta - MI05460 WL , which express Lsp1beta tagged with GFP . The immunolabelling was done with anti - GFP ( in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI and GFP , the right panels show the GFP alone . The scale bars represent 10 µm . C ) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC - Lsp1beta - MI05460 larvae . The OregonR value indicates the background level . The number of cells included in the analysis is displayed on the x - axis label and were quantified from two independent preparation , p - values were estimated after variance analysis with bilateral student test for equal variance . D ) Resident hemocytes located around the oenocytes or along the muscles ( top and bottom panels , respectively ) from WL srp ( hemo ) - moesin - RFP , that express moesin tagged with RFP in hemocytes . The immunolabelling was done with anti - RFP ( in red ) , anti - Pendulin ( Pen , in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI , Pen and RFP , the middle panels RFP alone and the right panels Pen alone . Arrow heads in the top panels indicate cells co - expressing RFP and Pen . The scale bars represent 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A) Identification of the position (circulating/resident) of the clusters within the larva by qPCR. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment. Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment (n=5, mean +/- SD are represented on the graph). The p-values are estimated by bilateral paired student test  B) Circulating and resident hemocytes (top and bottom panels, respectively) from Mimic-Lsp1beta-MI05460 WL, which express Lsp1beta tagged with GFP. The immunolabelling was done with anti-GFP (in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI and GFP, the right panels show the GFP alone. The scale bars represent 10 µm. C) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC-Lsp1beta-MI05460 larvae. The OregonR value indicates the background level. The number of cells included in the analysis is displayed on the x-axis label and were quantified from two independent preparation, p-values were estimated after variance analysis with bilateral student test for equal variance. D) Resident hemocytes located around the oenocytes or along the muscles (top and bottom panels, respectively) from WL srp(hemo)-moesin-RFP, that express moesin tagged with RFP in hemocytes. The immunolabelling was done with anti-RFP (in red), anti-Pendulin (Pen, in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, Pen and RFP, the middle panels RFP alone and the right panels Pen alone. Arrow heads in the top panels indicate cells co-expressing RFP and Pen. The scale bars represent 50 µm.


Figure 4A ) Heatmap representing the z - score for the top 5 regulons of each cluster determined with SCENIC . The dendrogram on the left side of the panel indicates the correlation between the regulons across the dataset . The z - score is indicated with a gradient from magenta ( z - score < 0 , the regulon is repressed ) to yellow ( z - score > 0 , the regulon is active ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Heatmap representing the z-score for the top 5 regulons of each cluster determined with SCENIC. The dendrogram on the left side of the panel indicates the correlation between the regulons across the dataset. The z-score is indicated with a gradient from magenta (z-score < 0, the regulon is repressed) to yellow (z-score > 0, the regulon is active).


Figure 5A ) UMAP projection representing the 16 clusters of cells identified in the WI sample . Note the presence of two additional clusters , LMLM - 1 and LM - 2 , compared to the NI sample . The two clusters correspond to the lamellocytes . B ) Estimation of the number of cells of each cluster per larva in normal condition ( NI , orange ) and after wasp infestation ( WI , blue ) . For each cluster , the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets . D ) Dot plot representing the expression levels ( gradient of red levels ) and the percentage of cells ( size of the dots ) that express the top 5 markers of each cluster in the WI dataset .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) UMAP projection representing the 16 clusters of cells identified in the WI sample. Note the presence of two additional clusters, LMLM-1 and LM-2, compared to the NI sample. The two clusters correspond to the lamellocytes. B) Estimation of the number of cells of each cluster per larva in normal condition (NI, orange) and after wasp infestation (WI, blue). For each cluster, the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets. D) Dot plot representing the expression levels (gradient of red levels) and the percentage of cells (size of the dots) that express the top 5 markers of each cluster in the WI dataset.


Figure 6 A ) Scatter plot comparing the transcriptome of the two lamellocyte clusters , deduced from the WI dataset . The x - axis is the average expression levels of the genes and the y - axis the log2 fold change ( LM - 1 / LM - 2 ) . The genes significantly enriched in the LM - 1 cluster are highlighted in red ( p < 10 - 6 ) , those significantly enriched in the LM - 2 cluster in orange ( p < 10 - 6 ) . C ) Expression levels ( gradient of color ) and the percentage of cells ( size of the dots ) for the top 10 markers of lamellocytes ( in black ) , of markers specific to LM - 1 compared to LM - 2 ( in red ) and specific to LM - 2 compared to LM - 1 ( in orange ) . Note that most markers enriched in LM - 1 compared to LM - 2 are exclusively expressed in LM - 1 while most markers enriched in LM - 2 compared to LM - 1 are also expressed in the plasmatocyte and crystal cell clusters . D ) Hemocytes from WL OregonR infested by wasp . The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea ( in gray , top panels ) and Shot ( in gray , lower panels ) . Phalloidin - FITC ( in green ) labels the actin filament particularly abundant in lamellocytes ( Tokusumi et al . , 2009 ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI , FITC and the lamellocyte markers , the middle panels show the FITC alone and the right panels the lamellocyte markers alone . The scale bars represent 20µm .

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

Figure 6 A) Scatter plot comparing the transcriptome of the two lamellocyte clusters, deduced from the WI dataset. The x-axis is the average expression levels of the genes and the y-axis the log2 fold change (LM-1/LM-2). The genes significantly enriched in the LM-1 cluster are highlighted in red (p<10-6), those significantly enriched in the LM-2 cluster in orange (p<10-6).  C) Expression levels (gradient of color) and the percentage of cells (size of the dots) for the top 10 markers of lamellocytes (in black), of markers specific to LM-1 compared to LM-2 (in red) and specific to LM-2 compared to LM-1 (in orange). Note that most markers enriched in LM-1 compared to LM-2 are exclusively expressed in LM-1 while most markers enriched in LM-2 compared to LM-1 are also expressed in the plasmatocyte and crystal cell clusters.  D) Hemocytes from WL OregonR infested by wasp. The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea (in gray, top panels) and Shot (in gray, lower panels). Phalloidin-FITC (in green) labels the actin filament particularly abundant in lamellocytes (Tokusumi et al., 2009) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, FITC and the lamellocyte markers, the middle panels show the FITC alone and the right panels the lamellocyte markers alone. The scale bars represent 20µm.


Figure 8 A ) Expression levels of the cluster markers in the hemolymph of WI larvae compared to NI larvae . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the graph on the right panel , indicates the Log2 of the ratio between the expression level in WI versus NI . Positive values indicate an up - regulation upon wasp infestation and negative values a down - regulation ( n = 3 , pool of 10 larvae per replicate , mean + / - SD are represented on the graph ) . The p - value are estimated by bilateral student test and indicated as follow : ns = not - significant ( > 0 . 05 ) , " * " = p [ 0 . 05 - 0 . 01 [ , " * * " = p [ 0 . 01 - 0 . 001 [ . B , C ) Expression levels of the cluster markers increasing in WI compared to NI according to ( A ) during the progression of the immune response to wasp infestation . Collections were carried out at t0 ( time of infestation , L2 ) , 24 h ( early L3 ) , 48 h ( mid L3 ) and 72 h ( WL ) after wasp infestation ( n = 3 ) . The time line is on the x - axis and the normalised expression levels on the y - axis . The expression levels were normalised to the levels at t0 . The dotted lines indicate t0 relative level ( = 1 ) . The markers that do not display strong variability during the timeline are presented in ( B ) and the ones showing a strong up - regulation are in ( C ) . D ) Expression levels during the infestation timeline as in ( B , C ) for the markers of proliferation . E ) Weighted dot plot representing the enrichment ( Log2 fold change ) of the markers in hemocytes originating from the lymph gland ( DotGFP - WI ) compared to embryonic hemocytes ( srpGFP - WI ) from WI larva . The lymph gland hemocytes and embryonic hemocytes were traced using the lymph gland driver DotGal4 and the embryonic driver Srp ( hemo ) Gal4 respectively combined with a lineage tracing construct . The relative expression levels are indicated by the size of the dots and the pvalue of the log2 fold change with the gradient dark blue ( not significant ) to light blue ( p < 10 - 3 ) . The p - value are estimated by bilateral student test and indicated as follow : ns = not - significant ( > 0 . 05 ) , " * " = p [ 0 . 05 - 0 . 01 [ , " * * " = p [ 0 . 01 - 0 . 001 [ , " * * * " = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 A) Expression levels of the cluster markers in the hemolymph of WI larvae compared to NI larvae. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the graph on the right panel, indicates the Log2 of the ratio between the expression level in WI versus NI. Positive values indicate an up-regulation upon wasp infestation and negative values a down-regulation (n=3, pool of 10 larvae per replicate, mean +/- SD are represented on the graph). The p-value are estimated by bilateral student test and indicated as follow: ns = not-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[.  B,C) Expression levels of the cluster markers increasing in WI compared to NI according to (A) during the progression of the immune response to wasp infestation. Collections were carried out at t0 (time of infestation, L2), 24 h (early L3), 48 h (mid L3) and 72 h (WL) after wasp infestation (n=3). The time line is on the x-axis and the normalised expression levels on the y-axis. The expression levels were normalised to the levels at t0. The dotted lines indicate t0 relative level (=1). The markers that do not display strong variability during the timeline are presented in (B) and the ones showing a strong up-regulation are in (C). D) Expression levels during the infestation timeline as in (B,C) for the markers of proliferation.  E) Weighted dot plot representing the enrichment (Log2 fold change) of the markers in hemocytes originating from the lymph gland (DotGFP-WI) compared to embryonic hemocytes (srpGFP-WI) from WI larva. The lymph gland hemocytes and embryonic hemocytes were traced using the lymph gland driver DotGal4 and the embryonic driver Srp(hemo)Gal4 respectively combined with a lineage tracing construct. The relative expression levels are indicated by the size of the dots and the pvalue of the log2 fold change with the gradient dark blue (not significant) to light blue (p<10-3). The p-value are estimated by bilateral student test and indicated as follow: ns = not-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[, "***" = p <0.001].


Figure 1B Western blots of whole - cell extracts showing efficient degradation of PDS5A or PDS5B ( left , right ) with dTAG treatment ( at different time points ) . D ChIP - seq signal for FLAG - PDS5A and FLAG - PDSB ( ChIP assays were performed with antibodies against Flag and PDS5A / B ) centered at TSS of promoters ( ± 3 kb ) across all peaks called for each of the proteins in DMSO treated PDS5A - dTAG or PDS5B - dTAG cells , showing the drastic reduction in PDS5A / B enrichment on chromatin after degradation . Top : normalized signal intensities for each protein . F ChIP - qPCR using Flag antibody demonstrating complete depletion of PDS5A or PDS5B ( left , right ) in H19 / IGF2 imprinting locus and the promoter region of PCDH10 , NC represents a negative binding site . Data are represented as mean ± SD from three biological replicates .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B Western blots of whole-cell extracts showing efficient degradation of PDS5A or PDS5B (left, right) with dTAG treatment (at different time points).D ChIP-seq signal for FLAG-PDS5A and FLAG-PDSB (ChIP assays were performed with antibodies against Flag and PDS5A/B) centered at TSS of promoters (± 3 kb) across all peaks called for each of the proteins in DMSO treated PDS5A-dTAG or PDS5B-dTAG cells, showing the drastic reduction in PDS5A/B enrichment on chromatin after degradation. Top: normalized signal intensities for each protein.F ChIP-qPCR using Flag antibody demonstrating complete depletion of PDS5A or PDS5B (left, right) in H19/IGF2 imprinting locus and the promoter region of PCDH10, NC represents a negative binding site. Data are represented as mean±SD from three biological replicates.


Figure 2B Heatmaps of signal intensities for FLAG - PDS5A , FLAG - PDS5B , CTCF , and RAD21 ChIP - seq data across all defined FLAG - PDS5A peaks . C Averaged ChIP - seq signal densities of FLAG - PDS5A , FLAG - PDS5B , RAD21 , and CTCF on PDS5A & PDS5B common sites and PDS5A / PDS5B unique sites . F Reciprocal Re - ChIP for PDS5A and PDS5B . The co - binding of PDS5A and PDS5B at PDS5A & PDS5B common sites were assayed by Re - ChIP - qPCR . The second ChIP for Re - ChIP was performed using antibodies against PDS5A , FLAG ( PDS5B ) , RAD21 , control IgG . RAD21 served as a positive control . Data are shown as fold enrichment versus IgG ( mean ± SD from three biological replicates ) .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B Heatmaps of signal intensities for FLAG-PDS5A, FLAG-PDS5B, CTCF, and RAD21 ChIP-seq data across all defined FLAG-PDS5A peaks.C Averaged ChIP-seq signal densities of FLAG-PDS5A, FLAG-PDS5B, RAD21, and CTCF on PDS5A & PDS5B common sites and PDS5A/PDS5B unique sites.F Reciprocal Re-ChIP for PDS5A and PDS5B. The co-binding of PDS5A and PDS5B at PDS5A&PDS5B common sites were assayed by Re-ChIP-qPCR. The second ChIP for Re-ChIP was performed using antibodies against PDS5A, FLAG(PDS5B), RAD21, control IgG. RAD21 served as a positive control. Data are shown as fold enrichment versus IgG (mean±SD from three biological replicates).


Figure 5A Validation of PDS5A ; PDS5B - dTAG cells for simultaneous depletion of PDS5A and PDS5B . B Chromatin fractionation assay showing the change of chromatin - bound level of RAD21 or CTCF after co - depletion of PDS5A and PDS5B . C Heatmaps displaying the ChIP - seq binding intensities for significantly changed RAD21 peaks after joint PDS5 proteins depletion ( left ) . Veen diagram showing RAD21 peaks identified in cells before and after co - depletion of PDS5A and PDS5B . D Changes of RAD21 signals at all loop anchors defined in PDS5A - dTAG cells or PDS5B - dTAG cells with DMSO treatment after PDS5A and PDS5B co - depletion , or either depletion . For PDS5A ; PDS5B - dTAG cells , we used the genomic positions of loop anchors identified in DMSO treated PDS5A - dTAG cells . E Differential RAD21 ChIP - seq signals of representative regions showing significantly down - regulated peaks after co - depletion of PDS5A and PDS5B . F Differential RAD21 ChIP - seq signals of representative regions showing significantly up - regulated peaks after co - depletion of PDS5A and PDS5B .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Validation of PDS5A;PDS5B-dTAG cells for simultaneous depletion of PDS5A and PDS5B.B Chromatin fractionation assay showing the change of chromatin-bound level of RAD21 or CTCF after co-depletion of PDS5A and PDS5B.C Heatmaps displaying the ChIP-seq binding intensities for significantly changed RAD21 peaks after joint PDS5 proteins depletion (left). Veen diagram showing RAD21 peaks identified in cells before and after co-depletion of PDS5A and PDS5B.D Changes of RAD21 signals at all loop anchors defined in PDS5A-dTAG cells or PDS5B-dTAG cells with DMSO treatment after PDS5A and PDS5B co-depletion, or either depletion. For PDS5A;PDS5B-dTAG cells, we used the genomic positions of loop anchors identified in DMSO treated PDS5A-dTAG cells. E Differential RAD21 ChIP-seq signals of representative regions showing significantly down-regulated peaks after co-depletion of PDS5A and PDS5B. F Differential RAD21 ChIP-seq signals of representative regions showing significantly up-regulated peaks after co-depletion of PDS5A and PDS5B.


Figure 6A , B Volcano plots showing gene expression changes after PDS5A ( A ) and PDS5B ( B ) degradation . Genes with significant changes ( padj < 0 . 05 ) are colored blue ( upregulated ) or red ( downregulated ) . C The overlap and discrepancy of PDS5A and PDS5B misregulated genes ( left ) . Heatmap displaying Benjamini - Hochberg adjusted p - value of shared genes down - regulated and up - regulated after PDS5A or PDS5B depletion ( right ) .

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Figure 6A, B Volcano plots showing gene expression changes after PDS5A (A) and PDS5B (B) degradation. Genes with significant changes (padj < 0.05) are colored blue (upregulated) or red (downregulated).C The overlap and discrepancy of PDS5A and PDS5B misregulated genes (left). Heatmap displaying Benjamini-Hochberg adjusted p-value of shared genes down-regulated and up-regulated after PDS5A or PDS5B depletion (right).