Figure 3 ( G ) Tumour growth ( in mm3 ) of FBXW7 + / + and FBXW7 - / - xenografts in nude mice ( n = 10 animals per group ) . Treatment with either vehicle , paclitaxel ( 1 , 5mg / kg ) or tigecycline ( 50mg / kg ) started at day 6 post - tumour - injection , and was administered three times per week . Error bars indicate SEM . ( H ) Representative images of the xenografts defined in ( G ) at day 15 .

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Figure 3(G) Tumour growth (in mm3) of FBXW7+/+ and FBXW7-/- xenografts in nude mice (n=10 animals per group). Treatment with either vehicle, paclitaxel (1,5mg/kg) or tigecycline (50mg/kg) started at day 6 post-tumour-injection, and was administered three times per week. Error bars indicate SEM. (H) Representative images of the xenografts defined in (G) at day 15.


Figure 5 ( D ) WB illustrating the levels of ATF4 in DLD - 1 cells treated as in ( C ) . GAPDH levels are shown as a loading control .

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Figure 5(D) WB illustrating the levels of ATF4 in DLD-1 cells treated as in (C). GAPDH levels are shown as a loading control.


Figure 1B ) Localization of the GFP - tagged PE synthesizing enzyme ( Psd ) targeted to the indicated organelles in choppΔ cells , grown in SD medium supplemented with 10 mM choline . Images shown are either a single Z - slice ( ER construct ) or a maximum intensity projection of several Z - sections ( other constructs ) ; LD , lipid droplets ; MM , mitochondrial matrix . A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image . C ) Localization of the GFP - tagged PC synthesizing enzyme ( Pmt ) targeted to the indicated organelles in choppΔ cells , grown in SD medium supplemented with 10 mM ethanolamine . Images shown are either a single Z - slice ( ER construct ) or a maximum intensity projection of several Z - sections ( other constructs ) . A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image .

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Figure 1B) Localization of the GFP-tagged PE synthesizing enzyme (Psd) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM choline. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs); LD, lipid droplets; MM, mitochondrial matrix. A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image. C) Localization of the GFP-tagged PC synthesizing enzyme (Pmt) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM ethanolamine. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs). A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image.


Figure 2B ) Five - fold serial dilutions of strains of the indicated genotypes ( wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes ) . Cells were grown on SD - HIS - LEU ( - HL ) medium ( KennedyOFF ) or SD - HL containing ethanolamine and choline ( KennedyON ) . C ) Thin layer chromatography ( TLC ) analysis of steady - state phospholipid profiles of cells of the indicated genotypes . Where Psd and / or Pmt are missing ( - ) , choppΔ cells were grown in the presence of ethanolamine and / or choline . Bands corresponding to the phospholipids PA , PS , PE , PI and PC are indicated .

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Figure 2B) Five-fold serial dilutions of strains of the indicated genotypes (wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes). Cells were grown on SD-HIS-LEU (-HL) medium (KennedyOFF) or SD-HL containing ethanolamine and choline (KennedyON).C) Thin layer chromatography (TLC) analysis of steady-state phospholipid profiles of cells of the indicated genotypes. Where Psd and/or Pmt are missing (-), choppΔ cells were grown in the presence of ethanolamine and/or choline. Bands corresponding to the phospholipids PA, PS, PE, PI and PC are indicated.


Figure 3C ) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes . Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions . This comparison included all six libraries where the enzymes are targeted to endosomes and / or mitochondria ( top left panel ) or six libraries with enzymes targeted to other organelles ( bottom left panel ) . Data - points for VPS13 are highlighted in red . Schematic illustrating Vps13 - mediated lipid transport at endosomes and mitochondria contact sites ( right panel ) .

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Figure 3C) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes. Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions. This comparison included all six libraries where the enzymes are targeted to endosomes and/or mitochondria (top left panel) or six libraries with enzymes targeted to other organelles (bottom left panel). Data-points for VPS13 are highlighted in red. Schematic illustrating Vps13-mediated lipid transport at endosomes and mitochondria contact sites (right panel).


Figure 4B ) Volcano plot comparing the number of transposon insertions per gene of all 12 libraries in KennedyON and OFF conditions . Genes significantly required in KennedyOFF conditions are highlighted in red ( p - value < 0 . 05 , log2 ( fold change ) < - 0 . 5 ) . Genes highlighted in the clusterogram in A ( top inset ) are indicated with blue arrows .

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Figure 4B) Volcano plot comparing the number of transposon insertions per gene of all 12 libraries in KennedyON and OFF conditions. Genes significantly required in KennedyOFF conditions are highlighted in red (p-value < 0.05, log2(fold change) < -0.5). Genes highlighted in the clusterogram in A (top inset) are indicated with blue arrows.


Figure 5B ) Requirement for mitochondrial lipid transport and biosynthesis genes in rewired libraries . Left panel , schematic depicting PE and cardiolipin ( CL ) synthesis in mitochondria . The inner mitochondrial putative lipid transporter Mdm31 is depicted in yellow . Grey arrows indicate the need for lipid transport from the ER or other organelles to mitochondria and transport between the MOM and MIM . Right panel , transposon numbers ( normalized to wt libraries ) for the indicated ( set of ) libraries for MDM31 , PGS1 , and CRD1 .

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Figure 5B) Requirement for mitochondrial lipid transport and biosynthesis genes in rewired libraries. Left panel, schematic depicting PE and cardiolipin (CL) synthesis in mitochondria. The inner mitochondrial putative lipid transporter Mdm31 is depicted in yellow. Grey arrows indicate the need for lipid transport from the ER or other organelles to mitochondria and transport between the MOM and MIM. Right panel, transposon numbers (normalized to wt libraries) for the indicated (set of) libraries for MDM31, PGS1, and CRD1.


Figure 6B ) Five - fold serial dilutions of strains of the indicated genotypes grown at 30 ° C on SD medium supplemented with 10 mM ethanolamine and / or 10 mM choline . Growth assays are representative images of biological replicates .

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Figure 6B) Five-fold serial dilutions of strains of the indicated genotypes grown at 30°C on SD medium supplemented with 10 mM ethanolamine and/or 10 mM choline. Growth assays are representative images of biological replicates.


Figure 7A ) Top : Co - localization of Csf1 - GFP cells in log phase with genomically integrated Pex10 - mCherry ( peroxisome marker ) and mtBFP plasmid ( mito marker ) . Bottom : Co - localization of log growing Csf1 - GFP cells with mCherry - Ubc6 ( tail - anchor ) ( ER marker ) and mtBFP plasmid ( mito marker ) . Scale bar , 2 µm . C ) Five - fold serial dilutions of strains of the indicated genotypes grown at indicated temperatures , in the presence or absence of 10 mM ethanolamine ( etn ) . E ) Bar graphs depicting the fraction of more saturated species ( defined as fatty acid chains with one double bond for ≤ 30 carbon chains or with two double bonds for ≥ 32 ) , and less unsaturated species ( defined as fatty acid chains with zero double bond for ≤ 30 carbon chains or with one double bond for ≥ 32 ) for each phospholipid class with different head groups . Bars represent the means of two or three biological replicates ( individual measurements are shown as points and triangles ) . p - values are calculated with a two - sample t - test .

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Figure 7A) Top: Co-localization of Csf1-GFP cells in log phase with genomically integrated Pex10-mCherry (peroxisome marker) and mtBFP plasmid (mito marker). Bottom: Co-localization of log growing Csf1-GFP cells with mCherry-Ubc6 (tail-anchor) (ER marker) and mtBFP plasmid (mito marker). Scale bar, 2 µm.C) Five-fold serial dilutions of strains of the indicated genotypes grown at indicated temperatures, in the presence or absence of 10 mM ethanolamine (etn).E) Bar graphs depicting the fraction of more saturated species (defined as fatty acid chains with one double bond for ≤30 carbon chains or with two double bonds for ≥32), and less unsaturated species (defined as fatty acid chains with zero double bond for ≤30 carbon chains or with one double bond for ≥32) for each phospholipid class with different head groups. Bars represent the means of two or three biological replicates (individual measurements are shown as points and triangles). p-values are calculated with a two-sample t-test.


Figure 1Figure 1 . Cytoplasmic conical structures in Thermococcus kodakaraensis . ( A ) A tomographic slice shows a side view of a conical structure ( c ) in the cytoplasm , rotated and enlarged in ( B ) . ( C ) A tomographic slice shows a side view of the cone in another cell , highlighting the subunit texture along the edge of the cone ( arrowheads ) . ( D , E ) Top views of a cone at different heights show the inner ring ( r ; enlarged in inset to highlight 19 - subunit structure ) and outer cone . ( F - H ) Sequential slices through a side view of a cone in a lysed cell show the relative location of the ring in the cone . ( I , J ) Different views of a 3D segmentation of the cone shown in ( A ) , embedded in a tomographic slice . s , S - layer ; m , membrane ; a , archaella ; rib , ribosomes ; rez , ribosome - excluding zone . Scale bars 100 nm ; scale bar in ( D ) applies to ( D , E ) ; scale bar in ( F ) applies to ( F - H ) ; segmentation not to scale .

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Figure 1Figure 1. Cytoplasmic conical structures in Thermococcus kodakaraensis. (A) A tomographic slice shows a side view of a conical structure (c) in the cytoplasm, rotated and enlarged in (B). (C) A tomographic slice shows a side view of the cone in another cell, highlighting the subunit texture along the edge of the cone (arrowheads). (D, E) Top views of a cone at different heights show the inner ring (r; enlarged in inset to highlight 19-subunit structure) and outer cone. (F-H) Sequential slices through a side view of a cone in a lysed cell show the relative location of the ring in the cone. (I, J) Different views of a 3D segmentation of the cone shown in (A), embedded in a tomographic slice. s, S-layer; m, membrane; a, archaella; rib, ribosomes; rez, ribosome-excluding zone. Scale bars 100 nm; scale bar in (D) applies to (D, E); scale bar in (F) applies to (F-H); segmentation not to scale.


Figure 2Figure 2 . Cones are associated with chemosensory arrays , ribosome - excluding zones and filament bundles . Tomographic slices show side ( A ) and top ( B , C ) views of cones ( c ) in three cells , highlighting associated chemosensory arrays ( ca ) , ribosome - excluding zones ( rez ) and filament bundles ( f ) . For a slice - by - slice view through the tomogram shown in ( A ) , see Movie S1 . ( D ) and ( E ) show different views of a 3D segmentation of the structures shown in ( C ) , with the conical structure in blue and the filament bundle in red . s , S - layer ; m , membrane ; a , archaellum ; o , other filaments . Scale bar 100 nm ( applies to A - C ) ; segmentation not to scale .

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Figure 2Figure 2. Cones are associated with chemosensory arrays, ribosome-excluding zones and filament bundles. Tomographic slices show side (A) and top (B, C) views of cones (c) in three cells, highlighting associated chemosensory arrays (ca), ribosome-excluding zones (rez) and filament bundles (f). For a slice-by-slice view through the tomogram shown in (A), see Movie S1. (D) and (E) show different views of a 3D segmentation of the structures shown in (C), with the conical structure in blue and the filament bundle in red. s, S-layer; m, membrane; a, archaellum; o, other filaments. Scale bar 100 nm (applies to A-C); segmentation not to scale.


Figure 3Figure 3 . Archaellum orientation with respect to the cell envelope . ( A - D ) show tomographic slices through side views of cones . In the bottom row , white lines show the angle of the archaellum with respect to the surface layer , and red dashed lines show the conserved distance from the archaellum at the membrane to the cone . Scale bar 100 nm ( applies to A - D ) .

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Figure 3Figure 3. Archaellum orientation with respect to the cell envelope. (A-D) show tomographic slices through side views of cones. In the bottom row, white lines show the angle of the archaellum with respect to the surface layer, and red dashed lines show the conserved distance from the archaellum at the membrane to the cone. Scale bar 100 nm (applies to A-D).


Figure 4Figure 4 . Structure of the T . kodakaraensis archaellum . ( A ) A sub - tomogram average of the archaellum reveals structural features , including four layers of density in the cytoplasm ( L1 - L3 , cone ) . CM , cytoplasmic membrane . The speculated identity of densities in the archaellum is proposed : archaellum fiber = FlaA / B flagellins ; integral membrane density = FlaJ ; L1 = FlaI ; L2 / L3 / cone = FlaH / FlaC / D / E . Scale bar 10 nm ( applies to A , B ) . Figure 4 . Structure of the T . kodakaraensis archaellum . ( B ) For comparison , a subtomogram average of the type IVa pilus machine from Myxococcus xanthus is shown ( adapted with permission from [ 8 ] ) . Arrows indicate components with recognized homology . OM , outer membrane ; IM , inner membrane . Scale bar 10 nm ( applies to A , B ) .

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Figure 4Figure 4. Structure of the T. kodakaraensis archaellum. (A) A sub-tomogram average of the archaellum reveals structural features, including four layers of density in the cytoplasm (L1-L3, cone). CM, cytoplasmic membrane. The speculated identity of densities in the archaellum is proposed: archaellum fiber = FlaA/B flagellins; integral membrane density = FlaJ; L1 = FlaI; L2/L3/cone = FlaH/FlaC/D/E.Scale bar 10 nm (applies to A, B).Figure 4. Structure of the T. kodakaraensis archaellum.(B) For comparison, a subtomogram average of the type IVa pilus machine from Myxococcus xanthus is shown (adapted with permission from [8]). Arrows indicate components with recognized homology. OM, outer membrane; IM, inner membrane. Scale bar 10 nm (applies to A, B).


Figure 4B . Growth of DiCre - 3D7 transgenic lines following LDH1 , HAD4 , and Lipin depletion with 100 nM rapamycin and 2 . 5 mM glucosamine was assessed relative to uninduced parasite cultures . The untransfected DiCre - 3D7 parasite line was used as a negative control . Data are presented as the mean ± standard error of the mean ( SEM ) from three independent replicates . C . Untargeted LC - MS analysis of LDH1 depletion ( one cycle of rapamycin and glucosamine treatment ) indicated minimal but selective metabolic changes . m / z feature intensities are plotted as the log2 ratio of treated / untreated and the Benjamini - Hochberg corrected P values across six biological replicates plotted as - log10 ( P ) . Below , LDH1 substrate and product ( pyruvate and lactate respectively ) abundance plotted as the ratio of treated / untreated from biological replicates ( mean ± SEM ) and the parental DiCre line presented as the negative control . D . Loss of Lipin ( PF3D7 _ 0303200 ) leads to accumulation of various lipid species ( three cycles of rapamycin and glucosamine treatment ) . Data are presented as the mean log2 fold change Lipin depleted ( treated ) enriched trophozoite - stage iRBCs to untreated controls from three to six biological replicates ( ± SD ) . The schematic depicts the proposed lipid phosphatase activity of Lipin . E . Loss of HAD4 leads to increases in intracellular levels of several nucleotides and intermediates in lower glycolysis . Data are represented as the mean log2 fold change of enriched trophozoite - stage iRBCs treated for three cycles compared to untreated controls from three / four biological replicates . Three nucleotide - nucleoside pairs are depicted in the top inset panels , HAD4 disruption leads to accumulation of the nucleotide - monophosphate and depletion of the nucleoside . The schematic depicts lower glycolysis and triose - phosphate interconversion , with the corresponding metabolite levels following HAD4 disruption depicted in the lower right inset .

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Figure 4B. Growth of DiCre-3D7 transgenic lines following LDH1, HAD4, and Lipin depletion with 100 nM rapamycin and 2.5 mM glucosamine was assessed relative to uninduced parasite cultures. The untransfected DiCre-3D7 parasite line was used as a negative control. Data are presented as the mean ± standard error of the mean (SEM) from three independent replicates.C. Untargeted LC-MS analysis of LDH1 depletion (one cycle of rapamycin and glucosamine treatment) indicated minimal but selective metabolic changes. m/z feature intensities are plotted as the log2 ratio of treated/untreated and the Benjamini-Hochberg corrected P values across six biological replicates plotted as -log10(P). Below, LDH1 substrate and product (pyruvate and lactate respectively) abundance plotted as the ratio of treated/untreated from biological replicates (mean±SEM) and the parental DiCre line presented as the negative control.D. Loss of Lipin (PF3D7_0303200) leads to accumulation of various lipid species (three cycles of rapamycin and glucosamine treatment). Data are presented as the mean log2 fold change Lipin depleted (treated) enriched trophozoite-stage iRBCs to untreated controls from three to six biological replicates (± SD). The schematic depicts the proposed lipid phosphatase activity of Lipin.E. Loss of HAD4 leads to increases in intracellular levels of several nucleotides and intermediates in lower glycolysis. Data are represented as the mean log2 fold change of enriched trophozoite-stage iRBCs treated for three cycles compared to untreated controls from three/four biological replicates. Three nucleotide-nucleoside pairs are depicted in the top inset panels, HAD4 disruption leads to accumulation of the nucleotide-monophosphate and depletion of the nucleoside. The schematic depicts lower glycolysis and triose-phosphate interconversion, with the corresponding metabolite levels following HAD4 disruption depicted in the lower right inset.


Figure 5B . Relative parasitemia was assessed for the untransfected DiCre 3D7 parental line and transfectant parasite lines with AMR1 under inducible disruption . Rapamycin / glucosamine treatment began on cycle zero and parasitemia determined by flow cytometry and compared to identical lines that were left untreated . Data represents the mean relative parasitemia ± SEM from three biological replicates ( y - axis ) across seven replication cycles ( x - axis ) . C . Metabolite profile following three cycles of rapamycin and glucosamine treatment . The abundance of isoprenoid biosynthestic intermediates ( 1 - deoxy - xylulose - 5 - P ( DOXP ) , methyl - erithritol - 4 - P ( MEP ) , methyl - erithritol - cyclo - 2 , 4 - PP ( MEcPP ) , and isopentyl - PP ) , and hemoglobin - like peptides are presented as the mean log2 fold change from four biological replicates ( ± SD ) .

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Figure 5B. Relative parasitemia was assessed for the untransfected DiCre 3D7 parental line and transfectant parasite lines with AMR1 under inducible disruption. Rapamycin/glucosamine treatment began on cycle zero and parasitemia determined by flow cytometry and compared to identical lines that were left untreated. Data represents the mean relative parasitemia ± SEM from three biological replicates (y-axis) across seven replication cycles (x-axis).C. Metabolite profile following three cycles of rapamycin and glucosamine treatment. The abundance of isoprenoid biosynthestic intermediates (1-deoxy-xylulose-5-P (DOXP), methyl-erithritol-4-P (MEP), methyl-erithritol-cyclo-2,4-PP (MEcPP), and isopentyl-PP), and hemoglobin-like peptides are presented as the mean log2 fold change from four biological replicates (±SD).


Figure 6C . HA - tagged SHMT - M detection via immunofluorescence microscopy . DAPI was used to visualise the nucleus , and MitoTracker CMXros used to illuminate the mitochondrion . Anti - HA signal partially colocalises with the mitochondrion .

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Figure 6C. HA-tagged SHMT-M detection via immunofluorescence microscopy. DAPI was used to visualise the nucleus, and MitoTracker CMXros used to illuminate the mitochondrion. Anti-HA signal partially colocalises with the mitochondrion.


Figure 7D . Pyrimidine biosynthetic intermediates accumulate when SHMT - M is disrupted compared to the DiCre parental line ( C ) following rapamycin / glucosamine treatment ( mean ± SEM ) . Pyrimidine biosynthetic disruption islinked to dihydroorotate dehydrogenase ( DHODH ) inhibition and blockage of the mitochondrial electron transport chain ( mETC ) . Ubiquinone ( Q ) is the necessary electron carrier for the mETC . E . Asexual growth when SHMT - M is depleted . Parasitemia was determined across seven replication cycles ( x - axis ) via flow cytometry and presented as the mean ± SEM from three independent experiments . Decylubiquinone ( Dub ) was added at 10 µM to determine if the growth defect observed was due to impaired ubiquinone synthesis or recycling .

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Figure 7D. Pyrimidine biosynthetic intermediates accumulate when SHMT-M is disrupted compared to the DiCre parental line (C) following rapamycin/glucosamine treatment (mean ±SEM). Pyrimidine biosynthetic disruption islinked to dihydroorotate dehydrogenase (DHODH) inhibition and blockage of the mitochondrial electron transport chain (mETC). Ubiquinone (Q) is the necessary electron carrier for the mETC.E. Asexual growth when SHMT-M is depleted. Parasitemia was determined across seven replication cycles (x-axis) via flow cytometry and presented as the mean ± SEM from three independent experiments. Decylubiquinone (Dub) was added at 10 µM to determine if the growth defect observed was due to impaired ubiquinone synthesis or recycling.


Figure 1IES retention PCR ( cropped inverted images ) . Four maternally - controlled IES and four non - maternally controlled IESs are shown . The IES + band represents retained IES ; the IES - band represents an excised IES ; additional bands are likely PCR artefacts . IRS is IES retention Score for the IESs calculated after whole genome sequencing .

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Figure 1IES retention PCR (cropped inverted images). Four maternally-controlled IES and four non-maternally controlled IESs are shown. The IES+ band represents retained IES; the IES- band represents an excised IES; additional bands are likely PCR artefacts. IRS is IES retention Score for the IESs calculated after whole genome sequencing.


Figure 3 ( A ) Schematic drawing of the life cycle stages of Paramecium tetraurelia . MIC and parental MAC are represented in red , representing the DAPI signal , and developing MAC ( dm ) is represented in green until fully developed , representing the GFP signal . ( B ) ISWI1 - tagged C - terminally with GFP localizes in the developing MAC as soon as developing new MACs ( panel Early Development ) become visible and remain there throughout late MAC development ( panel Late Development ) . Red : DAPI , Green : ISWI1 - GFP . Blue arrows identify developing MAC ; ( C ) Western blot analysis using anti - HA antibody after coimmunoprecipitation of ISWI1 - 3XFlagHA fusion protein . Non - transformed cells ( WT ) of the same strain were used as the negative control . 1 % of the total lysate was loaded as Input and 20 % of co - immunoprecipitated samples were loaded on 12 % SDS gel . ( D ) Volcano plot illustrating the distribution of proteins identified in label - free MS in WT Vs ISWI1 - 3XFlagHA . Significantly abundant proteins ( fold change ≥ 4 ) are highlighted in orange . ( E ) Western blot analysis using anti - HA and anti - GFP antibodies after coimmunoprecipitation of Ptiwi01 - 3XFlagHA fusion protein co - transformed with ISWI1 - GFP . Non - transformed cells ( WT ) of the same strain and ISWI1 - GFP fusion protein transformation were used as negative controls . 1 % of the total lysate was loaded as Input and 20 % of co - immunoprecipitated samples were loaded on 10 % SDS gel .

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 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 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 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) Schematic drawing of the life cycle stages of Paramecium tetraurelia. MIC and parental MAC are represented in red, representing the DAPI signal, and developing MAC (dm) is represented in green until fully developed, representing the GFP signal. (B) ISWI1-tagged C-terminally with GFP localizes in the developing MAC as soon as developing new MACs (panel Early Development) become visible and remain there throughout late MAC development (panel Late Development). Red: DAPI, Green: ISWI1-GFP. Blue arrows identify developing MAC;(C) Western blot analysis using anti-HA antibody after coimmunoprecipitation of ISWI1-3XFlagHA fusion protein. Non-transformed cells (WT) of the same strain were used as the negative control. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 12% SDS gel.(D) Volcano plot illustrating the distribution of proteins identified in label-free MS in WT Vs ISWI1-3XFlagHA. Significantly abundant proteins (fold change ≥ 4) are highlighted in orange.(E) Western blot analysis using anti-HA and anti-GFP antibodies after coimmunoprecipitation of Ptiwi01-3XFlagHA fusion protein co-transformed with ISWI1-GFP. Non-transformed cells (WT) of the same strain and ISWI1-GFP fusion protein transformation were used as negative controls. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 10%SDS gel.


figf1 ( b ) A53T α - synuclein ( α - syn ) expression was induced in stable PC12 cells for 48 h , then switched off for 24 h . Cells were treated with DMSO ( control ) , or with 0 . 94 μM , 4 . 7 μM or 47 μM of SMER10 ; 0 . 86 μM , 4 . 3 μM or 43 μM of SMER18 ; or 0 . 9 μM , 4 . 7 μM or 47 μM of SMER28 added in the switch - off period . A53T α - syn levels were analyzed by immunoblotting with antibody against HA ( left ) and densitometry analysis relative to actin ( right ) . Error bars denote s . e . m . P = 0 . 0917 , P = 0 . 009 , P = 0 . 0001 ( for increasing concentrations of SMER10 ) ; P = 0 . 0068 , P = 0 . 0023 , P = 0 . 0002 ( for increasing concentrations of SMER18 ) ; P = 0 . 0016 , P 0 . 0001 , P 0 . 0001 ( for increasing concentrations of SMER28 ) . ( c ) COS - 7 cells transfected with EGFP - HDQ74 construct were treated with DMSO ( control ) , 0 . 2 μM rapamycin ( Rap ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 48 h , and we assessed the proportions of apoptotic or aggregate - containing transfected cells . Error bars : 95 % confidence interval . P 0 . 0001 ( Rap and SMER28 ) , P = 0 . 013 ( SMER10 ) , P = 0 . 019 ( SMER18 ) ( aggregation ) ; P 0 . 0001 ( Rap , SMER18 and SMER28 ) , P = 0 . 004 ( SMER10 ) ( cell death ) . ( d ) Wild - type ( Atg5 + / + ) and knockout ( Atg5 − / − ) Atg5 MEFs were transfected with EGFP - HDQ74 and treated with DMSO ( control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 24 h , and we assessed the percentages of aggregate - containing transfected cells . The control ( DMSO - treated ) values were fixed at 1 for both cell lines . Error bars : 95 % confidence interval . P = 0 . 039 ( SMER10 ) , P 0 . 0001 ( SMER18 and SMER28 ) ( in Atg5 + / + cells ) ; P = 0 . 092 ( SMER10 ) , P = 0 . 271 ( SMER18 ) , P = 0 . 358 ( SMER28 ) ( in Atg5 − / − cells ) . Note that EGFP - HDQ74 aggregation was higher in Atg5 − / − than in Atg5 + / + cells ( Supplementary Fig . 3a ) . e ) The percentages of EGFP - positive COS - 7 cells with EGFP - HDQ74 aggregates as in c , treated with DMSO ( control ) , 10 μM lactacystin ( proteasome inhibitor ) or both 10 μM lactacystin along with either 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 48 h , expressed as odds ratios . Error bars : 95 % confidence interval . P 0 . 0001 ( control versus Lact ) , P = 0 . 014 ( SMER10 versus Lact ) , P 0 . 0001 ( SMER18 versus Lact ) , P = 0 . 001 ( SMER28 versus Lact ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

figf1(b) A53T α-synuclein (α-syn) expression was induced in stable PC12 cells for 48 h, then switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM or 47 μM of SMER10; 0.86 μM, 4.3 μM or 43 μM of SMER18; or 0.9 μM, 4.7 μM or 47 μM of SMER28 added in the switch-off period. A53T α-syn levels were analyzed by immunoblotting with antibody against HA (left) and densitometry analysis relative to actin (right). Error bars denote s.e.m. P = 0.0917, P = 0.009, P = 0.0001 (for increasing concentrations of SMER10); P = 0.0068, P = 0.0023, P = 0.0002 (for increasing concentrations of SMER18); P = 0.0016, P 0.0001, P 0.0001 (for increasing concentrations of SMER28).(c) COS-7 cells transfected with EGFP-HDQ74 construct were treated with DMSO (control), 0.2 μM rapamycin (Rap), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, and we assessed the proportions of apoptotic or aggregate-containing transfected cells. Error bars: 95% confidence interval. P 0.0001 (Rap and SMER28), P = 0.013 (SMER10), P = 0.019 (SMER18) (aggregation); P 0.0001 (Rap, SMER18 and SMER28), P = 0.004 (SMER10) (cell death).(d) Wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs were transfected with EGFP-HDQ74 and treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h, and we assessed the percentages of aggregate-containing transfected cells. The control (DMSO-treated) values were fixed at 1 for both cell lines. Error bars: 95% confidence interval. P = 0.039 (SMER10), P 0.0001 (SMER18 and SMER28) (in Atg5+/+ cells); P = 0.092 (SMER10), P = 0.271 (SMER18), P = 0.358 (SMER28) (in Atg5−/− cells). Note that EGFP-HDQ74 aggregation was higher in Atg5−/− than in Atg5+/+ cells (Supplementary Fig. 3a).e) The percentages of EGFP-positive COS-7 cells with EGFP-HDQ74 aggregates as in c, treated with DMSO (control), 10 μM lactacystin (proteasome inhibitor) or both 10 μM lactacystin along with either 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, expressed as odds ratios. Error bars: 95% confidence interval. P 0.0001 (control versus Lact), P = 0.014 (SMER10 versus Lact), P 0.0001 (SMER18 versus Lact), P = 0.001 (SMER28 versus Lact). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf2 ( a ) COS - 7 cells transfected with EGFP - LC3 construct for 4 h were treated with DMSO ( control ) , 0 . 2 μM rapamycin ( positive control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 16 h , and analyzed by fluorescence microscopy . The effects of treatment on the percentage of EGFP - positive cells with > 5 EGFP - LC3 vesicles are shown . Error bars denote s . e . m . P 0 . 0001 ( all SMERs ) . ( b ) HeLa cells stably expressing EGFP - LC3 were treated with DMSO ( control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 24 h . Confocal sections show cells containing EGFP - positive autophagic vesicles . Nuclei are stained with DAPI . Bar , 20 μM . ( c ) HeLa cells stably expressing EGFP - LC3 were treated for 4 h with DMSO ( control ) or 200 nM bafilomycin A1 ( Baf ) , or with 200 nM bafilomycin A1 and 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 . Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1 . Levels of EGFP - LC3 - II were determined by immunoblotting with antibody against EGFP ( above ) and densitometry analysis relative to actin ( below ) . Error bars denote s . e . m . P = 0 . 0259 ( baf ) , P 0 . 0001 ( SMER10 ) , P = 0 . 0003 ( SMER18 and SMER28 ) versus control ; P = 0 . 0025 ( SMER10 ) , P = 0 . 0218 ( SMER18 ) , P = 0 . 0195 ( SMER28 ) versus bafilomycin A1 . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

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

figf2(a) COS-7 cells transfected with EGFP-LC3 construct for 4 h were treated with DMSO (control), 0.2 μM rapamycin (positive control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 16 h, and analyzed by fluorescence microscopy. The effects of treatment on the percentage of EGFP-positive cells with >5 EGFP-LC3 vesicles are shown. Error bars denote s.e.m. P 0.0001 (all SMERs).(b) HeLa cells stably expressing EGFP-LC3 were treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h. Confocal sections show cells containing EGFP-positive autophagic vesicles. Nuclei are stained with DAPI. Bar, 20 μM.(c) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin A1 (Baf), or with 200 nM bafilomycin A1 and 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (above) and densitometry analysis relative to actin (below). Error bars denote s.e.m. P = 0.0259 (baf), P 0.0001 (SMER10), P = 0.0003 (SMER18 and SMER28) versus control; P = 0.0025 (SMER10), P = 0.0218 (SMER18), P = 0.0195 (SMER28) versus bafilomycin A1. ***P 0.001; **P 0.01; *P 0.05.


figf3 ( a - c ) Flies treated with 100 μM SMER10 ( a ) , 200 μM SMER18 ( b ) or 100 μM SMER28 ( c ) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO ( control ) alone ( 2 d after eclosion ) . Rhabdomere counts from all three independent experiments are included . n = 600 ommatidia ( SMER10 ) , n = 1 , 500 ommatidia ( SMER18 ) and n = 600 ommatidia ( SMER28 ) . Mann - Whitney test values P 0 . 0001 ( all SMERs ) . Student ' s t - test ( 1 - tailed ) P = 0 . 005 ( SMER10 ) , P = 0 . 004 ( SMER18 ) , P = 0 . 03 ( SMER28 ) comparing means of distributions from independent experiments . These SMER concentrations cause no overt toxicity to flies ( see Supplementary Methods ) . Distributions of DMSO - treated flies may vary when SMERs are treated in different experiments at different times . For instance , an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time - dependent .

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

figf3(a-c) Flies treated with 100 μM SMER10 (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO (control) alone (2 d after eclosion). Rhabdomere counts from all three independent experiments are included. n = 600 ommatidia (SMER10), n = 1,500 ommatidia (SMER18) and n = 600 ommatidia (SMER28). Mann-Whitney test values P 0.0001 (all SMERs). Student's t-test (1-tailed) P = 0.005 (SMER10), P = 0.004 (SMER18), P = 0.03 (SMER28) comparing means of distributions from independent experiments. These SMER concentrations cause no overt toxicity to flies (see Supplementary Methods). Distributions of DMSO-treated flies may vary when SMERs are treated in different experiments at different times. For instance, an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time-dependent.


figf4 ( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( a ) , 43 μM SMER18 ( b ) or 47 μM SMER28 ( c ) ) or both for the 8 h switch - off period - was analyzed by immunoblotting with antibody against HA ( above ) and densitometry analysis relative to actin ( below ) . The concentration of rapamycin is saturating for its effect on the clearance of A53T α - synuclein . Error bars denote s . e . m . P = 0 . 0025 ( Rap ) , P = 0 . 0018 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 0001 ( Rap or SMER10 versus SMER10 + Rap ) ( a ) ; P = 0 . 0069 ( Rap ) , P = 0 . 0498 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 0038 ( Rap versus SMER18 + Rap ) , P = 0 . 0007 ( SMER18 versus SMER18 + Rap ) ( b ) ; P 0 . 0001 ( Rap , SMER28 , Rap versus SMER28 + Rap , SMER28 versus SMER28 + Rap ) ( c ) . ( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates ( above ) and cell death ( below ) in COS - 7 cells , as in Figure 1c - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( d ) , 43 μM SMER18 ( e ) or 47 μM SMER28 ( f ) ) or both for 24 h - were expressed as odds ratios . Error bars : 95 % confidence interval . Aggregation ( top panels ) : P = 0 . 248 ( Rap ) , P = 0 . 217 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 001 ( Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 248 ( Rap ) , P = 0 . 543 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 008 ( Rap versus SMER18 + Rap ) , P = 0 . 002 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 248 ( Rap ) , P = 0 . 002 ( SMER28 ) , P 0 . 0001 ( SMER28 + Rap ) , P 0 . 0001 ( Rap versus SMER28 + Rap ) , P = 0 . 012 ( SMER28 versus SMER28 + Rap ) ( f ) . Cell death ( bottom panels ) : P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER10 , SMER10 + Rap , Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 002 ( Rap ) , P = 0 . 948 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 015 ( Rap versus SMER18 + Rap ) , P 0 . 0001 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER28 , SMER28 + Rap , Rap or SMER28 versus SMER28 + Rap ) ( f ) . Note that we have treated cells for a shorter time in this experiment ( 24 h ) , compared with Figure 1c ( 48 h ) . This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

figf4(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (a), 43 μM SMER18 (b) or 47 μM SMER28 (c)) or both for the 8 h switch-off period-was analyzed by immunoblotting with antibody against HA (above) and densitometry analysis relative to actin (below). The concentration of rapamycin is saturating for its effect on the clearance of A53T α-synuclein. Error bars denote s.e.m. P = 0.0025 (Rap), P = 0.0018 (SMER10), P 0.0001 (SMER10 + Rap), P 0.0001 (Rap or SMER10 versus SMER10 + Rap) (a); P = 0.0069 (Rap), P = 0.0498 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.0038 (Rap versus SMER18 + Rap), P = 0.0007 (SMER18 versus SMER18 + Rap) (b); P 0.0001 (Rap, SMER28, Rap versus SMER28 + Rap, SMER28 versus SMER28 + Rap) (c).(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (above) and cell death (below) in COS-7 cells, as in Figure 1c-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (d), 43 μM SMER18 (e) or 47 μM SMER28 (f)) or both for 24 h-were expressed as odds ratios. Error bars: 95% confidence interval. Aggregation (top panels): P = 0.248 (Rap), P = 0.217 (SMER10), P 0.0001 (SMER10 + Rap), P 0.001 (Rap or SMER10 versus SMER10 + Rap) (d); P = 0.248 (Rap), P = 0.543 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.008 (Rap versus SMER18 + Rap), P = 0.002 (SMER18 versus SMER18 + Rap) (e); P = 0.248 (Rap), P = 0.002 (SMER28), P 0.0001 (SMER28 + Rap), P 0.0001 (Rap versus SMER28 + Rap), P = 0.012 (SMER28 versus SMER28 + Rap) (f). Cell death (bottom panels): P = 0.002 (Rap), P 0.0001 (SMER10, SMER10 + Rap, Rap or SMER10 versus SMER10 + Rap) (d); P = 0.002 (Rap), P = 0.948 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.015 (Rap versus SMER18 + Rap), P 0.0001 (SMER18 versus SMER18 + Rap) (e); P = 0.002 (Rap), P 0.0001 (SMER28, SMER28 + Rap, Rap or SMER28 versus SMER28 + Rap) (f). Note that we have treated cells for a shorter time in this experiment (24 h), compared with Figure 1c (48 h). This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf5 ( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated for 24 h with either DMSO ( control ) , or with 47 μM SMER10 and its analogs ( SMER10a - c ) ( a ) , 43 μM SMER18 and its analogs ( SMER18a - l ) ( b ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( c ) - was analyzed by immunoblotting with anti - HA antibody ( above ) and densitometry analysis relative to actin ( below ) . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars denote s . e . m . P = 0 . 0058 ( SMER10a ) , P = 0 . 6736 ( SMER10b ) , P = 0 . 9507 ( SMER10c ) , P = 0 . 0481 ( SMER10 ) ( a ) ; P = 0 . 0006 ( SMER18a ) , P = 0 . 0249 ( SMER18b ) , P = 0 . 0167 ( SMER18c ) , P = 0 . 0117 ( SMER18d ) , P = 0 . 0269 ( SMER18e ) , P = 0 . 0165 ( SMER18f ) , P = 0 . 0148 ( SMER18g ) , P = 0 . 0011 ( SMER18h ) , P = 0 . 7369 ( SMER18i ) , P = 0 . 0012 ( SMER18j ) , P = 0 . 1531 ( SMER18k ) , P = 0 . 0006 ( SMER18l ) , P = 0 . 0001 ( SMER18 ) ( b ) ; P = 0 . 0014 ( SMER28a ) , P = 0 . 0002 ( SMER28b ) , P = 0 . 0001 ( SMER28d ) , P = 0 . 0048 ( SMER28h ) , P = 0 . 0002 ( SMER28i ) , P = 0 . 0162 ( SMER28j ) , P 0 . 0001 ( SMER28c , SMER28e - g , SMER28k , SMER28 ) ( c ) . ( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates in COS - 7 cells as in Figure 1c - treated for 48 h with either DMSO ( control ) , or with 47 μM SMER10 and its analog ( SMER10a ) ( d ) , 43 μM SMER18 and its analogs ( SMER18a , SMER18c - h , SMER18j , SMER18l ) ( e ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( f ) - were expressed as odds ratios . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars : 95 % confidence interval . P = 0 . 003 ( SMER10a ) , P = 0 . 004 ( SMER10 ) ( d ) ; P 0 . 0001 ( SMER18a , SMER18c , SMER18d , SMER18f , SMER18h ) , P = 0 . 009 ( SMER18e ) , P = 0 . 001 ( SMER18g ) , P = 0 . 382 ( SMER18j ) , P = 0 . 067 ( SMER18l ) , P = 0 . 031 ( SMER18 ) ( e ) ; P 0 . 0001 ( SMER28a , SMER28c , SMER28e - g , SMER28i - k , SMER28 ) , P = 0 . 574 ( SMER28b ) , P = 0 . 041 ( SMER28d ) , P = 0 . 002 ( SMER28h ) ( f ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

figf5(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a-c) (a), 43 μM SMER18 and its analogs (SMER18a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-k) (c)-was analyzed by immunoblotting with anti-HA antibody (above) and densitometry analysis relative to actin (below). All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars denote s.e.m. P = 0.0058 (SMER10a), P = 0.6736 (SMER10b), P = 0.9507 (SMER10c), P = 0.0481 (SMER10) (a); P = 0.0006 (SMER18a), P = 0.0249 (SMER18b), P = 0.0167 (SMER18c), P = 0.0117 (SMER18d), P = 0.0269 (SMER18e), P = 0.0165 (SMER18f), P = 0.0148 (SMER18g), P = 0.0011 (SMER18h), P = 0.7369 (SMER18i), P = 0.0012 (SMER18j), P = 0.1531 (SMER18k), P = 0.0006 (SMER18l), P = 0.0001 (SMER18) (b); P = 0.0014 (SMER28a), P = 0.0002 (SMER28b), P = 0.0001 (SMER28d), P = 0.0048 (SMER28h), P = 0.0002 (SMER28i), P = 0.0162 (SMER28j), P 0.0001 (SMER28c, SMER28e-g, SMER28k, SMER28) (c).(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Figure 1c-treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, SMER18c-h, SMER18j, SMER18l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f)-were expressed as odds ratios. All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars: 95% confidence interval. P = 0.003 (SMER10a), P = 0.004 (SMER10) (d); P 0.0001 (SMER18a, SMER18c, SMER18d, SMER18f, SMER18h), P = 0.009 (SMER18e), P = 0.001 (SMER18g), P = 0.382 (SMER18j), P = 0.067 (SMER18l), P = 0.031 (SMER18) (e); P 0.0001 (SMER28a, SMER28c, SMER28e-g, SMER28i-k, SMER28), P = 0.574 (SMER28b), P = 0.041 (SMER28d), P = 0.002 (SMER28h) (f). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


Figure 1 ( B ) Immunofluorescence ( IF ) validation of mintbody specificity . Mouse MC12 cells , which stably express H3K27me3 - mintbody ( sfGFP ) , are labelled with antibodies specific for H3K27me3 ( Cy5 ) and H3K9me3 ( Cy3 ) . DNA is stained with Hoechst33342 . Single confocal sections are shown . Arrows mark Xi . Scale bar = 10 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 O O O O O O O O O O O O O

Figure 1(B) Immunofluorescence (IF) validation of mintbody specificity. Mouse MC12 cells, which stably express H3K27me3-mintbody (sfGFP), are labelled with antibodies specific for H3K27me3 (Cy5) and H3K9me3 (Cy3). DNA is stained with Hoechst33342. Single confocal sections are shown. Arrows mark Xi. Scale bar = 10 μm.


Figure 2 ( D ) ESCs as in ( B ) were engineered to express H3K27me3 - mintbody ( SNAP / JF646 ) and H4K20me1 - mintbody ( mCherry ) . Cells were cultured in the absence of LIF for 2 days . Time - lapse images for 12 z - plane confocal stack were acquired every 1 h . Maximum intensity projection images are shown with elapse time ( hh : mm ) . Arrowheads mark Xi . Scale bar = 10

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 2(D) ESCs as in (B) were engineered to express H3K27me3-mintbody (SNAP/JF646) and H4K20me1-mintbody (mCherry). Cells were cultured in the absence of LIF for 2 days. Time-lapse images for 12 z-plane confocal stack were acquired every 1 h. Maximum intensity projection images are shown with elapse time (hh:mm). Arrowheads mark Xi. Scale bar = 10 μm


Figure 4 ( E ) H4K20me1 ( blue ) and H3K27me3 ( red ) accumulation across the Xi after 12 h of DOX treatment . The black line is a locally estimated scatterplot smoothing ( LOESS ) regression on all 10 - kb windows ( dots ) . Below each plot shown is the Xist locus ( green bar ) and Xist entry sites ( black bars ) .

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

Figure 4(E) H4K20me1 (blue) and H3K27me3 (red) accumulation across the Xi after 12 h of DOX treatment. The black line is a locally estimated scatterplot smoothing (LOESS) regression on all 10-kb windows (dots). Below each plot shown is the Xist locus (green bar) and Xist entry sites (black bars).


Figure 5 ( A ) Genome browser tracks showing H3K27me3 ( top ) and H4K20me1 ( bottom ) accumulation at a gene silenced rapidly ( Rnf12 ) or more slowly ( Pgk1 ) . Allele - specific tracks were overlaid ( B6 in red and Cast in blue ) . Note strong H4K20me1 bi - allelic pre - marking at gene bodies . ( B ) Average H3K27me3 ( top ) and H4K20me1 ( bottom ) enrichment at the B6 allele over initially active or inactive genes ± 30kb at the X chromosome . Shown is data for all time points . Data information : TSS : transcription start site ; TES : transcript end site .

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 O O O O O O O O O O O O O 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(A) Genome browser tracks showing H3K27me3 (top) and H4K20me1 (bottom) accumulation at a gene silenced rapidly (Rnf12) or more slowly (Pgk1). Allele-specific tracks were overlaid (B6 in red and Cast in blue). Note strong H4K20me1 bi-allelic pre-marking at gene bodies.(B) Average H3K27me3 (top) and H4K20me1 (bottom) enrichment at the B6 allele over initially active or inactive genes ± 30kb at the X chromosome. Shown is data for all time points. Data information: TSS : transcription start site ; TES : transcript end site.


Figure 6 ( B ) Representative image of IF / RNA FISH for Xist RNA and H4K20me1 in cells expressing XistFL or XistΔBC . Arrowheads point to the Xist RNA domains . Scale bar = 5 μm . ( C ) Graph represents the mean % + / - StDev of Xist RNA domains enriched for H4K20me1 ( left ) or H3K27me3 ( right ) in cells expressing XistWT or XistΔBC . Shown are averages from at least 2 independent experiments ; minimum of 50 Xist RNA domains were counted per experiment ; only P - values corresponding to significant differences from unpaired Student ' s t - test comparing mutants to Xist FL are indicated as * ( P - value < 0 . 05 ) . Data for H3K27me3 was extracted from published data ( Bousard et al . , 2019 ) .

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 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

Figure 6(B) Representative image of IF/RNA FISH for Xist RNA and H4K20me1 in cells expressing XistFL or XistΔBC. Arrowheads point to the Xist RNA domains. Scale bar = 5 μm.(C) Graph represents the mean % +/- StDev of Xist RNA domains enriched for H4K20me1 (left) or H3K27me3 (right) in cells expressing XistWT or XistΔBC. Shown are averages from at least 2 independent experiments; minimum of 50 Xist RNA domains were counted per experiment; only P-values corresponding to significant differences from unpaired Student's t-test comparing mutants to Xist FL are indicated as * (P-value < 0.05). Data for H3K27me3 was extracted from published data (Bousard et al., 2019).


Figure 1 ( A ) Morphological phenotypes of Wild type ( WT ) , mkk1 mkk2 and summ3 - 1 mkk1 mkk2 plants . Photos were taken on three - week - old soil - grown plants . ( B ) Trypan Blue staining of cell death in the indicated genotypes . ( C ) DAB staining of H2O2 accumulation in the indicated genotypes . ( D - E ) Expression of PR1 ( D ) and PR2 ( E ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results .

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 B-PANEL_START 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 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

Figure 1(A) Morphological phenotypes of Wild type (WT), mkk1 mkk2 and summ3-1 mkk1 mkk2 plants. Photos were taken on three-week-old soil-grown plants.(B) Trypan Blue staining of cell death in the indicated genotypes.(C) DAB staining of H2O2 accumulation in the indicated genotypes.(D-E) Expression of PR1 (D) and PR2 (E) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results.


Figure 3 ( A ) Morphological phenotypes of WT , summ3 - 17 , mekk1 - 1 and summ3 - 17 mekk1 - 1 plants grown on soil for three weeks . ( B - C ) Expression levels of PR1 ( B ) and PR2 ( C ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results . Two - week - old seedlings grown on half MS plates were used for assays in ( B ) - ( C ) . ( D ) Morphological phenotypes of WT , mpk4 - 3 , summ3 - 1 mpk4 - 3 and summ3 - 2 mpk4 - 3 plants . Photos were taken on three - week - old soil grown plants . ( E - F ) Expression levels of PR1 ( E ) and PR2 ( F ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results . Two - week - old seedlings grown on half MS plates were used for assays in ( E ) - ( F ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Morphological phenotypes of WT, summ3-17, mekk1-1 and summ3-17 mekk1-1 plants grown on soil for three weeks.(B-C) Expression levels of PR1 (B) and PR2 (C) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results. Two-week-old seedlings grown on half MS plates were used for assays in (B)-(C).(D) Morphological phenotypes of WT, mpk4-3, summ3-1 mpk4-3 and summ3-2 mpk4-3 plants. Photos were taken on three-week-old soil grown plants.(E-F) Expression levels of PR1 (E) and PR2 (F) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results. Two-week-old seedlings grown on half MS plates were used for assays in (E)-(F).


Figure 4 ( A ) Morphological phenotypes of transgenic plants over - expressing MEKK2 - FLAG protein in WT and summ3 - 17 background . Line 1 and 2 express MEKK2 - FLAG in WT background . Line 3 - 5 express MEKK2 - FLAG in the summ3 - 17 mutant background . The photograph shows four - week - old soil grown plants . Expression of MEKK2 - FLAG was detected by western blot using an anti - FLAG antibody . ( B - C ) Trypan Blue staining of cell death ( B ) in the indicated genotypes . ( B - C ) DAB staining of H2O2 accumulation ( C ) in the indicated genotypes . ( D - E ) Expression levels of PR1 ( D ) and PR2 ( E ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results .

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 B-PANEL_START 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(A) Morphological phenotypes of transgenic plants over-expressing MEKK2-FLAG protein in WT and summ3-17 background. Line 1 and 2 express MEKK2-FLAG in WT background. Line 3-5 express MEKK2-FLAG in the summ3-17 mutant background. The photograph shows four-week-old soil grown plants. Expression of MEKK2-FLAG was detected by western blot using an anti-FLAG antibody.(B-C) Trypan Blue staining of cell death (B) in the indicated genotypes.(B-C) DAB staining of H2O2 accumulation (C) in the indicated genotypes.(D-E) Expression levels of PR1 (D) and PR2 (E) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results.


Figure 5 ( A ) Quantification of luciferase activity in N . benthamiana leaves expressing CRCK3NLuc and MPK4CLuc or MPK6CLuc . Data were collected three days after infiltration . The data are shown as mean ± SD ( n = 8 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated twice with similar results . ( B ) Phosphorylation of CRCK3G390R by MPK4 . MPK4 was immunoprecipitated from wild type or MPK4 : : MPK4 - FLAG . After incubation with [ γ - 32P ] ATP and the immunoprecipitated MPK4 - FLAG protein in protein kinase buffer , CRCK3G390R was separated by SDS - PAGE . The autoradiograph of the gel is shown in the top panel , and immunoblot analysis of MPK4 - FLAG levels is shown in the bottom panel . Myelin basic protein ( MBP ) was used as a positive control . This experiment was repeated three times with similar results . ( C ) Western blot analysis of CRCK3 - FLAG proteins in wild type and mpk4 - 3 . Total protein was extracted from two - week - old CRCK3 - FLAG transgenic plants in WT and mpk4 background grown on ½ MS medium plates . The protein extracts were treated with or without λ - PMPase phosphatase . The proteins were separated by Phos - tagTM acrylamide gel electrophoresis and CRCK - FLAG proteins were detected by western blot analysis using an anti - FLAG antibody . Similar results were obtained in two independent experiments . pCRCK3 , phosphorylated CRCK3 - FLAG protein . ( D ) Western blot analysis of CRCK3 - FLAG and CRCK35S - 5A - FLAG proteins in transgenic plants of wild type background . Total protein was extracted from two - week - old plants grown on ½ MS medium plates . The protein extracts were treated with or without λ - PMPase phosphatase . The proteins were separated by Phos - tagTM acrylamide gel electrophoresis and the FLAG - tagged proteins were detected by western blot analysis using an anti - FLAG antibody . Similar results were obtained in two independent experiments . 5S - 5A stands for a change in five amino acids ( S172A , S181A , S188A , S195A , S202A ) .

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 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 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

Figure 5(A) Quantification of luciferase activity in N. benthamiana leaves expressing CRCK3NLuc and MPK4CLuc or MPK6CLuc. Data were collected three days after infiltration. The data are shown as mean ± SD (n=8) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated twice with similar results.(B) Phosphorylation of CRCK3G390R by MPK4. MPK4 was immunoprecipitated from wild type or MPK4::MPK4-FLAG. After incubation with [γ-32P] ATP and the immunoprecipitated MPK4-FLAG protein in protein kinase buffer, CRCK3G390R was separated by SDS-PAGE. The autoradiograph of the gel is shown in the top panel, and immunoblot analysis of MPK4-FLAG levels is shown in the bottom panel. Myelin basic protein (MBP) was used as a positive control. This experiment was repeated three times with similar results.(C) Western blot analysis of CRCK3-FLAG proteins in wild type and mpk4-3. Total protein was extracted from two-week-old CRCK3-FLAG transgenic plants in WT and mpk4 background grown on ½ MS medium plates. The protein extracts were treated with or without λ-PMPase phosphatase. The proteins were separated by Phos-tagTM acrylamide gel electrophoresis and CRCK-FLAG proteins were detected by western blot analysis using an anti-FLAG antibody. Similar results were obtained in two independent experiments. pCRCK3, phosphorylated CRCK3-FLAG protein.(D) Western blot analysis of CRCK3-FLAG and CRCK35S-5A-FLAG proteins in transgenic plants of wild type background. Total protein was extracted from two-week-old plants grown on ½ MS medium plates. The protein extracts were treated with or without λ-PMPase phosphatase. The proteins were separated by Phos-tagTM acrylamide gel electrophoresis and the FLAG-tagged proteins were detected by western blot analysis using an anti-FLAG antibody. Similar results were obtained in two independent experiments. 5S-5A stands for a change in five amino acids (S172A, S181A, S188A, S195A, S202A).


Figure 6 ( A ) In vivo pull - down of HA - tagged SUMM2 by FLAG - tagged CRCK3 . FLAG - tagged full - length or truncated CRCK3 and HA - tagged SUMM2 proteins were transiently expressed in N . bethamiana by agro - infiltration . FLAG - tagged CRCK3 proteins were immunoprecipitated with anti - FLAG conjugated beads ( Sigma ) and detected by Western blot using anti - FLAG antibody . HA - tagged SUMM2 protein co - immunoprecipitated with FLAG - tagged CRCK3 was detected by Western blot using anti - HA antibody . Similar results were obtained in two independent experiments . ( B ) In vivo pull - down of HA - tagged SUMM2 by FLAG - tagged CRCK3 kinase domain . KD represents a truncation of SUMM3 with only the kinase domain ( amino acid 201 - 510 ) . FLAG - tagged full - length or truncated CRCK3 and HA - tagged SUMM2 proteins were transiently expressed in N . bethamiana by agro - infiltration . FLAG - tagged CRCK3 proteins were immunoprecipitated with anti - FLAG conjugated beads ( Sigma ) and detected by Western blot using anti - FLAG antibody . HA - tagged SUMM2 protein co - immunoprecipitated with FLAG - tagged CRCK3 was detected by Western blot using anti - HA antibody . Similar results were obtained in two independent experiments .

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 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

Figure 6(A) In vivo pull-down of HA-tagged SUMM2 by FLAG-tagged CRCK3.FLAG-tagged full-length or truncated CRCK3 and HA-tagged SUMM2 proteins were transiently expressed in N. bethamiana by agro-infiltration. FLAG-tagged CRCK3 proteins were immunoprecipitated with anti-FLAG conjugated beads (Sigma) and detected by Western blot using anti-FLAG antibody. HA-tagged SUMM2 protein co-immunoprecipitated with FLAG-tagged CRCK3 was detected by Western blot using anti-HA antibody. Similar results were obtained in two independent experiments.(B) In vivo pull-down of HA-tagged SUMM2 by FLAG-tagged CRCK3 kinase domain. KD represents a truncation of SUMM3 with only the kinase domain (amino acid 201-510).FLAG-tagged full-length or truncated CRCK3 and HA-tagged SUMM2 proteins were transiently expressed in N. bethamiana by agro-infiltration. FLAG-tagged CRCK3 proteins were immunoprecipitated with anti-FLAG conjugated beads (Sigma) and detected by Western blot using anti-FLAG antibody. HA-tagged SUMM2 protein co-immunoprecipitated with FLAG-tagged CRCK3 was detected by Western blot using anti-HA antibody. Similar results were obtained in two independent experiments.


Figure 1RT - qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR + / + and LXR − / − primary BMMs . Fold changes are shown relative to LXR + / + 0 h . Error bars represent ± SEM for n = 4 - 8 ( * * P = 0 . 001 at 8 h and * * P = 0 . 00004 at 16 h versus LXR − / − ; * * P = 0 . 0003 at 0 h versus LXR + / + ) ; bottom , proposed model explaining the ligand ‐ dependent induction of Abca1 . Following ligand stimulation , LXR - corepressor complexes are exchanged for LXR - coactivator complexes , which promote gene expression . The LXR - cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown .

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 O O O O O O O

Figure 1RT-qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR+/+ and LXR−/− primary BMMs. Fold changes are shown relative to LXR+/+ 0 h. Error bars represent ± SEM for n = 4-8 (**P = 0.001 at 8 h and **P = 0.00004 at 16 h versus LXR−/−; **P = 0.0003 at 0 h versus LXR+/+); bottom, proposed model explaining the ligand‐dependent induction of Abca1. Following ligand stimulation, LXR-corepressor complexes are exchanged for LXR-coactivator complexes, which promote gene expression. The LXR-cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown.


Figure 2Luciferase reporter assays from RAW 264 . 7 macrophages expressing reporter alone , or together with full ‐ length SND1 , SART1 , or HMBOX1 . LXR ligand stimulations with 1 μM T0901317 were performed for 18 h . A diagram of the reporter construct is shown above . Fold changes are shown relative to vehicle ‐ stimulated reporter alone ( first lane ) . Error bars represent ± SEM for n = 9 ( * * P = 0 . 001 , * P = 0 . 011 for SND1 , * P = 0 . 046 for SART1 ) . Luciferase reporter assays from RAW 264 . 7 macrophages expressing reporter alone , or together with full ‐ length NCOA5 or an NCOA5 mutant lacking the NH2 ‐ terminus . LXR ligand stimulations with 1 μM T0901317 were performed for 18 h . Diagrams of the reporter constructs are shown above . Fold changes are shown relative to vehicle ‐ stimulated reporter alone ( first lane ) . Error bars represent ± SEM for n = 6 - 12 ( * * P 0 . 001 , * P = 0 . 035 ) . RT - qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus . LXR ligand stimulations were performed with 1 μM T0901317 for 18 h . Fold changes are shown relative to vehicle ‐ stimulated control . Error bars represent ± SEM for n = 4 ( * P = 0 . 029 ) .

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 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 2Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length SND1, SART1, or HMBOX1. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. A diagram of the reporter construct is shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 9 (**P = 0.001, *P = 0.011 for SND1, *P = 0.046 for SART1).Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length NCOA5 or an NCOA5 mutant lacking the NH2‐terminus. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. Diagrams of the reporter constructs are shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 6-12 (**P 0.001, *P = 0.035).RT-qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus. LXR ligand stimulations were performed with 1 μM T0901317 for 18 h. Fold changes are shown relative to vehicle‐stimulated control. Error bars represent ± SEM for n = 4 (*P = 0.029).


Figure 3In vitro pulldown assays using recombinant GST ‐ LXRα to isolate the indicated in vitro translated NCOA5 constructs . Assays were performed for 2 h in the presence or absence of 2 μM T0901317 . Samples were immunoblotted as indicated . Note the requirement of the NCOA5 NH2 ‐ terminus for interacting with LXRα . Immunoprecipitation of Protein C ‐ tagged LXRα from stable RAW 264 . 7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h , followed by immunoblotting for endogenous NCOA5 or over ‐ expressed LXRα . Protein immunoblots of nuclear extracts are shown below . NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control . qPCR was performed for the Abca1 proximal LXRE . Note the ligand ‐ stimulated association of NCOA5 . Error bars represent ± SEM for n = 4 - 9 ( * P = 0 . 02 ) . NCOA5 ChIP assays from LXR − / − BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h . qPCR was performed for the Abca1 proximal LXRE . Error bars represent ± SEM for n = 5 - 6 ( * * P = 0 . 0003 for vehicle and * * P = 0 . 002 for T0901317 ) .

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 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 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 3In vitro pulldown assays using recombinant GST‐LXRα to isolate the indicated in vitro translated NCOA5 constructs. Assays were performed for 2 h in the presence or absence of 2 μM T0901317. Samples were immunoblotted as indicated. Note the requirement of the NCOA5 NH2‐terminus for interacting with LXRα.Immunoprecipitation of Protein C‐tagged LXRα from stable RAW 264.7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h, followed by immunoblotting for endogenous NCOA5 or over‐expressed LXRα. Protein immunoblots of nuclear extracts are shown below.NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the ligand‐stimulated association of NCOA5. Error bars represent ± SEM for n = 4-9 (*P = 0.02).NCOA5 ChIP assays from LXR−/− BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6 (**P = 0.0003 for vehicle and **P = 0.002 for T0901317).


Figure 4RT - qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non ‐ silencing or Ncoa5 ‐ specific shRNA . Fold changes are shown relative to shControl 0 h . Note the elevated Abca1 expression at 24 - 30 h following loss of NCOA5 . Error bars represent ± SEM for n = 4 - 6 ( * * P = 0 . 0007 at 24 h and * * P = 0 . 004 at 30 h , * P = 0 . 02 ) . Quantification of ABCA1 immunoblots from primary BMMs infected as in ( A ) . Representative immunoblot is shown in Supplementary Fig S7E . LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h . Error bars represent ± SEM for n = 2 ( * * P = 0 . 007 ) .

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 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

Figure 4RT-qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non‐silencing or Ncoa5‐specific shRNA. Fold changes are shown relative to shControl 0 h. Note the elevated Abca1 expression at 24-30 h following loss of NCOA5. Error bars represent ± SEM for n = 4-6 (**P = 0.0007 at 24 h and **P = 0.004 at 30 h, *P = 0.02).Quantification of ABCA1 immunoblots from primary BMMs infected as in (A). Representative immunoblot is shown in Supplementary Fig S7E. LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h. Error bars represent ± SEM for n = 2 (**P = 0.007).


Figure 5A , B RT - qPCR of Abca1 expression from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 alone or together with 6 μg / ml PolyIC ( A ) or 10 ng / ml LPS ( B ) . Fold changes are shown relative to vehicle ‐ stimulated shControl . Note only the loss of TLR3 ‐ mediated repression following Ncoa5 silencing . Error bars represent ± SEM for n = 4 - 10 ( * * P = 0 . 0003 for A , * * P = 0 . 0004 for shControl in B , * * P = 0 . 0002 for shNcoa5 in B versus T0901317 ) . C , D RT - qPCR of Abca1 expression from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h unstimulated , or with 6 μg / ml PolyIC ( C ) or 10 ng / ml LPS ( D ) . Fold changes are shown relative to unstimulated shControl . Error bars represent ± SEM for n = 4 - 6 ( * * P = 0 . 0006 for C , * * P = 0 . 00000001 for shControl in D , * * P = 0 . 000001 for shNcoa5 in D , * P = 0 . 013 versus T0901317 ) . E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317 , 1 μM T0901317 + 6 μg / ml PolyIC , or vehicle control . qPCR was performed for the Abca1 proximal LXRE . Note the increased occupancy only in the presence of both lipid and inflammatory ligands . Error bars represent ± SEM for n = 8 - 12 ( * * P = 0 . 0007 versus T0901317 ) . F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg / ml PolyIC or left unstimulated . qPCR was performed for the Abca1 proximal LXRE . Error bars represent ± SEM for n = 5 - 6 . G Cholesterol efflux assays to APOA1 from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Agonist stimulations were performed for 6 h . Error bars represent ± SEM for n = 3 ( * P = 0 . 02 versus T0901317 ) .

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 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 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 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

Figure 5A, B RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317 alone or together with 6 μg/ml PolyIC (A) or 10 ng/ml LPS (B). Fold changes are shown relative to vehicle‐stimulated shControl. Note only the loss of TLR3‐mediated repression following Ncoa5 silencing. Error bars represent ± SEM for n = 4-10 (**P = 0.0003 for A, **P = 0.0004 for shControl in B, **P = 0.0002 for shNcoa5 in B versus T0901317).C, D RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h unstimulated, or with 6 μg/ml PolyIC (C) or 10 ng/ml LPS (D). Fold changes are shown relative to unstimulated shControl. Error bars represent ± SEM for n = 4-6 (**P = 0.0006 for C, **P = 0.00000001 for shControl in D, **P = 0.000001 for shNcoa5 in D, *P = 0.013 versus T0901317).E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the increased occupancy only in the presence of both lipid and inflammatory ligands. Error bars represent ± SEM for n = 8-12 (**P = 0.0007 versus T0901317).F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg/ml PolyIC or left unstimulated. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6.G Cholesterol efflux assays to APOA1 from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Agonist stimulations were performed for 6 h. Error bars represent ± SEM for n = 3 (*P = 0.02 versus T0901317).


Figure 6A , B Unmodified / pSer5 RNAPII ( A ) or RNAPII pSer2 ( B ) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317 , 1 μM T0901317 + 6 μg / ml PolyIC , or vehicle control . qPCR was performed for the Abca1 TSS . Error bars represent ± SEM for n = 6 - 9 ( * * P = 0 . 0001 in A , * * P = 0 . 002 in B versus T0901317 ) . C , D RNAPII ChIP assays as in ( A , B ) but performed from LXR − / − BMMs . Error bars represent ± SEM for n = 5 - 9 ( * * P = 0 . 000000001 , * P = 0 . 02 versus T0901317 ) . E RT - qPCR of Abca1 expression from LXR + / + versus LXR − / − BMMs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 , or 1 μM T0901317 together with 6 μg / ml PolyIC . Fold changes are shown relative to vehicle ‐ stimulated LXR + / + . Error bars represent ± SEM for n = 4 ( * * P = 0 . 004 , * P = 0 . 012 versus T0901317 ) . F RNAPII pSer2 ChIP assays from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 , or 1 μM T0901317 + 6 μg / ml PolyIC . qPCR was performed for the Abca1 TSS . Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs ( * * P = 0 . 004 versus baseline ) . Error bars represent ± SEM for n = 3 from 11 mice .

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 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 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

Figure 6A, B Unmodified/pSer5 RNAPII (A) or RNAPII pSer2 (B) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 TSS. Error bars represent ± SEM for n = 6-9 (**P = 0.0001 in A, **P = 0.002 in B versus T0901317).C, D RNAPII ChIP assays as in (A, B) but performed from LXR−/− BMMs. Error bars represent ± SEM for n = 5-9 (**P = 0.000000001, *P = 0.02 versus T0901317).E RT-qPCR of Abca1 expression from LXR+/+ versus LXR−/− BMMs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 together with 6 μg/ml PolyIC. Fold changes are shown relative to vehicle‐stimulated LXR+/+. Error bars represent ± SEM for n = 4 (**P = 0.004, *P = 0.012 versus T0901317).F RNAPII pSer2 ChIP assays from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 + 6 μg/ml PolyIC. qPCR was performed for the Abca1 TSS. Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs (**P = 0.004 versus baseline). Error bars represent ± SEM for n = 3 from 11 mice.