( A - C ) Expression of Pfkp mRNA ( top panels ) Stat3 Pfkp expression ( bottom panels ) after knockdown of Stat3 using three independent shRNAs in either R1 ( A ) or E14 ( B ) or after overexpression of Stat3 in R1 ( C ) , as measured by qPCR Western blot , respectively . β - actin was employed as a loading control throughout .

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(A-C) Expression of Pfkp mRNA levels (top panels) and Stat3 and Pfkp protein expression (bottom panels) after knockdown of Stat3 using three independent shRNAs in either R1 cells (A) or E14 cells (B) or after overexpression of Stat3 in R1 cells (C), as measured by qPCR and Western blot, respectively. β-actin was employed as a loading control throughout.


ChIP assays against Stat3 p - Stat3 ( Y705 ) or isotype - matched immunoglobulin G ( IgG ) ( F ) , as measured by PCR , were representative of three independent experiments . Nanog promoter is shown as a positive control .

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ChIP assays against Stat3 p-Stat3 (Y705) or isotype-matched immunoglobulin G (IgG) (F), as measured by PCR, and images were representative of three independent experiments. Nanog promoter is shown as a positive control.


( G ) Transcriptional activity of indicated pGL3 - Pfkp promoter or mutant constructs expressed in HEK293T with or without overexpression of Stat3 , as measured by luciferase reporter ( top panel ) . Western blotting was used to verify Stat3 expression ( bottom panel ) .

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(G) Transcriptional activity of the indicated pGL3-based Pfkp promoter or mutant constructs expressed in HEK293T cells with or without overexpression of Stat3, as measured by luciferase reporter assay (top panel). Western blotting was used to verify Stat3 expression (bottom panel).


( B ) Representative of alkaline phosphatase - stained colonies formed by R1 after overexpression of Pfkp . were representative of three independent experiments .

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(B) Representative images of alkaline phosphatase-stained colonies formed by R1 cells after overexpression of Pfkp. Images were representative of three independent experiments.


( K , L ) Representative of immunofluorescence in control Pfkp knockdown R1 for Sox17 following definitive endoderm differentiation ( K ) or Foxa2 following definitive endoderm differentiation ( L ) . Green indicates positive with nuclei counterstained with DAPI . were representative of three independent experiments . ( M , N ) Quantitative of Sox17 ( M ) Foxa2 ( N ) in R1 from ( K ) ( L ) , respectively as determined by flow cytometry . Representative profiles ( left panels ) quantitation ( right panels ) as a of total .

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(K, L) Representative images of immunofluorescence staining in control and Pfkp knockdown R1 cells for Sox17 following definitive endoderm differentiation (K) or Foxa2 following definitive endoderm differentiation (L). Green staining indicates positive cells with nuclei counterstained with DAPI. Images were representative of three independent experiments. (M, N) Quantitative analysis of Sox17 (M) and Foxa2 (N) in R1 cells from (K) and (L), respectively as determined by flow cytometry. Representative profiles (left panels) and quantitation (right panels) as a percentage of total cells.


( A , B ) interactome of Pfkp in R1 . SDS - PAGE gel comparing Pfkp control ( IgG ) immunoprecipitates ( left ) with candidate Pfkp - interacting identified by mass spectrometry ( right ) ( A ) . Validation of candidate Pfkp - interacting using Western blotting ( B ) .

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(A, B) Protein interactome of Pfkp in R1 cells. SDS-PAGE gel comparing Pfkp and control (IgG) immunoprecipitates (left) with candidate Pfkp-interacting proteins identified by mass spectrometry (right) (A). Validation of candidate Pfkp-interacting proteins using Western blotting (B).


( E - F ) Co - IP assays mapping interactions between Pfkp fragments of Lin41 ( E ) , interactions between Lin41 fragments of Pfkp ( F ) in HEK293T .

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(E-F) Co-IP assays mapping interactions between Pfkp and fragments of Lin41 (E), interactions between Lin41 and fragments of Pfkp (F) in HEK293T cells.


( J , K ) Expression of Lin41 in control versus Pfkp knockdown R1 without or with MG132 treatment ( J ) or with Lactacyctin treatment ( K ) , as determined by Western blot .

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(J, K) Expression of Lin41 in control versus Pfkp knockdown R1 cells without or with MG132 treatment (J) or with Lactacyctin treatment (K), as determined by Western blot.


( F ) HEK293T were co - transfected with indicated vectors to overexpress Pfkp in combination with HA - ubiquitin ( Ub ) wildtype Flag - Lin41 or serine substitution mutants . Lin41 ubiquitination were determined using Western blotting against HA ( top panels ) with input samples analyzed in parallel to verify transfection efficiency ( bottom panels ) .

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(F) HEK293T cells were co-transfected with the indicated vectors to overexpress Pfkp in combination with HA-ubiquitin (Ub) and the individual wildtype Flag-Lin41 or serine substitution mutants. Lin41 ubiquitination levels were determined using Western blotting against HA (top panels) with input samples analyzed in parallel to verify transfection efficiency (bottom panels).


( G ) Control or Pfkp knockdown R1 were co - transfected with wildtype Flag - Lin41 or serine substitution mutants Lin41 ubiquitination determined as per ( F ) using Western blotting against Flag . ( H ) In vitro ubiquitination assays employing products from in vitro kinase assays employing recombinant GST - Lin41 Flag - Pfkp , with Lin41 ubiquitination measured by Western blotting against Lin41 . (

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(G) Control or Pfkp knockdown R1 cells were co-transfected with the individual wildtype Flag-Lin41 or serine substitution mutants and Lin41 ubiquitination levels determined as per (F) using Western blotting against Flag. (H) In vitro ubiquitination assays employing the products from in vitro kinase assays employing recombinant GST-Lin41 and Flag-Pfkp, with Lin41 ubiquitination measured by Western blotting against Lin41. (


( C - E ) Expression of selected expression of pluripotency lineage specification markers in from ( B ) determined by qPCR ( C ) with parallel Western blotting of a panel of ectodermal ( D ) endodermal ( E ) markers .

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(C-E) Expression of selected expression of pluripotency and lineage specification markers in the cells from (B) determined by qPCR (C) with parallel Western blotting analysis of a panel of ectodermal (D) and endodermal (E) markers.


( G - J ) pull - down assays measuring interactions between Fgf5 ( G ) , Otx2 ( H ) , Sox1 ( I ) , Pax2 ( J ) mRNAs endogenous Lin41 in R1 . Recovery of each mRNA was compared in sense anti - sense probes using qPCR ( top ) with Western blotting used to detect Lin41 ( bottom ) .

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(G-J) RNA pull-down assays measuring interactions between Fgf5 (G), Otx2 (H), Sox1 (I), and Pax2 (J) mRNAs and endogenous Lin41 in R1 cells. Recovery of each mRNA was compared in sense and anti-sense probes using qPCR (top) with Western blotting used to detect Lin41(bottom).


( K ) Relative stability of mRNAs for ectodermal genes Fgf5 , Otx2 , Sox1 , Pax2 R1 in control Pfkp knockdown EBs at Day 6 , as determined in actinomycin D chase assays . ( L ) Relative stability of ectodermal gene mRNAs measured as per ( K ) in Day 6 Pfkp knockdown EBs transfected with or without Lin41 .

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(K) Relative stability of mRNAs for the ectodermal genes Fgf5, Otx2, Sox1, and Pax2 R1 cells in control and Pfkp knockdown EBs at Day 6, as determined in actinomycin D chase assays. (L) Relative stability of ectodermal gene mRNAs measured as per (K) in Day 6 Pfkp knockdown EBs transfected with or without Lin41.


( A ) indicated yeast strains ( wild type , pex19Δ , pex3Δ , msp1Δ , pex19Δmsp1Δ , pex3Δmsp1Δ ) expressing RFP - SKL ( peroxisomal marker ) mitochondria - targeted green ﬂuorescent ( mGFP ) were grown to mid - log phase imaged by ﬂuorescence microscopy . Representative are shown , scale bar , 2 μm . DIC , differential interference contrast . Enhanced : red signal was set to ' best - fit ' in ZENPro microscopy software .

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(A) The indicated yeast strains (wild type, pex19Δ, pex3Δ, msp1Δ, pex19Δmsp1Δ, and pex3Δmsp1Δ) expressing RFP-SKL (peroxisomal marker) and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and imaged by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast. Enhanced: the red signal intensity was set to 'best-fit' in ZENPro microscopy software analysis.


( B ) Volcano plot showing most enriched / decreased in mitochondrial proteome of pex19Δ versus pex19Δmsp1Δ strains as detected by quantitative mass spectrometry ( n = 7 , biological replicates ; n = 2 , technical replicates ) . Effect sizes were calculated for Cohen ' s d A ' s relevance was determined using a p - value threshold of < 0 . 05 with an absolute effect threshold of 1 or greater ( large effect ) . Gray dots represent measured red dots highlight peroxisomal - associated .

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(B) Volcano plot showing the most enriched/decreased proteins in the mitochondrial proteome of pex19Δ versus pex19Δmsp1Δ strains as detected by quantitative mass spectrometry (n=7, biological replicates; n=2, technical replicates). Effect sizes were calculated for Cohen's d A protein's relevance was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins and red dots highlight peroxisomal-associated proteins.


( D ) indicated yeast strains expressing PEX13 - RFP mitochondria - targeted green ﬂuorescent ( mGFP ) were grown to mid - log phase visualized by ﬂuorescence microscopy . Representative are shown , scale bar , 2 μm . DIC , differential interference contrast .

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(D) The indicated yeast strains expressing PEX13-RFP and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and visualized by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast.


( F ) Total yeast lysate , post mitochondrial supernatant , HistodenzTM - purified mitochondria from indicated ( wild type , msp1Δ , pex19Δ , pex19Δmsp1Δ , pex3Δ , pex3Δmsp1Δ ) expressing Pex13 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE immunoblotted for Pex13 ( α - V5 ) , cytochrome c ( α - cyt c ) , HSP70 ( α - hsp70 ) . ( G ) Total yeast lysate , post mitochondrial supernatant , HistodenzTM - purified mitochondria from indicated ( wild type , msp1Δ , pex19Δ , pex19Δmsp1Δ , pex3Δ , pex3Δmsp1Δ ) expressing Pex11 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE immunoblotted for Pex11 ( α - V5 ) porin ( α - porin ) , cytochrome c ( α - cyt c ) , HSP70 ( α - hsp70 ) .

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(F) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex13 (α-V5), cytochrome c (α-cyt c), and HSP70 (α-hsp70). (G) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex11-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex11 (α-V5) and porin (α-porin), cytochrome c (α-cyt c), and HSP70 (α-hsp70).


( A ) Volcano plot ( log10 ) of most enriched / decreased in mitochondrial proteome of pex3Δ versus pex3Δmsp1Δ strains as detected by quantitative mass spectrometry ( n = 3 , biological replicates , n = 1 , technical replicate ) . 2010 ) . Effect sizes were calculated for Cohen ' s d A ' s relevance to model was determined using a p - value threshold of < 0 . 05 with an absolute effect threshold of 1 or greater ( large effect ) . Gray dots represent measured . Red dots highlight peroxisomal - associated .

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(A) Volcano plot (log10) of the most enriched/decreased proteins in the mitochondrial proteome of pex3Δ versus pex3Δmsp1Δ strains as detected by quantitative mass spectrometry (n=3, biological replicates, n=1, technical replicate). 2010). Effect sizes were calculated for Cohen's d A protein's relevance to the model was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins. Red dots highlight peroxisomal-associated proteins.


( C ) Mitochondria from indicated yeast strains ( wild type , pex19Δ , pex19Δmsp1Δ ) , expressing Pex13 - V5 ( under its endogenous promotor ) were either left untreated ( lane 1 , 2 , 3 ) or exposed to Na2CO3 separated into pellet ( 4 , 5 , 6 ) or supernatant fraction ( 7 , 8 , 9 ) . After separation by SDS - PAGE fractions were immunoblotted for Pex13 - V5 ( α - V5 ) porin ( α - porin ) .

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(C) Mitochondria from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ), expressing Pex13-V5 (under its endogenous promotor) were either left untreated (lane 1,2,3) or exposed to Na2CO3 and separated into the membrane pellet (4,5,6) or supernatant fraction (7,8,9). After separation by SDS-PAGE the fractions were immunoblotted for Pex13-V5 (α-V5) and porin (α-porin).


( B ) Digitonin - solubilized ( 1 or 4 g / g digitonin / , as indicated ) mitochondrial membranes from indicated yeast strains ( wild type , pex19Δ , pex19Δmsp1Δ ) expressing Pex13 - V5 ( under its endogenous promotor ) were separated by BN - PAGE with a 3 - 18 % gradient stained with Coomassie brilliant blue . BHM , bovine heart mitochondria ; V2 , V - dimer ; SL , supercomplex III2IV2 ; Ss , supercomplex III2IV1 ; V1 , V - monomer .

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(B) Digitonin-solubilized (1 or 4 g/g digitonin/protein ratio, as indicated) mitochondrial membranes from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by BN-PAGE with a 3-18% gradient and stained with Coomassie brilliant blue. BHM, bovine heart mitochondria; V2, complex V-dimer; SL,supercomplex III2IV2; Ss, supercomplex III2IV1; V1, complex V-monomer.


( F ) Mitochondria from indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , pex13Δmsp1Δpex19Δ ) expressing Pex22 - V5 ( under its endogenous promotor ) ( HistodenzTM - purified ) were separated by SDS - PAGE immunoblotted for Pex22 - V5 ( α - Pex22 - V5 ) porin ( α - porin ) . ( G ) Mitochondria from indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , pex13Δmsp1Δpex19Δ ) expressing Pot1 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE immunoblotted for Pot1 - V5 ( α - Pot1 - V5 ) porin ( α - porin ) . ( H ) Mitochondria from indicated yeast strains ( pex19Δ , pex19Δmsp1 Δ , pex13Δmsp1Δpex19Δ ) expressing Mdh3 - V5 ( under its endogenous promotor ) ( HistodenzTM - purified ) were separated by SDS - PAGE immunoblotted for Mdh3 - V5 ( α - Mdh3 - V5 ) porin ( α - porin ) .

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(F) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ) expressing Pex22-V5 (under its endogenous promotor) (HistodenzTM-purified) were separated by SDS-PAGE and immunoblotted for Pex22-V5 (α-Pex22-V5) and porin (α-porin). (G) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ ) expressing Pot1-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pot1-V5 (α-Pot1-V5) and porin (α-porin). (H) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1 Δ, and pex13Δmsp1Δpex19Δ) expressing Mdh3-V5 (under its endogenous promotor) (HistodenzTM-purified) were separated by SDS-PAGE and immunoblotted for Mdh3-V5 (α-Mdh3-V5) and porin (α-porin).


( I ) Mitochondria from indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , pex13Δmsp1Δpex19Δ ) expressing Pot1 - V5 ( under its endogenous promotor ) were either left untreated ( nt ) ( lane 1 , 4 , 7 ) or were treated with proteinase K in high fidelity buffer ( PK - HF ) ( cutting control , lane 2 , 5 , 8 ) or low fidelity buffer ( PK - LF ) ( protease protection , lane 3 , 6 , 9 ) separated by SDS - PAGE immunoblotted for Mia40 ( intermembrane space ) ( α - Mia40 ) , Pot1 - V5 ( α - V5 ) , porin ( outer ) ( α - porin ) Tom22 ( outer but partially proteinase exposed ) ( α - Tom22 ) .

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(I) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ) expressing Pot1-V5 (under its endogenous promotor) were either left untreated (nt) (lane 1,4,7) or were treated with proteinase K in high fidelity buffer (PK-HF) (cutting control, lane 2,5,8) or low fidelity buffer (PK-LF) (protease protection assay, lane 3,6,9) and separated by SDS-PAGE and immunoblotted for Mia40 (intermembrane space) (α-Mia40), Pot1-V5 (α-V5), porin (outer membrane) (α-porin) and Tom22 (outer membrane but partially proteinase exposed) (α-Tom22).


( A ) microscopy of human fibroblast lines derived from ZSD - cWT , cWT + ATAD1 , PEX3 - / - , PEX3 - / - + ATAD1 - expressing PEX13 - GFP stained with Mitotracker  Deep Red FM . Representative are shown , scale bar , 10 μm .

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(A) Fluorescence microscopy of human fibroblast cell lines derived from ZSD patients-cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1-expressing PEX13-GFP and stained with Mitotracker Deep Red FM. Representative images are shown, scale bar, 10 μm.


( B ) Electron microscopy of human fibroblast lines cWT , cWT + ATAD1 , PEX3 - / - , PEX3 - / - + ATAD1 . Representative are shown , scale bar 500 nm .

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(B) Electron microscopy of human fibroblast cell lines cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1. Representative images are shown, scale bar 500 nm.


( A ) Bioenergetic profile of human fibroblast lines . OCR ( pmol / min / norm . unit ) for cWT , cWT + ATAD1 , PEX3 - / - , PEX3 - / - + ATAD1 , cWT - ATAD1 - / - , PEX3 - / - atad1 - / - plotted against time ( XF96e Extracellular Flux Analyzer , Mito - Stress - ) . 1 μM Oligomycin A , 0 . 15 μM FCCP , 1 μM rotenone + 1 μM antimycin A ( final concentrations ) were sequentially delivered to XF96e medium through injection ports in sensor cartridge . graph is compilation of two independent assays , error bars show mean + / - s . d . ( n = 4 , biological replicates ) .

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(A) Bioenergetic profile of human fibroblast cell lines. OCR (pmol/min/norm.unit) for cWT, cWT+ATAD1, PEX3-/-, PEX3-/-+ATAD1, cWT-ATAD1-/-, and PEX3-/- atad1-/- cells plotted against time (XF96e Extracellular Flux Analyzer, Mito-Stress-Test). 1 μM Oligomycin A, 0.15 μM FCCP, and 1 μM rotenone + 1 μM antimycin A (final concentrations) were sequentially delivered to the XF96e assay medium through injection ports in the sensor cartridge. The graph is the compilation of two independent assays, error bars show mean +/- s.d. (n=4, biological replicates).


( E ) Scatter plot representation of cardiolipin lipidomics . average ( n = 4 , biological replicates ) normalized peak of each of 11 detected cardiolipin species , log10 pareto - scaled is visualized . horizontal lines represent mean peak intesity of 11 caridolipin species in each . ( F ) Scatter plot representation of a subpopulation of phosphoethanolamine ( PE ) lipidomics . average ( n = 4 , biological replicates ) normalized peak of each of 20 phosphoethanolamine ( PE ) species ( decreased in PEX3 - / - ) , log10 pareto - scaled , is visualized . horizontal lines represent mean peak intesity of 20 PE species in each . ( G ) Scatter plot representation of ether - phospholipid lipidomics . average ( n = 4 , biological replicates ) normalized peak of each of 21 detected ether - phospholipid ( plasmalogen ) species , log10 pareto - scaled , is visualized . horizontal lines represent mean peak intesity of 21 plasmalogen species in each .

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(E) Scatter plot representation of cardiolipin lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 11 detected cardiolipin species, log10 pareto-scaled is visualized. The horizontal lines represent the mean peak intesity of all 11 caridolipin species in each cell line. (F) Scatter plot representation of a subpopulation of the phosphoethanolamine (PE) lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 20 phosphoethanolamine (PE) species (decreased in PEX3-/-), log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 20 PE species in each cell line. (G) Scatter plot representation of ether-phospholipid lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 21 detected ether-phospholipid (plasmalogen) species, log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 21 plasmalogen species in each cell line.


Time - series measurements of ( B ) E . coli abundance , ( C ) B . subtilis abundance ,

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Time-series measurements of (B) E. coli abundance, (C) B. subtilis abundance,


( H ) Bar plot of measured extracellular pBB275 plasmid at 0 hr 6 hr in co - culture composed of E . coli MG1655 - rfp B . subtilis .

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(H) Bar plot of measured extracellular pBB275 plasmid concentration at 0 hr and 6 hr in the co-culture composed of E. coli MG1655-rfp and B. subtilis.


( B , C ) ( B ) Extracellular pBB275 plasmid or ( C ) extracellular E . coli genomic at 0 or 6 hr in co - cultures composed of a recA + or recA - E . coli plasmid donor engineered B . subtilis .

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(B, C) (B) Extracellular pBB275 plasmid concentration or (C) extracellular E. coli genomic DNA concentration at 0 or 6 hr in the co-cultures composed of a recA+ or recA- E. coli plasmid donor and engineered B. subtilis.


( E ) Extracellular pBB275 or pUC19 plasmid in E . coli MG1655 monoculture or or co - culture with engineered B . subtilis .

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(E) Extracellular pBB275 or pUC19 plasmid concentration in the E. coli MG1655 monoculture or or co-culture with engineered B. subtilis.


( F ) HGT efficiencies of pBB275 plasmid ( with or without a f1 ori ) at 6 hr in co - culture composed of E . coli MG1655 engineered B . subtilis .

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(F) HGT efficiencies of pBB275 plasmid (with or without a f1 ori) at 6 hr in the co-culture composed of E. coli MG1655 and engineered B. subtilis.


( B ) E . coli B . subtilis abundances in co - culture in presence of 0 , 0 . 02 , 0 . 2 or 2 mM IPTG at 6 hr .

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

(B) E. coli and B. subtilis abundances in the co-culture in the presence of 0, 0.02, 0.2 or 2 mM IPTG at 6 hr.


Time - series measurements of extracellular plasmid or E . coli gDNA concentrations in co - culture in presence of ( D ) 0 . 02 mM , ( E ) 0 . 2 mM or ( F ) 2 mM IPTG .

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

Time-series measurements of extracellular plasmid or E. coli gDNA concentrations in the co-culture in the presence of (D) 0.02 mM, (E) 0.2 mM or (F) 2 mM IPTG.


( H ) Abundance of transformants at 6 hr in co - culture in presence of 0 , 0 . 02 , 0 . 2 or 2 mM IPTG .

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

(H) Abundance of transformants at 6 hr in the co-culture in the presence of 0, 0.02, 0.2 or 2 mM IPTG.


( C ) Scatter plot of initial abundance of heat - killed E . coli MG1655 - rfp extracellular pBB275 plasmid at 0h or 6 hr in co - culture .

(C) Scatter plot of the initial abundance of heat-killed E. coli MG1655-rfp and extracellular pBB275 plasmid concentration at 0h or 6 hr in the co-culture.


Time - series measurements of ( E ) abundance of E . coli MG1655 - rfp , ( F ) abundance of engineered B . subtilis Unpaired t - was used to determine if abundances in presence absence of chloramphenicol were statistically different . Stars ( * ) indicate p - value of 0 . 0237 0 . 0283 for 4 hr 8 hr , respectively .

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

Time-series measurements of (E) the abundance of E. coli MG1655-rfp, (F) the abundance of engineered B. subtilis Unpaired t-test was used to determine if the abundances in the presence and absence of chloramphenicol were statistically different. Stars (*) indicate p-value of 0.0237 and 0.0283 for 4 hr and 8 hr, respectively.


Time - series measurements of ( G ) extracellular pBB275 plasmid , Unpaired t - was used to determine if abundances in presence absence of

Time-series measurements of (G) extracellular pBB275 plasmid concentration, Unpaired t-test was used to determine if the abundances in the presence and absence of chloramphenicol


( C ) Histogram of number of E . coli plasmid replication origins ColE1 , p15A , CloDF13 or pSC101 identified in Bacillus or non - Bacillus sequences with a larger than 106 bp ( indicating potential genomic integration ) . ColE1 was found in 6 Bacillus genomes 43 non - Bacillus genomes . CloDF13 was found in 23 non - Bacillus genomes . p15A pSC101 were not detected in this NCBI RefSeq dataset .

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

(C) Histogram of the number of E. coli plasmid replication origins ColE1, p15A, CloDF13 or pSC101 identified in Bacillus or non-Bacillus DNA sequences with a sequence length larger than 106 bp (indicating potential genomic integration). ColE1 was found in 6 Bacillus genomes and 43 non-Bacillus genomes. CloDF13 was found in 23 non-Bacillus genomes. p15A and pSC101 were not detected in this NCBI RefSeq dataset.


D of toxicity viability of HCT116 , U87 BxPC3 grown in spheroids ( 3D ) treated as indicated for 24h ( BxPC3 ) or 48h ( HCT116 , U87 ) ( NEN 1 . 2 µM ; Domperidone ( Domp ) , Imipramine ( Imi ) , Desipramine ( Desi ) Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM ) . Data information : Data are presented as mean ( SD ) ( N = 3 ) were analyzed by two - way ANOVA with Tukey post - hoc Significance is indicated for multiple comparisons as indicated . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

D Ratio of toxicity and viability of HCT116, U87 and BxPC3 cells grown in spheroids (3D) and treated as indicated for 24h (BxPC3) or 48h (HCT116, U87) (NEN 1.2 µM; Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM). Data information: Data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test Significance is indicated for multiple comparisons as indicated. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Immunoblot of CHOP , ATF4 , phosphorylated ( p - ) t - ) eIF2alpha ( eIF2a ) of HCT116 treated as indicated for 16h ( NEN ( N ) 1 . 2 µM ; Domperidone ( Domp ) , Amitriptyline ( Ami ) , each 30 µM ) .

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B Immunoblot analysis of CHOP, ATF4, phosphorylated (p-) and t-) eIF2alpha (eIF2a) of HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM; Domperidone (Domp), Amitriptyline (Ami), each 30 µM).


F of toxicity viability in HCT116 spheroids treated as indicated for 48h ( NEN 1 . 2 µM , Domperidone ( Domp ) , Imipramine ( Imi ) , Desipramine ( Desi ) Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM , ISRIB 1 µM ) .

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

F Ratio of toxicity and viability in HCT116 spheroids treated as indicated for 48h (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM, ISRIB 1 µM).


A Relative mRNA expression of indicated CLEAR network genes in HCT116 treated as indicated ( ( NEN ( N ) 1 . 2 µM ; Domperidone ( Domp , D ) , Imipramine ( Imi ) , Desipramine ( Desi ) Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM ) , determined by qPCR . Data information : In ( A ) ( first ( TFE3 ) third ( CD68 ) graph ( N = 3 ) ; second ( TFE3 ) fourth ( CD68 ) graph ( N = 5 , besides NEN + Clomi ( N = 4 ) NEN + Desi ( N = 4 ) ) data are presented as mean ( SD ) were analyzed by a one - way ANOVA with Tukey post - hoc . In second fourth graph , significance is indicated for comparison of combinatorial treatments ( NEN + TCAs ) to controls ( Ctrl ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

A Relative mRNA expression levels of indicated CLEAR network genes in HCT116 cells treated as indicated ((NEN (N) 1.2 µM; Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM), determined by qPCR. Data information: In (A) (first (TFE3) and third (CD68) graph (N=3); second (TFE3) and fourth (CD68) graph (N=5, besides NEN+Clomi (N=4) and NEN+Desi (N=4)) data are presented as mean (SD) and were analyzed by a one-way ANOVA with Tukey post-hoc test. In the second and the fourth graph, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls (Ctrl). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Immunofluorescence ( IF ) of HCT116 treated with DMSO ( Control ( Ctrl ) ) , NEN , Domperidone ( Domp ) or NEN + Domp as indicated ( NEN , 1 . 2 µM ; Domp , 30 µM ) for 16 h stained with anti - Lamp1 ( top panel ) , Alexa - 555 LC3 ( middle panel ) co - stained with Alexa - 647 p62 ( bottom panel ) . Nuclei , Blue ( dapi ) . Scale bar 20 µm . Mean ( ) of Lamp1 ( Ctrl , NEN , Domp ( N = 20 ) ; NEN + Domp ( N = 24 ) ) , LC3 - II ( Ctrl , NEN + Domp ( N = 16 ) ) ; NEN , Domp ( N = 16 ) ) or P62 ( Ctrl ( N = 38 ) ; NEN ( N = 27 ) ; Domp ( N = 28 ) ; NEN + Domp ( N = 21 ) ) was quantified analyzed by one - way ANOVA with Tukey post - hoc ( for LC3 - II ) or Kruskal - Wallis with Dunn ´ s post - hoc ( for Lamp1 p62 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

B Immunofluorescence (IF) of HCT116 cells treated with DMSO (Control (Ctrl)), NEN, Domperidone (Domp) or NEN+Domp as indicated (NEN, 1.2 µM; Domp, 30 µM) for 16 h and stained with anti-Lamp1 (top panel), Alexa-555 LC3 (middle panel) co-stained with Alexa-647 p62 (bottom panel). Nuclei, Blue (dapi). Scale bar 20 µm. Mean (SEM) fluorescence of Lamp1 (Ctrl, NEN, Domp (N=20 cells); NEN+Domp (N=24 cells)), LC3-II (Ctrl, NEN+Domp (N=16 cells)); NEN, Domp (N=16 cells)) or P62 (Ctrl (N=38 cells); NEN (N=27 cells); Domp (N=28 cells); NEN+Domp (N=21 cells)) was quantified and analyzed by one-way ANOVA with Tukey post-hoc test (for LC3-II) or Kruskal-Wallis with Dunn´s post-hoc test (for Lamp1 and p62). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


C Relative UPP1 mRNA expression in HCT116 either transfected with control ( siCtrl ) or combined TFE3 - MITF - targeting ( siTFE3 / siMITF ) siRNAs treated as indicated ( NEN ( N ) 1 . 2 µM , Domperidone ( D ) 30 µM , ISRIB 1 µM ) , determined by qPCR . Data information : data ( N = 3 ) are presented as mean ( SD ) were analyzed by two - way ANOVA with Tukey post - hoc

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

C Relative UPP1 mRNA expression levels in HCT116 cells either transfected with control (siCtrl) or combined TFE3- and MITF- targeting (siTFE3/siMITF) siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM, ISRIB 1 µM), determined by qPCR. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test


F Immunoblot of HCT116 whole lysate using antibodies against indicated upon treatment of as indicated for 16h ( NEN ( low ) 0 . 6 µM or NEN ( High ) 1 . 2 µM , Domperidone ( Domp ) 30 µM ) were transfected either with control ( siCtrl ) or UPP1 - targeting ( siUPP1 ) siRNAs 48h prior to treatment .

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F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN (low) 0.6 µM or NEN (High) 1.2 µM, Domperidone (Domp) 30 µM) Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 48h prior to treatment.


H of toxicity viability of HCT116 spheroids ( 3D ) treated as indicated ( NEN 1 . 2 µM , Imipramine ( Imi ) , Desipramine ( Desi ) , Amitriptyline ( Ami ) , each 30 µM ) . were transfected with control ( siCtrl ) or UPP1 - targeting ( siUPP1 ) siRNAs 24h prior to spheroid formation . Data information : data ( N = 3 ) are presented as mean ( SD ) were analyzed by two - way ANOVA with Tukey post - hoc

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

H Ratio of toxicity and viability of HCT116 spheroids (3D) treated as indicated (NEN 1.2 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM). Cells were transfected with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 24h prior to spheroid formation. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test


A Relative mRNA expression of DHODH in HCT116 treated as indicated for 16h ( NEN ( N ) 1 . 2 µM , Domperidone ( Domp , D ) , Imipramine ( Imi ) , Desipramine ( Desi ) , Amitriptyline ( Ami ) , each 30 µM , Clomipramine ( Clomi ) 20 µM ) . Data information : , data are presented as mean ( SD ) ( N = 3 ) were analyzed by Kruskal - Wallis with Dunn ´ s post - hoc ( A , left graph ) , one - way ANOVA with Tukey post - hoc ( A , right graph ) In ( A ) , significance is indicated for comparison of different treatments to controls . * * p < 0 , 01 ; * * * * p < 0 , 0001 .

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

A Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM, Clomipramine (Clomi) 20 µM). Data information: , data are presented as mean (SD) (N=3) and were analyzed by Kruskal-Wallis with Dunn´s post-hoc test (A, left graph), one-way ANOVA with Tukey post-hoc test (A, right graph) In (A), significance is indicated for the comparison of the different treatments to controls. ** p < 0,01; **** p < 0,0001.


D Caspase activity in HCT116 treated as indicated for 24h ( NEN 0 . 6 µM , Imipramine ( Imi ) , Desipramine ( Desi ) Amitriptyline ( Ami ) , each 20 µM , Clomipramine ( Clomi ) 10 µM , A77126 ( A77 ) 50 µM ) . Data information : data are presented as mean ( SD ) ( N = 3 ) were analyzed by two - way ANOVA with Tukey post - hoc

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D Caspase activity in HCT116 cells treated as indicated for 24h (NEN 0.6 µM, Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 20 µM, Clomipramine (Clomi) 10 µM, A77126 (A77) 50 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test


A Immunofluorescence ( IF ) of HCT116 treated as indicated for 12h ( Imipramine Domperidone , each 30 µM ) stained for cholesterol content using filipin III . B Immunofluorescence ( IF ) of HCT116 treated with Domperidone co - stained for cholesterol Lamp1 .

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

A Immunofluorescence (IF) images of HCT116 cells treated as indicated for 12h (Imipramine and Domperidone, each 30 µM) and stained for cholesterol content using filipin III. B Immunofluorescence (IF) images of HCT116 cells treated with Domperidone and co-stained for cholesterol and Lamp1.


E Relative mRNA expression of specified genes in HCT116 , treated as indicated for 16h ( NEN ( N ) 1 . 2 µM , Domperidone ( D ) 30 µM , Cyclodextrin ( CyD ) 0 . 75 % ) . F Immunoblot of HCT116 whole lysates using antibodies against gammaH2AX treated as indicated for 16h ( NEN 1 . 2 µM , Domperidone ( Domp ) 30 µM , Cyclodextrin ( CD ) 0 . 75 % ) . Data information : data are presented as mean ( SD ) ( N = 3 ) were analyzed by a one - way ANOVA with Tukey post - hoc * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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E Relative mRNA expression levels of the specified genes in HCT116 cells, treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (D) 30 µM, Cyclodextrin (CyD) 0.75%). F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h (NEN 1.2 µM, Domperidone (Domp) 30 µM, Cyclodextrin (CD) 0.75%). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


A of toxicity viability determined in pancreatic cancer - derived organoids from two ( ( left ) PDO - 42 ( right ) PDO - 48 ) treated as indicated for 3 ( PDO - 42 ) or 5 ( PDO - 48 ) days . Significant differences are shown for comparison between single combined drug treatments as indicated ( NEN 1 . 2 µM or 2 . 5 µM , Amitriptyline ( Ami , A ) , Imipramine ( Imi , I ) , Desipramine ( Desi , D ) , each 30 µM , Clomipramine ( Clomi , C ) 20 µM ) . Data information : data are presented as mean ( SD ) ( N = 3 ) were analyzed by a one - way ANOVA with Tukey post - hoc . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

A Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between single and combined drug treatments as indicated (NEN 1.2 µM or 2.5 µM, Amitriptyline (Ami, A), Imipramine (Imi, I), Desipramine (Desi, D), each 30 µM, Clomipramine (Clomi, C) 20 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B of toxicity viability determined in pancreatic cancer - derived organoids from two ( ( left ) PDO - 42 ( right ) PDO - 48 ) treated as indicated for 3 ( PDO - 42 ) or 5 ( PDO - 48 ) days . Significant differences are shown for comparison between drug treatments as indicated to control condition ( NEN ( N ) 1 . 2 µM or 2 . 5 µM , Amitriptyline ( A ) , Imipramine ( I ) 20 µM , Paclitaxel ( Pacli ) , 2 , 20 or 200nM ) . As references for sensitization effects to standard chemotherapy , bars showing single Paclitaxel treatments using indicated concentrations are highlighted in blue . Bars for controls ( Ctrl ) , Paclitaxel ( Pacli ) , NEN ( N ) alone combined NEN + Paclitaxel treatment are identical in graphs for comparison to combinations with respective TCA drugs as indicated . Data information : data are presented as mean ( SD ) ( N = 3 ) were analyzed by a one - way ANOVA with Tukey post - hoc . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

B Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between drug treatments as indicated to the control condition (NEN (N) 1.2 µM or 2.5 µM, Amitriptyline (A), Imipramine (I) 20 µM, Paclitaxel (Pacli), 2, 20 or 200nM). As references for sensitization effects to standard chemotherapy, the bars showing single Paclitaxel treatments using the indicated concentrations are highlighted in blue. Bars for controls (Ctrl), Paclitaxel (Pacli), NEN (N) alone and combined NEN+Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated. Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


A . Immunoblot of extracts following depletion of WDR26 , RanBP9 , ARMC8 , GID4 using pools of siRNAs for 72 hr in HeLa Kyoto . Endogenous of indicated were monitored by western blotting ( n = 3 ) .

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

A. Immunoblot of cell extracts following depletion of WDR26, RanBP9, ARMC8, and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells. Endogenous levels of the indicated proteins were monitored by western blotting (n=3).


Western blotting of samples after ectopic overexpression of HBP1 ( B ) alone , or together with WDR26 or GID4 in HEK - 293T . HBP1 were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

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

Western blotting of samples after ectopic overexpression of HBP1 (B) alone, or together with WDR26 or GID4 in HEK-293T cells. HBP1 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


Western blotting of samples after ectopic overexpression of ZMYND19 ( C ) alone , or together with WDR26 or GID4 in HEK - 293T . ZMYND19 were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

Western blotting of samples after ectopic overexpression of ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK-293T cells. ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


C . , D . Western blot of in vitro ubiquitinated HBP1 , which was performed by mixing purified HBP1 with ubiquitin E1 , UBCH5a , ubiquitin in presence of indicated hGID sub - complexes ( n = 3 ) .

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

C., D. Western blot analysis of in vitro ubiquitinated HBP1, which was performed by mixing purified HBP1 with ubiquitin E1, UBCH5a, and ubiquitin in the presence of the indicated hGID sub-complexes (n=3).


E . Immunoblots of in vitro ubiquitinated ZMYND19 , which was performed by mixing purified ZMYND19 with ubiquitin E1 , UBE2H , ubiquitin 6 - subunit hGID ( ARCM8 , RanBP9 , WDR26 , MAEA , RMND5a TWA1 ) in presence or absence of GID4 a 10 - fold excess of PGLV GID4 - specific peptide ( n = 2 )

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

E. Immunoblots of in vitro ubiquitinated ZMYND19, which was performed by mixing purified ZMYND19 with ubiquitin E1, UBE2H, ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide (n=2)


D . Western blotting of samples following ectopic overexpression of HBP1 either alone , or with full - ( FL ) or WD40 - truncated WDR26 ( ΔWD40 ) in HEK - 293T . HBP1 were monitored in treated with MG132 or DMSO for 12 - 14 h ( n = 3 ) .

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

D. Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full-length (FL) or WD40-truncated WDR26 (ΔWD40) in HEK-293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12-14 h (n=3).


E . Western blot of in vitro ubiquitinated HBP1 in presence of wild type WDR26 ( WT ) or WDR26 ( ΔWD40 ) mutant ( n = 2 ) .

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

E. Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 (WT) or the WDR26 (ΔWD40) mutant (n=2).


C . Transiently expressed FLAG - GID4 HSS - tagged ARMC8 isoforms ( α or β ) in HEK293T . presence of GID4 in isoform - specific ARMC8 immunoprecipitates was visualized by immunoblotting ( left panels ) . right panel shows a western blot of transiently expressed immunoprecipitated HSS - WDR26 from HEK - 293T presence of endogenous ARMC8 isoforms ( α or β ) was probed by immunoblotting ( n = 2 ) .

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

C. Transiently expressed FLAG-GID4 and HSS-tagged ARMC8 isoforms (α or β) in HEK293T cells. The presence of GID4 in isoform-specific ARMC8 immunoprecipitates was visualized by immunoblotting (left panels). The right panel shows a western blot of transiently expressed and immunoprecipitated HSS-WDR26 from HEK-293T cells and the presence of endogenous ARMC8 isoforms (α or β) was probed by immunoblotting (n=2).


D . Baculoviral co - expression in Sf9 of 5 - subunit hGID ( 5mer ; Strep - RanBP9 , His - WDR26 , FLAG - MAEA , His - RMND5a His - TWA1 ) along with His - GID4 in presence of ARMC8α or ARMC8β . Strep - or His - pulldowns revealed presence of GID4 in ARMC8α , but not in ARMC8β , complexes ( n = 3 ) .

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

D. Baculoviral co-expression in Sf9 cells of the 5-subunit hGID complex (5mer; Strep-RanBP9, His-WDR26, FLAG-MAEA, His-RMND5a and His-TWA1) along with His-GID4 in the presence of ARMC8α or ARMC8β. Strep- or His-pulldowns revealed the presence of GID4 in ARMC8α, but not in ARMC8β, complexes (n=3).


E . Immunoblot of in vitro ubiquitinated GID4 - hGID complexes containing either ARMC8α or ARMC8β ( 6mer ; ARMC8 , RanBP9 , WDR26 , MAEA , RMND5a TWA1 ) . Where indicated , reaction was carried out in presence of 20 - fold molar excess of PGLV GID4 - binding peptide ( n = 3 ) .

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

E. Immunoblot analysis of in vitro ubiquitinated GID4-hGID complexes containing either ARMC8α or ARMC8β (6mer; ARMC8, RanBP9, WDR26, MAEA, RMND5a and TWA1) . Where indicated, the reaction was carried out in the presence of 20-fold molar excess of the PGLV GID4-binding peptide (n=3).


F . Western blot of in vitro ubiquitinated HBP1 by hGID 5 - subunit in presence of different ARMC8 isoforms ( α or β ) ( n = 2 ) .

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

F. Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5-subunit complex in the presence of the different ARMC8 isoforms (α or β) (n=2).


B , of log10 scaled PBMC community frequencies for samples ( n = 42 × 2 timepoints = 84 samples ) . Clinical variables are depicted for samples across top of ; convalescent post - symptom onset anti - CHIKV antibody titer viral titer ( which was measured during acute phase ) are both in units of log10 dilutions . Hierarchical clustering ( using complete linkage ) was applied to both samples ( X axis ) communities ( Y axis ) . Four major clusters of communities two major clusters of samples ( largely separating acute convalescent samples ) are highlighted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, heatmap of log10 scaled PBMC community frequencies for all samples (n = 42 patients × 2 timepoints = 84 samples). Clinical variables are depicted for all samples across the top of the heatmap; convalescent post-symptom onset anti-CHIKV antibody titer and viral titer (which was measured during the acute phase) are both in units of log10 dilutions. Hierarchical clustering (using complete linkage) was applied to both samples (X axis) and communities (Y axis). Four major clusters of communities and two major clusters of samples (largely separating acute and convalescent samples) are highlighted.


C , fold changes in log10 frequencies for PBMC communities between acute convalescent phase samples , filtered to a significance threshold of FDR < 0 . 05 ( moderated paired t - , mixed effects model ) . Error bars represent 95 % confidence interval . acute phase frequency for each community is depicted with purple . * , FDR - adjusted P ( FDR P ) < 0 . 05 ; * * , FDR P < 0 . 01 , * * * , FDR 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

C, fold changes in log10 frequencies for PBMC communities between acute and convalescent phase samples, filtered to a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model). Error bars represent the 95% confidence interval. The acute phase frequency for each community is depicted with the purple heatmap. *, FDR-adjusted P (FDR P) < 0.05; **, FDR P < 0.01, ***, FDR P < 0.001.


E , differences in CHIKV E2 expression between acute - phase convalescent - phase samples per MetaHybridLouvain sub - community . Sub - communities are ordered by largest to smallest difference filtered to sub - communities where median of channel means per sample was higher in acute - phase samples at a significance threshold of FDR < 0 . 05 . Sub - communities are named by their parent canonical community name plus an arbitrary

E, differences in CHIKV E2 protein expression between acute-phase and convalescent-phase samples per MetaHybridLouvain sub-community. Sub-communities are ordered by largest to smallest difference and filtered to sub-communities where the median of the channel means per sample was higher in the acute-phase samples at a significance threshold of FDR < 0.05. Sub-communities are named by their parent canonical community name plus an arbitrary number


F , of log10 scaled PBMC sub - community frequencies for samples ( n = 42 × 2 timepoints = 84 samples ) . Clinical variables are depicted for samples across top of ; convalescent post - symptom onset anti - CHIKV antibody titer viral titer ( which was measured during acute phase ) are both in units of log10 dilutions . Hierarchical clustering ( using complete linkage ) was applied to both samples ( X axis ) sub - communities ( Y axis ) . Four major clusters of sub - communities two major clusters of samples ( largely separating acute convalescent samples ) are highlighted .

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

F, heatmap of log10 scaled PBMC sub-community frequencies for all samples (n = 42 patients × 2 timepoints = 84 samples). Clinical variables are depicted for all samples across the top of the heatmap; convalescent post-symptom onset anti-CHIKV antibody titer and viral titer (which was measured during the acute phase) are both in units of log10 dilutions. Hierarchical clustering (using complete linkage) was applied to both samples (X axis) and sub-communities (Y axis). Four major clusters of sub-communities and two major clusters of samples (largely separating acute and convalescent samples) are highlighted.


, fold changes in log10 frequencies for PBMC sub - communities contrasted between acute convalescent phase samples , filtered to a significance threshold of FDR < 0 . 05 ( moderated paired t - , mixed effects model ) . Error bars represent 95 % confidence interval . acute phase frequency for each sub - community is depicted with purple . * , FDR - adjusted P ( FDR P ) < 0 . 05 ; * * , FDR P < 0 . 01 , * * * , FDR 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

, fold changes in log10 frequencies for PBMC sub-communities contrasted between acute and convalescent phase samples, filtered to a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model). Error bars represent the 95% confidence interval. The acute phase frequency for each sub-community is depicted with the purple heatmap. *, FDR-adjusted P (FDR P) < 0.05; **, FDR P < 0.01, ***, FDR P < 0.001.


A , relative expression of CD14 + CD16 + in CD14 + CD16 + sub - communities , depicted as boxplots of mean expression for samples ( n = 42 × 2 timepoints = 84 samples ) , indicates that sub - community 1 has a CD14 + CD16 + + ( aka " non - classical " ) phenotype , while sub - community 2 has a CD14 + + CD16 + ( aka " intermediate " ) phenotype . Differences shown here are significant at FDR < 0 . 05 ( moderated paired t - , mixed effects model ) ; for a view of differences significant at this

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

A, relative expression of CD14+ and CD16+ in CD14+CD16+ sub-communities, depicted as boxplots of the mean expression levels for all samples (n = 42 patients × 2 timepoints = 84 samples), indicates that sub-community 1 has a CD14+CD16++ (aka "non-classical") phenotype, while sub-community 2 has a CD14++CD16+ (aka "intermediate") phenotype. Differences shown here are significant at FDR < 0.05 (moderated paired t-test, mixed effects model); for a view of all differences significant at this threshold


B , of relative expression of markers that most differentiate sub - communities of CD14 + monocytes by difference in mean expression for samples ( n = 42 × 2 timepoints = 84 samples ) . Markers are filtered to those different at a significance threshold of FDR < 0 . 05 ( moderated paired t - , mixed effects model ) fold change > 1 . 3 ; for a view of differences significant at FDR < 0 . 05 mean of each channel across sub - communities is shown by adjacent purple .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, heatmap of relative expression of markers that most differentiate the sub-communities of CD14+ monocytes by difference in mean expression levels for all samples (n = 42 patients × 2 timepoints = 84 samples). Markers are filtered to those different at a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model) and fold change > 1.3; for a view of all differences significant at FDR < 0.05 The mean intensity of each channel across all sub-communities is shown by the adjacent purple heatmap.


( n = 42 pairs of samples , 1 pair per ) . Wilcoxon signed - rank was used to determine statistical significance , with a Benjamini - Hochberg adjustment for FDR ( 40 comparisons ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A , CXC chemokines . B , CC chemokines . C , interferons . D , TNF superfamily cytokines . E , interleukins . F , colony - stimulating factors .

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

(n = 42 pairs of samples, 1 pair per patient). Wilcoxon signed-rank test was used to determine statistical significance, with a Benjamini-Hochberg adjustment for FDR (40 comparisons). *P < 0.05, **P < 0.01, ***P < 0.001. A, CXC chemokines. B, CC chemokines. C, interferons. D, TNF superfamily cytokines. E, interleukins. F, colony-stimulating factors.


Human whole blood transcriptomic signatures for timepoint across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . A , volcano plot of differentially expressed host transcripts between acute - convalescent - phase samples , with negative log10 - scaled q values on Y axis , fitted β coefficient for each transcript ( corresponding to modeled log2 fold change ) on X axis . Transcripts to right of vertical dashed were comparatively upregulated in acute - phase samples , while transcripts to left were upregulated during convalescent phase . Transcripts that pass FDR < 0 . 05 fold change > 2 are colored salmon turquoise for acute - associated convalescent - associated transcripts , respectively . Top transcripts by q value or fold change are individually labeled by their corresponding gene symbol .

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

Human whole blood transcriptomic signatures for timepoint across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. A, volcano plot of differentially expressed host transcripts between acute- and convalescent-phase samples, with negative log10-scaled q values on the Y axis, and the fitted β coefficient for each transcript (corresponding to modeled log2 fold change) on the X axis. Transcripts to the right of the vertical dashed line were comparatively upregulated in acute-phase samples, while transcripts to the left were upregulated during the convalescent phase. Transcripts that pass FDR < 0.05 and fold change > 2 are colored salmon and turquoise for acute-associated and convalescent-associated transcripts, respectively. Top transcripts by q value or fold change are individually labeled by their corresponding gene symbol.


Human whole blood transcriptomic signatures for timepoint across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . B , of expression in units of Z - scores per transcript for top 50 differentially expressed host transcripts between acute convalescent phase samples . Clinical variables are depicted for samples across top of ; convalescent post symptom onset anti - CHIKV antibody titer viral titer ( which was measured during acute phase ) are both in units of log10 dilutions . Hierarchical clustering ( using complete linkage ) was applied to both samples ( X axis ) transcripts ( Y axis ) . Two major clusters of samples ( largely separating acute convalescent samples ) are highlighted .

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

Human whole blood transcriptomic signatures for timepoint across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. B, heatmap of expression in units of Z-scores per transcript for the top 50 differentially expressed host transcripts between acute and convalescent phase samples. Clinical variables are depicted for all samples across the top of the heatmap; convalescent post symptom onset anti-CHIKV antibody titer and viral titer (which was measured during the acute phase) are both in units of log10 dilutions. Hierarchical clustering (using complete linkage) was applied to both samples (X axis) and transcripts (Y axis). Two major clusters of samples (largely separating acute and convalescent samples) are highlighted.


Human whole blood transcriptomic signatures for CHIKV viral titer at acute phase of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . C , volcano plot as in A but for differentially expressed host transcripts between samples with higher lower CHIKV viral titer . Transcripts to right of vertical dashed are associated with higher viral titer , while transcripts to left are associated with lower viral titer . Transcripts significant at FDR < 0 . 05 are colored red .

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

Human whole blood transcriptomic signatures for CHIKV viral titer at the acute phase of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. C, volcano plot as in A but for differentially expressed host transcripts between samples with higher and lower CHIKV viral titer. Transcripts to the right of the vertical dashed line are associated with higher viral titer, while transcripts to the left are associated with lower viral titer. Transcripts significant at FDR < 0.05 are colored red.


Human whole blood transcriptomic signatures for symptom severity , across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . volcano plot for differentially expressed host transcripts between with more severe less severe acute - phase symptoms . Transcripts to right of vertical dashed were comparatively upregulated in more severe cases , while transcripts to left were upregulated in less severe cases .

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

Human whole blood transcriptomic signatures for symptom severity, across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. volcano plot for differentially expressed host transcripts between patients with more severe and less severe acute-phase symptoms. Transcripts to the right of the vertical dashed line were comparatively upregulated in more severe cases, while transcripts to the left were upregulated in less severe cases.


Human whole blood transcriptomic signatures for symptom severity across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . of expression for 20 differentially expressed host transcripts ( significant at FDR < 0 . 05 ) under a model that includes a severity - timepoint interaction . Transcripts have been filtered to those differentially expressed between timepoints in more severe cases but not in less severe cases .

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

Human whole blood transcriptomic signatures for symptom severity across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. heatmap of expression for 20 differentially expressed host transcripts (all significant at FDR < 0.05) under a model that includes a severity-timepoint interaction. Transcripts have been filtered to those differentially expressed between the timepoints in more severe cases but not in less severe cases.


Human whole blood transcriptomic signatures for convalescent ( 15d post - symptom onset ) anti - CHIKV antibody ( Ab ) titer across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . F , volcano plot for differentially expressed host transcripts between with higher lower convalescent ( 15d post symptom onset ) anti - CHIKV antibody ( Ab ) titers . Transcripts to right of vertical dashed were comparatively upregulated in with a higher convalescent anti - CHIKV antibody titer , while transcripts to left were upregulated in with a lower convalescent anti - CHIKV antibody titer .

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Human whole blood transcriptomic signatures for convalescent (15d post-symptom onset) anti-CHIKV antibody (Ab) titer across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. F, volcano plot for differentially expressed host transcripts between patients with higher and lower convalescent (15d post symptom onset) anti-CHIKV antibody (Ab) titers. Transcripts to the right of the vertical dashed line were comparatively upregulated in patients with a higher convalescent anti-CHIKV antibody titer, while transcripts to the left were upregulated in patients with a lower convalescent anti-CHIKV antibody titer.


( A ) detected in Vps34 - TAP immunoprecipitations by MS are listed . number of identified peptides of each peptide coverage are also shown .

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(A) Proteins detected in Vps34-TAP immunoprecipitations by MS analysis are listed. The number of identified peptides of each protein and the peptide coverage are also shown.


( B ) expressing TAP - tagged Atg38 indicated HA - tagged were grown in YPD . extracts were prepared were immunoprecipitated by IgG - Dynabeads as described in Materials methods . whole - extract ( left , " input " ) precipitated ( right ) were analyzed by immunoblotting with indicated antibodies .

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(B) Cells expressing TAP-tagged Atg38 and the indicated HA-tagged proteins were grown in YPD. The extracts were prepared and were immunoprecipitated by IgG-Dynabeads as described in Materials and methods. The whole-cell extract (left, "input") and the precipitated proteins (right) were analyzed by immunoblotting with the indicated antibodies.


( C ) Relative abundances of identified by LC - MS / MS of Atg14 - TAP Atg38 - TAP purifications from YPD - grown . Relative abundances are defined as emPAIprey / emPAIbait are shown as mean ± SD of two independent experiments .

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(C) Relative abundances of proteins identified by LC-MS/MS of Atg14-TAP and Atg38-TAP purifications from YPD-grown cells. Relative abundances are defined as emPAIprey/emPAIbait and are shown as mean ± SD of two independent experiments.


( D ) extracts were prepared from wild - type atg14Δ expressing Atg38 - TAP as well as HA - tagged of I components . IgG Dynabead - precipitated ( bottom ) , together with whole - extracts ( top ) , were immunoblotted with indicated antibodies .

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(D) Cell extracts were prepared from wild-type and atg14Δ cells expressing Atg38-TAP as well as HA-tagged proteins of complex I components. The IgG Dynabead-precipitated proteins (bottom), together with the whole-cell extracts (top), were immunoblotted with the indicated antibodies.


( E ) ATG38 - 2GFP ATG17 - 2mCherry in wild - type atg14Δ were cultured at 30 ° C with or without rapamycin . After 180 min , were analyzed by microscopy . Bar , 5 µm .

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(E) ATG38-2GFP ATG17-2mCherry cells in wild-type and atg14Δ were cultured at 30°C with or without rapamycin. After 180 min, cells were analyzed by fluorescence microscopy. Bar, 5 µm.


( A ) expressing Pho8Δ60 were grown in YPD shifted to SD ( - N ) medium for 3 h at 30 ° C . Lysates from each group of were tested for ALP activity . ALP activity is shown as mean ± SD of three independent experiments with activity of wild - type normalized to 1 .

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(A) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD(-N) medium for 3 h at 30°C. Lysates from each group of cells were tested for ALP activity. ALP activity is shown as mean ± SD of three independent experiments with the activity of wild-type cells normalized to 1.


( B ) with indicated genotypes were grown in YPD treated with rapamycin for 3 h at 30 ° C . Total was separated by SDS - PAGE detected by immunoblotting with anti - API .

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(B) Cells with the indicated genotypes were grown in YPD and treated with rapamycin for 3 h at 30°C. Total protein was separated by SDS-PAGE and detected by immunoblotting with anti-API.


( C ) were grown in YPD medium for 1 h at 30 ° C separated to intracellular ( I ; ) extracellular ( E ; medium ) fractions by centrifugation . fractions were treated as detailed in Materials methods aliquots of each were separated by SDS - PAGE . positions of precursor ( p2 - CPY ) mature ( m - CPY ) forms of CPY are indicated .

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(C) Cells were grown in YPD medium for 1 h at 30°C and separated to the intracellular (I; cells) and extracellular (E; medium) fractions by centrifugation. The fractions were treated as detailed in Materials and methods and aliquots of each were separated by SDS-PAGE. The positions of the precursor (p2-CPY) and mature (m-CPY) forms of CPY are indicated.


( A ) Wild - type atg38Δ expressing indicated GFP fusion Ape1 - mRFP were cultured at 30 ° C in presence of rapamycin . After 60 min , were analyzed by microscopy . Of total pool of Ape1 - mRFP signal , of Ape1 - mRFP colocalizing with GFP was calculated . This is shown as mean ± SD of two independent experiments , with number of counted indicated at bottom of graph . ( B ) Examples of wild - type atg38Δ expressing either Atg14 - 3 × GFP or Vps34 - 3 × GFP in A . Bar , 5 µm .

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(A) Wild-type and atg38Δ cells expressing the indicated GFP fusion proteins and Ape1-mRFP were cultured at 30°C in the presence of rapamycin. After 60 min, cells were analyzed by fluorescence microscopy. Of the total pool of Ape1-mRFP signal, the percentage of Ape1-mRFP colocalizing with GFP was calculated. This percentage is shown as mean ± SD of two independent experiments, with the number of cells counted indicated at the bottom of the graph. (B) Examples of wild-type and atg38Δ cells expressing either Atg14-3 × GFP or Vps34-3 × GFP in A. Bar, 5 µm.


( C ) extracts were prepared from in A , then analyzed by immunoblotting with anti - GFP anti - Pgk1 antibodies .

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(C) Cell extracts were prepared from the cells in A, then analyzed by immunoblotting with anti-GFP and anti-Pgk1 antibodies.


( A ) extracts were prepared from wild - type atg38Δ strains expressing TAP - tagged Atg38 indicated HA . extracts were immunoprecipitated by IgG - Dynabeads . whole - extracts ( top , " input " ) precipitates ( bottom ) were analyzed by immunoblotting with indicated antibodies . Relative binding efficiency was determined as described in Materials methods . To compare results from multiple experiments , value for reference sample ( wild - type ) was normalized to 1 . 0 . Relative binding efficiency is shown as mean ± SD of three independent experiments .

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

(A) Cell extracts were prepared from wild-type and atg38Δ strains expressing TAP-tagged Atg38 and the indicated HA proteins. The extracts were immunoprecipitated by IgG-Dynabeads. The whole-cell extracts (top, "input") and the precipitates (bottom) were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods. To compare results from multiple experiments, the value for the reference sample (wild-type cells) was normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.


( B ) Relative abundances of identified in TAP purifications from indicated strains were analyzed as described in Fig . 1 C are shown as mean ± SD of two independent experiments .

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(B) Relative abundances of proteins identified in TAP purifications from the indicated strains were analyzed as described in Fig. 1 C and are shown as mean ± SD of two independent experiments.


( C ) expressing Pho8Δ60 were grown in YPD shifted to SD ( - N ) medium for 3 h at 30 ° C . lysates from each group of were tested for ALP activity . ALP activity is shown as mean ± SD of three independent experiments with activity of wild - type normalized to 1 .

(C) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD(-N) medium for 3 h at 30°C. Cell lysates from each group of cells were tested for ALP activity. ALP activity is shown as mean ± SD of three independent experiments with the activity of wild-type cells normalized to 1.


( D ) expressing Vps34 - 6HA , Vps30 - 9myc , Atg38 - 3FLAG , Atg14 - TAP were lysed in a buffer containing 150 mM NaCl . Atg14 - TAP immunoprecipitate with IgG - Dynabeads from this lysate was split into eight fractions washed with lysis buffer containing indicated concentrations of NaCl . resulting precipitates were analyzed by immunoblotting with indicated antibodies . Relative binding efficiency was determined as described in Materials methods , although a 150 - mM NaCl sample was used as a control normalized to 1 . 0 . Relative binding efficiency is shown as mean ± SD of three independent experiments .

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(D) Cells expressing Vps34-6HA, Vps30-9myc, Atg38-3FLAG, and Atg14-TAP were lysed in a buffer containing 150 mM NaCl. The Atg14-TAP immunoprecipitate with IgG-Dynabeads from this lysate was split into eight fractions and washed with lysis buffer containing the indicated concentrations of NaCl. The resulting precipitates were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods, although a 150-mM NaCl sample was used as a control and normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.


( A ) AH109 containing indicated GAL4 activation domain fusions ( bait ) GAL4 - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . interaction of fusion was examined by plating on SD - Leu - Trp - His ( − LWH , middle ) SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

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(A) AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( B ) Interactions between indicated fusion were analyzed as in A . AH109 containing indicated GAL4 activation domain fusions ( bait ) GAL4 - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . interaction of fusion was examined by plating on SD - Leu - Trp - His ( − LWH , middle ) SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

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(B) Interactions between the indicated fusion proteins were analyzed as in A. AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( C ) TAP - tagged Vps34 was purified from vps30Δ atg14Δ vps38Δ . purified were separated on SDS - PAGE visualized by Coomassie . Recombinant GST - Atg38 ( WT ) , GST - Atg381 - 120 ( N ) , GST - Atg38121 - 226 ( C ) were incubated with Vps15 - Vps34 - TAP bound to IgG - Dynabeads . eluted were analyzed by immunoblotting .

(C) The TAP-tagged Vps34 protein complex was purified from the vps30Δ atg14Δ vps38Δ cells. The purified proteins were separated on SDS-PAGE and visualized by Coomassie staining. Recombinant GST-Atg38 (WT), GST-Atg381-120 (N), and GST-Atg38121-226 (C) were incubated with Vps15-Vps34-TAP complex bound to IgG-Dynabeads. The eluted proteins were analyzed by immunoblotting.


( A ) Interactions between indicated fusion were analyzed as in Fig . 4 A . AH109cells containing indicated GAL4 activation domain fusions ( bait ) GAL4 - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . interaction of fusion was examined by plating on SD - Leu - Trp - His ( − LWH , middle ) SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

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(A) Interactions between the indicated fusion proteins were analyzed as in Fig. 4 A. AH109cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( B ) of Atg38 by gel filtration . Recombinant Atg38 was fractionated over a gel filtration column . Each fraction was subjected to SDS - PAGE visualized by Coomassie .

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(B) Analysis of Atg38 by gel filtration. Recombinant Atg38 protein was fractionated over a gel filtration column. Each fraction was subjected to SDS-PAGE and visualized by Coomassie staining.


( A ) extracts were prepared from atg38Δ strains expressing Atg14 - TAP either Vps34 - 6HA or Vps15 - 6HA with Atg381 - 120 fused with GFP either GBP or GBP3A . extracts were immunoprecipitated with IgG - Dynabeads . precipitated , together with whole - extracts , were immunoblotted with indicated antibodies ( left ) . Relative binding efficiency was determined as described in Fig . 3 A was shown as mean ± SD of three independent experiments .

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(A) Cell extracts were prepared from atg38Δ strains expressing Atg14-TAP and either Vps34-6HA or Vps15-6HA with Atg381-120 fused with GFP and either GBP or GBP3A. The cell extracts were immunoprecipitated with IgG-Dynabeads. The precipitated proteins, together with the whole-cell extracts, were immunoblotted with the indicated antibodies (left). Relative binding efficiency was determined as described in Fig. 3 A and was shown as mean ± SD of three independent experiments.


B . Endogenous CUL4A CUL4B complexes were isolated from 293T lysates by immunoprecipitation ( IP ) associated were detected by immunoblotting ( IB ) . Mock includes beads only control .

B. Endogenous CUL4A and CUL4B complexes were isolated from 293T cell lysates by immunoprecipitation (IP) and associated proteins were detected by immunoblotting (IB). Mock includes beads only control.


C . Flag - tagged WT mutant WDTC1proteins were transiently expressed in 293T . Flag - WDTC1 complexes were immunoprecipitated with anti - FLAG their associated were detected by immunoblotting as indicated ; EV , empty vector control .

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C. Flag-tagged WT and mutant WDTC1proteins were transiently expressed in 293T cells. Flag-WDTC1 complexes were immunoprecipitated with anti-FLAG and their associated proteins were detected by immunoblotting as indicated; EV, empty vector control.


E . 293T were transfected with HA - ubiquitin along with various combinations of plasmid siRNA as indicated . Transfected were lysed under denaturing conditions to obtain whole extracts ( WCE ) immunoprecipitated with anti - FLAG . WDTC1 ubiquitylation was evaluated by immunoblotting as indicated .

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E. 293T cells were transfected with HA-ubiquitin along with various combinations of plasmid and siRNA as indicated. Transfected cells were lysed under denaturing conditions to obtain whole cell extracts (WCE) and immunoprecipitated with anti-FLAG. WDTC1 ubiquitylation was evaluated by immunoblotting as indicated.


F . 293T transiently expressing Flag - WDTC1 were transfected with scramble ( scrm ) siRNA or siRNAs against DDB1 , CUL4A CUL4B . Flag - WDTC1 knockdown efficiency were assessed by immunoblotting as indicated .

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F. 293T cells transiently expressing Flag-WDTC1 were transfected with scramble (scrm) siRNA or siRNAs against DDB1, CUL4A and CUL4B. Flag-WDTC1 levels and knockdown efficiency were assessed by immunoblotting as indicated.


A . 3T3 - L1preadipocytes were adipogenically induced lysates were prepared from collected at indicated days . Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti - WDTC1 followed by immunoblot analyses with indicated antibodies . Fatty acid synthase ( FAS ) were monitored to evaluate induction .

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A. 3T3-L1preadipocytes were adipogenically induced and lysates were prepared from cells collected at indicated days. Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti-WDTC1 followed by immunoblot analyses with indicated antibodies. Fatty acid synthase (FAS) levels were monitored to evaluate induction.


B - D . 3T3 - L1 stable expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 were adipogenically induced . Adipogenesis was assessed by Oil Red O ( ORO ) ( B ) ; plate ( top ) microscopic ( bottom ) views , 0 . 1 mm scale bar .

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B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenesis was assessed by Oil Red O staining (ORO) (B); plate (top) and microscopic (bottom) views, 0.1 mm scale bar.


B - D . 3T3 - L1 stable expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 were adipogenically induced . Triglyceride were quantified by an enzymatic ( C ) .

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B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Triglyceride levels were quantified by an enzymatic assay (C).


B - D . 3T3 - L1 stable expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 were adipogenically induced . Adipogenic gene expression was evaluated by RT - qPCR ( D ) .

B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenic gene expression was evaluated by RT-qPCR analysis (D).


A . Schematic of WDTC1 knockdown - rescue lentiviral vector ( top ) . 3T3 - L1preadipocytes were lentivirally transduced to generate stable expressing a non - specific ( NS ) shRNA control , shWDTC1 targeting endogenous Wdtc1mRNA , or simultaneously expressing shWDTC1 shRNA - resistant WDTC1 ; bottom , confirmation of knockdown WDTC1 ectopic expression .

A. Schematic of WDTC1 knockdown-rescue lentiviral vector (top). 3T3-L1preadipocytes were lentivirally transduced to generate stable cells expressing a non-specific (NS) shRNA control, shWDTC1 targeting endogenous Wdtc1mRNA, or cells simultaneously expressing shWDTC1 and shRNA-resistant WDTC1; bottom, confirmation of knockdown and WDTC1 ectopic expression.


B - D . 3T3 - L1 stable described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by ORO ( B )

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B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by ORO (B)


B - D . 3T3 - L1 stable described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by triglyceride quantitation ( C )

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B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by triglyceride quantitation (C)


B - D . 3T3 - L1 stable described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by adipogenic gene expression ( D ) .

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B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by adipogenic gene expression (D).


A . Induced 3T3 - L1 stably expressing Flag - WDTC1 were subjected to subcellular fractionation . Aliquots of total extract ( TCE ) subcellular fractions were analyzed by immunoblot as indicated ; fraction purity was assessed by α - Tubulin histone H3 antibodies .

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A. Induced 3T3-L1 cells stably expressing Flag-WDTC1 were subjected to subcellular fractionation. Aliquots of total cell extract (TCE) and subcellular fractions were analyzed by immunoblot as indicated; fraction purity was assessed by α-Tubulin and histone H3 antibodies.