Wild - type ( Wt ) , KRASG12D , Trp53f / f mice were intercrossed , possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad - Cre were sacrificed when moribund . In parallel , C57BL / 6 mice received ten consecutive weekly intraperitoneal injections of 1 g / Kg urethane were sacrificed after six months . Data summary ( ) representative of immunoreactivity of tissue sections of pleural pulmonary tissues tumors from these mice for different markers of human malignant pleural mesothelioma ( MPM ) lung adenocarcinoma ( LUAD ) . n = 10 mice / group were analyzed for each marker . Brown color indicates immunoreactivity blue color hematoxylin counterstaining . Note ubiquitous strong expression of Wilms ' 1 ( WT1 ) , patchy moderate expression of vimentin ( VIM ) , ubiquitous moderate expression of mesothelin ( MSLN ) , ubiquitous strong expression of calretinin ( CALB2 ) , podoplanin ( PDPN ) , osteopontin ( SPP1 ) , patchy moderate expression of cytokeratin 5 / 6 ( CK5 / 6 ) , absence of expression of surfactant C ( SFTPC ) in murine KRAS - driven mesotheliomas . Note also ubiquitous strong expression of WT1 , patchy moderate expression of VIM , ubiquitous low - level expression of MSLN , ubiquitous strong expression of CALB2 SPP1 , ubiquitous low - level expression of PDPN , variable moderate expression of CK5 / 6 , ubiquitous moderate expression of SFTPC in murine KRASG12D - driven urethane - induced LUAD .

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Wild-type (Wt), KRASG12D, and Trp53f/f mice were intercrossed, and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad-Cre and were sacrificed when moribund. In parallel, C57BL/6 mice received ten consecutive weekly intraperitoneal injections of 1 g/Kg urethane and were sacrificed after six months. Data summary (heatmap) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma (MPM) and lung adenocarcinoma (LUAD). n = 10 mice/group were analyzed for each marker. Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining. Note the ubiquitous strong expression of Wilms' tumor 1 (WT1), patchy moderate expression of vimentin (VIM), ubiquitous moderate expression of mesothelin (MSLN), ubiquitous strong expression of calretinin (CALB2), podoplanin (PDPN), and osteopontin (SPP1), patchy moderate expression of cytokeratin 5/6 (CK5/6), and the absence of expression of surfactant protein C (SFTPC) in murine KRAS-driven mesotheliomas. Note also the ubiquitous strong expression of WT1, the patchy moderate expression of VIM, the ubiquitous low-level expression of MSLN, the ubiquitous strong expression of CALB2 and SPP1, the ubiquitous low-level expression of PDPN, the variable moderate expression of CK5/6, and the ubiquitous moderate expression of SFTPC in murine KRASG12D-driven and urethane-induced LUAD.


C Data summaries from in vitro MTT reduction ( top ; 2 x 104 / well ; n = 6 independent experiments ) in vivo subcutaneous growth after injection of 106 per C57BL / 6 mouse ( bottom ; n = 5 / group ) .

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C Data summaries from in vitro MTT reduction (top; 2 x 104 cells/well; n = 6 independent experiments) and in vivo subcutaneous tumor growth after injection of 106 cells per C57BL/6 mouse (bottom; n = 5/group).


E , F RT - PCR ( E ) qPCR ( F ) of KPM parental tumors show Trp53f / f allele deletion ( Δ ) Bap1 Cdkn2a overexpression compared with PMC .

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E, F RT-PCR (E) and qPCR (F) of KPM cells and parental tumors show Trp53f/f allele deletion (Δ) and Bap1 and Cdkn2a overexpression compared with PMC.


A Kaplan - Meier survival plot with survival table .

A Kaplan-Meier survival plot with survival table.


F , G C57BL / 6 mice received pleural KPM1 followed by a single intrapleural injection of liposomes containing 1 % DMSO or 15 mg / Kg deltarasin in 1 % DMSO at day 9 post - . Shown are data summaries of MPE volume ( n = 8 7 DMSO deltarasin - treated mice / group , respectively ) pleural fluid nucleated at day 19 post - KPM1 ( F ) , as well as representative of pleural effusions ( dashed lines ) tumors ( t in ( G ) ) .

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F, G C57BL/6 mice received pleural KPM1 cells followed by a single intrapleural injection of liposomes containing 1% DMSO or 15 mg/Kg deltarasin in 1% DMSO at day 9 post-tumor cells. Shown are data summaries of MPE volume (n = 8 and 7 DMSO and deltarasin-treated mice/group, respectively) and pleural fluid nucleated cells at day 19 post-KPM1 cells (F), as well as representative images of pleural effusions (dashed lines) and tumors (t in (G)).


B Volcano plot showing some top KPM versus PMC differentially expressed genes .

B Volcano plot showing some top KPM versus PMC differentially expressed genes.


C KPM PMC expression of classic mesothelioma markers ( top ) top KPM versus PMC overexpressed genes ( bottom ) .

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C KPM and PMC expression of classic mesothelioma markers (top) and top KPM versus PMC overexpressed genes (bottom).


( A ) Flow - cytometry for CD1d expression ( top ) CD1d mean ( MFI ; bottom ) on CD45 - , ILCs ( Lin - CD45 + CD127 + ) , DCs ( CD11c + ) B ( B220 + ) of depicted tissues from WT mice ( n = 4 ) .

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(A) Flow-cytometry for CD1d expression (top) and CD1d mean fluorescence intensity (MFI; bottom) on CD45- cells, ILCs (Lin-CD45+CD127+), DCs (CD11c+) and B cells (B220+) of the depicted tissues from WT mice (n=4).


( B ) Flow - cytometry profiles showing gating strategy ( dot - plots ) , CD1d expression ( histograms ) CD1d MFI ( bars ) in T - bet + ( empty profile ) , GATA - 3 + ( grey ) , RORγt + ( grey ) T - bet + RORγt + ( dotted ) ILCs in mLN , spleen , PP SI - LP ( n = 5 ) . Numbers indicate of in depicted gates .

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(B) Flow-cytometry profiles showing gating strategy (dot-plots), CD1d expression (histograms) and CD1d MFI (bars) in T-bet+ (empty profile), GATA-3+ (dark grey), RORγt+ (light grey) and T-bet+RORγt+ (dotted line) ILCs in mLN, spleen, PP and SI-LP (n=5). Numbers indicate percentage of cells in the depicted gates.


( C ) Quantitative RT - PCR of mRNA encoding CD1d in freshly isolated ILC3s ( from mLN of CD1d - deficient mice mLN spleen of WT mice ) , B DCs ( n = 3 ) . Results are normalized to those of GAPDH ; RE = relative expression . * * p < 0 . 01 ( two - unpaired t - ) .

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(C) Quantitative RT-PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s (from mLN of CD1d-deficient mice and mLN and spleen of WT mice), B cells and DCs (n=3). Results are normalized to those of GAPDH; RE=relative expression. **p< 0.01 (two-tailed unpaired t-test).


( D ) Flow - cytometry profiles showing NKp46 RORγt expression in Lin - CD45 + ; CD1d CCR6 expression in NCR - ILC3s CD1d CD4 expression in NCR - CCR6 + ILC3s from mLN SI - LP ( n = 4 ) . Numbers indicate of in depicted gates .

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(D) Flow-cytometry profiles showing NKp46 and RORγt expression in Lin-CD45+cells; CD1d and CCR6 expression in NCR-ILC3s and CD1d and CD4 expression in NCR-CCR6+ILC3s from mLN and SI-LP (n=4). Numbers indicate percentage of cells in the depicted gates.


( E ) CD1d expression ( histograms ) CD1d MFI in NCR + ( empty profile ) , NCR - CCR6 - ( grey ) NCR - CCR6 + ( grey ) ILC3s from mLN SI - LP ( n = 4 ) .

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(E) CD1d expression (histograms) and CD1d MFI in NCR+ (empty profile), NCR-CCR6- (dark grey) and NCR-CCR6+ (light grey) ILC3s from mLN and SI-LP (n=4).


( A ) were cultured in presence or absence of BODIPY - αGalCer at 4oC or 37oC as indicated . Flow cytometry profiles percentages of BODIPY - αGalCer + DC or ILC3 from indicated tissues incubated at 4oC ( bars ) or 37oC ( grey bars ) are shown ( n = 3 ) .

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(A) Cells were cultured in the presence or absence of BODIPY-αGalCer at 4oC or 37oC as indicated. Flow cytometry profiles and percentages of BODIPY-αGalCer+ DC or ILC3 from the indicated tissues incubated at 4oC (white bars) or 37oC (grey bars) are shown (n=3).


( B ) WT mice were intravenously injected with BODIPY - αGalCer or PBS ( control ) ; lipid uptake was analysed by flow - cytometry in depicted populations from spleen 16 h after lipid injection ( n = 3 ) . Numbers indicate of in depicted gates .

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(B) WT mice were intravenously injected with BODIPY-αGalCer or PBS (control); lipid uptake was analysed by flow-cytometry in the depicted populations from the spleen 16 h after lipid injection (n=3). Numbers indicate percentage of cells in the depicted gates.


( C ) IL - 2 secretion by DN32 . D3 co - cultured with ILC3 sort - purified from mLN or spleen as indicated , pulsed ( + ) or not ( - ) with αGalCer with or without antiCD1d - blocking antibody .

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(C) IL-2 secretion by DN32.D3 cells co-cultured with ILC3 sort-purified from mLN or spleen as indicated, pulsed (+) or not (-) with αGalCer and with or without antiCD1d-blocking antibody.


( D - F ) Sort - purified ILC3s were preincubated with αGalCer ( ILC3 ( αGal ) ) or PBS ( ILC3 ( cont ) ) adoptively transferred into WT recipients . ( E ) Fold change in mRNA expression for indicated cytokines in iNKT sort - purified from spleen , mLN or SI - LP of mice injected with αGalCer - loaded ILC3s . Gene expression was measured by qPCR , normalized to GAPDH presented as expression relative to iNKT sort - purified from mice injected with control ILC3s ( n = 3 - 5 ) .

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(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (E) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort-purified from spleen, mLN or SI-LP of mice injected with αGalCer-loaded ILC3s. Gene expression was measured by qPCR, normalized to GAPDH and presented as expression relative to iNKT cells sort-purified from mice injected with control ILC3s (n=3-5).


( D - F ) Sort - purified ILC3s were preincubated with αGalCer ( ILC3 ( αGal ) ) or PBS ( ILC3 ( cont ) ) adoptively transferred into WT recipients . ( F ) Intracellular cytokine ( left ) of cytokine + ( right ) in spleniciNKT from ILC3 - recipient mice ( n = 3 ) . Numbers indicate of in depicted gates . * * p < 0 . 01 , two - unpaired t - .

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(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (F) Intracellular cytokine staining (left) and percentage of cytokine+ cells (right) in spleniciNKT cells from ILC3-recipient mice (n=3). Numbers indicate percentage of cells in the depicted gates. **p<0.01, two-tailed unpaired t-test.


( A - B ) Flow - cytometry plots showing gating strategy ( A ) of ILC populations ( from Lin - CD45 + CD127 + , B ) in mLN from WT ( n = 4 ) CD1d - deficient ( n = 3 ) mice .

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(A-B) Flow-cytometry plots showing gating strategy (A) and percentage of ILC populations (from Lin-CD45+CD127+cells, B) in mLN from WT (n=4) and CD1d-deficient (n=3) mice.


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( C ) Fold change in mRNA expression for indicated cytokines in ILC3s after antibody - mediated CD1d crosslink ( n = 3 ) . Gene expression was measured by qPCR normalized to GAPDH to mRNA expression in control ILC3s . * p < 0 . 05 two - unpaired t - .

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(C) Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody-mediated CD1d crosslink (n=3). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. *p<0.05 two-tailed unpaired t-test.


( D ) Fold change in IL22 mRNA expression in ILC3s after antibody - mediated CD1d crosslink / or IL - 23 stimulation ( n = 4 ) . Gene expression was measured by qPCR normalized to GAPDH to mRNA expression in control ILC3s . * * p < 0 . 01 two - unpaired t - .

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(D) Fold change in IL22 mRNA expression in ILC3s after antibody-mediated CD1d crosslink and/or IL-23 stimulation (n=4). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. **p<0.01 two-tailed unpaired t-test.


( E ) IL - 22 detection by ELISA in supernatant of ILC3s cultured in presence of αCD1d / or IL - 23 ( n = 3 ) . * p < 0 . 05 two - unpaired t -

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(E) IL-22 detection by ELISA in the supernatant of ILC3s cultured in the presence of αCD1d and/or IL-23 (n=3). *p<0.05 two-tailed unpaired t-test


( F - G ) Intracellular cytokine ( F ) of IL - 22 + ILC3s ( G ) after stimulation with αCD1d , IL - 23 / or IL - 1β . * p < 0 . 05 , * * p < 0 . 01 two - unpaired t - ( n = 4 - 8 )

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(F-G) Intracellular cytokine staining (F) and percentage of IL-22+ILC3s (G) after stimulation with αCD1d, IL-23 and/or IL-1β. *p<0.05, **p<0.01 two-tailed unpaired t-test (n=4-8)


( H ) WT mice were i . v . injected with αGalCer or PBS ( control ) activation of splenicILC3s was assessed by flow - cytometry as CD69 up - regulation at 6 h or 16 h after injection . Grey filled profile = ILC3 from αGalCer - injected mice ; Empty profile = ILC3 from PBS - injected mice ; Dotted = CD45 - . Right graph , of CD69 + ILC3s 6 h after injection of αGalCer ( grey ) or PBS ( ) . ( n = 3 ) * p < 0 . 05 two - unpaired t - .

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(H) WT mice were i.v. injected with αGalCer or PBS (control) and activation of splenicILC3s was assessed by flow-cytometry as CD69 up-regulation at 6 h or 16 h after injection. Grey filled profile= ILC3 from αGalCer-injected mice; Empty profile= ILC3 from PBS-injected mice; Dotted line= CD45-cells. Right graph, percentage of CD69+ILC3s 6 h after injection of αGalCer (grey) or PBS (white). (n=3) *p<0.05 two-tailed unpaired t-test.


( I - J ) Fold change in mRNA expression for indicated cytokines in ILC3s sort - purified from spleen ( I ) or SI - LP ( J ) 6 h after intravenous ( I ) or oral ( J ) αGalcer administration . Gene expression was measured by qPCR normalized to GAPDH to expression in ILC3s sort - purified from PBS - injected mice ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 two - unpaired t - .

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(I-J) Fold change in mRNA expression for the indicated cytokines in ILC3s sort-purified from spleen (I) or SI-LP (J) 6 h after intravenous (I) or oral (J) αGalcer administration. Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort-purified from PBS-injected mice (n=3). *p<0.05, **p<0.01 two-tailed unpaired t-test.


( A ) Neural precursors derived from human iPS ( iNPCs ) were mock or ZIKV - infected co - stained for Centrin ( green ) , Sox2 ( blue ) flavivirus envelope ( red ) . were fixed 16 hours post infection ( hpi ) . ( B ) Mock ZIKV - infected U87 H4 in S phase co - stained for Centrin ( green ) , flavivirus envelope ( red ) ( blue ) . ( C ) of of mock or ZIKV - infected iNPC , U87 or H4 in S phase with greater than four Centrin foci . Bars represent mean ± SD normalized to control . Asterisk denotes p < 0 . 005 ( Student ' s t ) . Three biological replicates are represented . Data information : Scale bars indicate 100nm .

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(A) Neural precursors derived from human iPS cells (iNPCs) were mock or ZIKV-infected and co-stained for Centrin (green), Sox2 (blue) and flavivirus envelope (red). All cells were fixed 16 hours post infection (hpi). (B) Mock and ZIKV-infected U87 and H4 cells in S phase co-stained for Centrin (green), flavivirus envelope (red) and DNA (blue). (C) Quantification of the percentage of mock or ZIKV-infected iNPC, U87 or H4 cells in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 100nm.


( A ) Mock or ZIKV - infected U87 in S phase co - stained for Centrin ( red ) centrosomal microcephaly - associated , CEP63 ( green ) . ( B ) ± s . d . of CEP63 at centrosome ( 0 . 5μm around Centrin signal ) around centrosome ( 1μm surrounding Centrin signal minus centrosomal CEP63 ) were quantified in mock ZIKV infected U87 . For quantifications 8 were analyzed per experiment ( n = 3 ) . Asterisk denotes p < 0 . 005 ( Student ' s t ) . Data information : Scale bars indicate 5μm for . were fixed 16 hours post infection ( hpi ) ,

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(A) Mock or ZIKV-infected U87 cells in S phase co-stained for Centrin (red) and the centrosomal microcephaly-associated protein, CEP63 (green). (B) The fluorescence intensity ± s.d. of CEP63 at the centrosome (0.5μm around the Centrin signal) and around the centrosome (1μm surrounding the Centrin signal minus the centrosomal CEP63) were quantified in mock and ZIKV infected U87 cells. For fluorescence quantifications 8 cells were analyzed per experiment (n=3). Asterisk denotes p<0.005 (Student's t test). Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),


( D ) Immunoblot of lysate from mock or ZIKV - infected U87 probed for CEP63 ZIKV NS3 . Actin served as a loading control . Asterisk denotes specific band for CEP63 .

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(D) Immunoblot analysis of lysate from mock or ZIKV-infected U87 cells probed for CEP63 and ZIKV NS3. Actin served as a loading control. Asterisk denotes specific band for CEP63.


( E ) S - phase mock ZIKV infected control Cep63gt / gt MEFs co - stained for Centrin ( green ) flavivirus envelope ( red ) . ( F ) of of mock or ZIKV - infected Het Cep63gt / gt MEFs in S phase with greater than four Centrin foci . Bars represent mean ± SD normalized to control . Asterisk denotes p < 0 . 005 ( Student ' s t ) . Three biological replicates are represented . Data information : Scale bars indicate 5μm for . were fixed 16 hours post infection ( hpi ) ,

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(E) S-phase mock and ZIKV infected control and Cep63gt/gt MEFs co-stained for Centrin (green) and flavivirus envelope (red). (F) Quantification of the percentage of mock or ZIKV-infected Het and Cep63gt/gt MEFs in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),


( B ) Mock or ZIKV - infected U87 lysates were immunoprecipitated for CEP63 or c - Myc , which served as a negative control for this other immunoprecipitations ( Control ) . Precipitated were immunoblotted for CEP63 NS3 . Asterisk denotes specific band .

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(B) Mock or ZIKV-infected U87 cell lysates were immunoprecipitated for CEP63 or c-Myc, which served as a negative control for this and other immunoprecipitations (Control). Precipitated proteins were immunoblotted for CEP63 and NS3. Asterisk denotes specific band.


( C ) We expressed Myc - tagged Brazilian Zika NS3 in 293T / 17 immunoprecipitated c - Myc . Precipitated were probed for Myc tag of NS3 endogenous CEP63 . centriole distal end component , CP110 served as a negative control . Asterisk denotes specific band for CEP63 .

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(C) We expressed Myc-tagged Brazilian Zika NS3 in 293T/17 cells and immunoprecipitated c-Myc. Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63. The centriole distal end component, CP110 served as a negative control. Asterisk denotes specific band for CEP63.


( G ) HeLa transfected with Control or Myc - tagged Brazilian ZIKV NS3 ( Br NS3 ) co - stained for Myc ( red ) Centrin ( green ) . Data information : Scale bars indicate 5μm for .

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(G) HeLa cells transfected with Control or Myc-tagged Brazilian ZIKV NS3 (Br NS3) co-stained for Myc (red) and Centrin (green). Data information: Scale bars indicate 5μm for all images.


( H ) of of control NS3 expressing HeLa with greater than four Centrin foci . Bars represent mean ± SD normalized to control . For quantifications at least 100 were counted per experiment ( biological replicates = 3 ) . p < 0 . 005 ( paired Student ' s t ) for comparison of NS3 to mock transfected was denoted with asterisk .

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(H) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. For all quantifications at least 100 cells were counted per experiment (biological replicates=3). p<0.005 (paired Student's t test) for the comparison of NS3 to mock transfected cells was denoted with asterisk.


( A ) HeLa lysates were immunoprecipitated with antibodies to CEP63 TBK1 . Precipitated were immunoblotted for CEP63 TBK1 .

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(A) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1. Precipitated proteins were immunoblotted for CEP63 and TBK1.


( B ) Mock ZIKV - infected U87 were co - stained for γ - tubulin ( red ) p - TBK1 ( green ) . Data information : Scale bars indicate 5μm for .

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(B) Mock and ZIKV-infected U87 cells were co-stained for γ-tubulin (red) and p-TBK1 (green). Data information: Scale bars indicate 5μm for all images.


( C ) Lysate from mock or ZIKV - infected U87 at 16 hpi were immunoblotted with antibodies to NS3 , TBK1 , p - TBK1 . Actin served as a loading control .

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(C) Lysate from mock or ZIKV-infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3, TBK1, and p-TBK1. Actin served as a loading control.


( D ) RT - ddPCR of IFNβ transcript at indicated time points in mock ZIKV - infected H4 . Bars represent mean ± SD normalized to control . Asterisk denotes p < 0 . 005 ( Student ' s t ) . Three biological replicates are represented .

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(D) RT-ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV-infected H4 cells. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented.


( E ) Control Cep63gt / gt MEFs were co - stained with antibodies to γ - tubulin ( red ) p - Tbk1 ( green ) . Data information : Scale bars indicate 5μm for .

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(E) Control and Cep63gt/gt MEFs were co-stained with antibodies to γ-tubulin (red) and p-Tbk1 (green). Data information: Scale bars indicate 5μm for all images.


( F ) Immunoblot of control Cep63gt / gt MEFs probed with antibodies to Tbk1 p - Tbk1 . Actin served as a loading control .

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(F) Immunoblot analysis of control and Cep63gt/gt MEFs probed with antibodies to Tbk1 and p-Tbk1. Actin served as a loading control.


( G ) HeLa lysate was immunoprecipitated with antibodies to CEP63 , DTX4 or c - Myc ( negative control ) . Precipitated were blotted with antibodies to CEP63 DTX4 .

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(G) HeLa cell lysate was immunoprecipitated with antibodies to CEP63, DTX4 or c-Myc (negative control). Precipitated proteins were blotted with antibodies to CEP63 and DTX4.


( H ) Mock ZIKV - infected U87 co - stained for Centrin ( red ) DTX4 ( green ) . Data information : Scale bars indicate 5μm for .

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(H) Mock and ZIKV-infected U87 cells co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.


( I ) Total lysate from mock ZIKV - transfected U87 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 DTX4 . Actin served as a loading control .

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(I) Total cell lysate from mock and ZIKV-transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4. Actin served as a loading control.


( J ) SC CEP63 siRNA treated HeLa were co - stained for Centrin ( red ) DTX4 ( green ) . Data information : Scale bars indicate 5μm for .

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(J) SC and CEP63 siRNA treated HeLa cells were co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.


( K ) TBK1 or c - Myc ( negative control ) were immunoprecipitated from Control or Cep63 mutant MEFs . Precipitating were probed with antibodies to TBK1 K48 - linked ubiquitin .

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(K) TBK1 or c-Myc (negative control) were immunoprecipitated from Control or Cep63 mutant MEFs. Precipitating proteins were probed with antibodies to TBK1 and K48-linked ubiquitin.


( L ) 16hpi Mock ZIKV - infected U87 lysate was immunoprecipitated for TBK1 or c - Myc . immunoprecipitations were probed using antibodies to TBK1 K - 48 - linked ubiquitin .

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(L) 16hpi Mock and ZIKV-infected U87 cell lysate was immunoprecipitated for TBK1 or c-Myc. The immunoprecipitations were probed using antibodies to TBK1 and K-48-linked ubiquitin.


A , B : serum of S1P ( A ) apoM ( B ) in HLT ( n = 47 ) COV ( n = 111 ) . box - plots represent interquartile range with median value ( central ) ; whiskers represent measured range of HLT COV . Each measurement was run in triplicate , performed at least twice . Two - Student ' s t - was used for statistical . * * * * p < 0 . 0001 .

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A, B: serum levels of S1P (A) and apoM (B) in HLT (n=47) and COV (n=111) patients. The box-plots represent the interquartile range with median value (central line); the whiskers represent the measured range of HLT and COV. Each measurement was run in triplicate, and performed at least twice. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.


Pearson correlation between S1P apoM ( C ) Scatter plots , together with fitted regression , are shown . Pearson correlation was performed for statistical . Exact p values or * * * * p < 0 . 0001 are reported .

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Pearson correlation between S1P and apoM (C) Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.


Pearson correlation between S1P RBC number ( D ) , HGB ( E ) , HCT value ( F ) . Scatter plots , together with fitted regression , are shown . Pearson correlation was performed for statistical . Exact p values or * * * * p < 0 . 0001 are reported .

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Pearson correlation between S1P and RBC number (D), HGB concentration (E), HCT value (F). Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.


A , B : correlations between S1P HDL - C ( A ) , albumin ( B ) . Data information : Scatter plots fitted regression are shown in each figure . Each S1P measurement was run in triplicate , performed at least twice in independent assays . Pearson correlation was performed for statistical . Exact p values or * * * * p < 0 . 0001 are reported in figure .

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A, B: correlations between S1P and HDL-C (A), and albumin (B). Data information: Scatter plots and fitted regression line are shown in each figure. Each S1P measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.


C , D : correlations between apoM HDL - C ( C ) albumin ( D ) . Data information : Scatter plots fitted regression are shown in each figure . Each apoM measurement was run in triplicate , performed at least twice in independent assays . Pearson correlation was performed for statistical . Exact p values or * * * * p < 0 . 0001 are reported in figure .

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C, D: correlations between apoM and HDL-C (C) and albumin (D). Data information: Scatter plots and fitted regression line are shown in each figure. Each apoM measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.


( A - D ) serum concentrations of S1P ( A ) , apoM ( B ) , albumin ( C ) HDL - C ( D ) in noICU ( n = 89 ) ICU ( n = 22 ) are shown . box - plots represent interquartile range with median ( central ) ; whiskers represent measured range of noICU ICU . Each S1P apoM measurement was run in triplicate , performed at least twice in independent assays . Two - Student ' s t - was used for statistical . * * * * p < 0 . 0001 .

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(A-D) The serum concentrations of S1P (A), apoM (B), albumin (C) and HDL-C (D) in noICU (n=89) and ICU (n= 22) patients are shown. The box-plots represent the interquartile range with median (central line); the whiskers represent the measured range of noICU and ICU patients. Each S1P and apoM measurement was run in triplicate, and performed at least twice in independent assays. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.


Pearson correlation between S1P ( A - C ) PSI days from admission NLR in COV ( n = 111 ) . Scatter plots fitted regression are shown in each figure . Exact p values or * * * * p < 0 . 0001 are reported .

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Pearson correlation between S1P (A-C) PSI days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.


Pearson correlation between apoM ( D - F ) PSI , days from admission NLR in COV ( n = 111 ) . Scatter plots fitted regression are shown in each figure . Exact p values or * * * * p < 0 . 0001 are reported .

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Pearson correlation between apoM (D-F) and PSI , days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.


( A , B ) Cumulative risk for ICU admission ( A ) cumulative survival ( B ) in COV ( n = 111 ) , grouped for cut - off value of S1P serum level of 0 . 60 μM . Statistical was performed by Cox regression . Exact p values are reported .

(A,B) Cumulative risk for ICU admission (A) and cumulative survival (B) in COV patients (n=111), grouped for cut-off value of S1P serum level of 0.60 μM. Statistical analysis was performed by Cox regression. Exact p values are reported.


b . Multiplexed Error Robust in situ hybridization ( MERFISH ) DAPI stained nuclei in mouse hippocampus . Each gene is represented by a different color . For entire hippocampus ( left ) , only mRNA spots are shown with a scale bar of 500 microns . On zoomed - in section ( right ) , each gene is represented by a different color dot , DAPI is displayed in . scale bar is 20 microns .

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b. Multiplexed Error Robust Fluorescent in situ hybridization (MERFISH) and DAPI stained nuclei in the mouse hippocampus. Each gene is represented by a different color. For the entire hippocampus (left), only the mRNA spots are shown with a scale bar of 500 microns. On the zoomed-in section (right), each gene is represented by a different color dot, and the DAPI intensity is displayed in white. The scale bar is 20 microns.


a . Normalized expression of Tox in CA1sp1 , Leng8 in CA3sp3 , Hecw1 in DG3 , Thra in Astro1 show variable expression throughout hippocampus . Scale bar is 500 microns . spDEGs were computed by comparing true variance in gene expression between subtype neighborhoods to that of randomly permuted ( sub ) type neighborhoods .

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a. Normalized expression of Tox in CA1sp1, Leng8 in CA3sp3, Hecw1 in DG3, and Thra in Astro1 show variable expression throughout the hippocampus. Scale bar is 500 microns. spDEGs were computed by comparing the true variance in gene expression between cell subtype neighborhoods to that of randomly permuted cell (sub)type neighborhoods.


A : HIF - 1α in HT29 tumors . Left : vehicle - treated controls Middle / left : Doublet LDM cyclophosphamide + capecitabine ( LDMCTX + LDMCPB ) Middle / right : LDM cyclophosphamide + MTD capecitabine ( LDMCTX + MTDCPB ) . Inset , high - magnification image of region marked with a green asterisk ( * ) . Right : of effect of monotherapies or doublet LDM / MTD regimens on HIF - 1α + . F ( 5 , 12 ) = 8 . 791 P = 0 . 001 for overall treatment by Brown - Forsythe ANOVA ; * , P = 0 . 046 vs vehicle ; # , P < 0 . 0001 vs LDMCTX + MTDCPB by Benjamini , Krieger Yekuteli post hoc .

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A: HIF-1α levels in HT29 primary tumors. Left: vehicle-treated controls Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB) Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-1α+ area. F(5,12) = 8.791 and P = 0.001 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.046 vs vehicle; #, P< 0.0001 vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test.


B : HIF - 2α in HT29 tumors . Left : Example of immunostaining in vehicle - treated controls . Inset , high - magnification image of region marked with a green asterisk ( * ) . Right : of effect of monotherapies or doublet LDM / MTD regimens on HIF - 2α + . F ( 5 , 18 ) = 1 . 215 P = 0 . 3424 ( not significant ) for overall treatment by Brown - Forsythe ANOVA .

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B: HIF-2α levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-2α+ area. F(5,18) = 1.215 and P = 0.3424 (not significant) for overall treatment by Brown-Forsythe ANOVA.


C : CA9 in HT29 tumors . Left : Example of immunostaining in vehicle - treated controls . Inset , high - magnification image of region marked with a green asterisk ( * ) . Right : of effect of monotherapies or doublet LDM / MTD regimens on CA9 + . F ( 5 , 11 ) = 2 . 466 P = 0 . 0961 ( not significant ) for overall treatment by Brown - Forsythe ANOVA .

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C: CA9 levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on CA9+ area. F(5,11) = 2.466 and P = 0.0961 (not significant) for overall treatment by Brown-Forsythe ANOVA.


D : Correlation between HIF - 1α proliferation indexes in HT29 tumors . Left middle / left : HIF - 1α Ki67 expression in vehicle - treated controls ; consecutive sections are shown . Middle / right : HIF - 1α + vs Ki67 + proliferation index scatterplot . Each point represents median values for each . Regression ( red ) 95 % CI ( shaded blue ) are indicated . F ( 1 , 27 ) = 34 . 45 P = 10 - 4 ; slope ≠ 0 by F - Right : Machine - learning of effect of monotherapies or doublet LDM / MTD regimens upon Ki67 + proliferative index . Median indexes per are indicated . F ( 5 , 12 ) = 1 . 609 P = 0 . 2312 ( not significant ) for overall treatment by Brown - Forsythe ANOVA .

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D: Correlation between HIF-1α levels and proliferation indexes in HT29 primary tumors. Left and middle/left: HIF-1α and Ki67 expression in vehicle-treated controls; consecutive sections are shown. Middle/right: HIF-1α+ vs Ki67+ proliferation index scatterplot. Each point represents median values for each tumor. Regression line (red) and 95% CI (shaded blue area) are indicated. F(1,27) = 34.45 and P = 10-4; slope ≠ 0 by F-test Right: Machine-learning quantification of the effect of monotherapies or doublet LDM/MTD regimens upon Ki67+ proliferative index. Median indexes per tumor are indicated. F(5,12) = 1.609 and P = 0.2312 (not significant) for overall treatment by Brown-Forsythe ANOVA.


A : Effect of LDM MTD chemotherapy on HT29 liver metastatic nodule . Left : Histogram of pooled cross - sectional metastatic diameter ( Ø ) . Middle : Metastatic diameter ( Ø ) by chemotherapy regimen . A decreasing linear trend for median metastatic diameter was observed ( left to right , slope = - 143 . 2 , P = 0 . 043 by post - for trend ) . Right : Dichotomized metastatic at median diameter of vehicle - treated tumors . Liver nodule was classified as small ( blue ) or large ( red ) . nodule per group category ( small / large ) are plotted on right ordinate . χ2 ( df = 5 ) = 6 . 80 ; P = 0 . 2361 ( not significant ) for overall effects on ; χ2 ( df = 1 ) = 4 . 71 ; P = 0 . 0299 ( LDMCTX + LDMCPB vs vehicle ) .

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A: Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size. Left: Histogram of pooled cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. A decreasing linear trend for median metastatic diameter was observed (left to right, slope = -143.2, P = 0.043 by post-test for trend). Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Liver nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 6.80; P= 0.2361 (not significant) for overall effects on size; χ2 (df=1) = 4.71; P= 0.0299 (LDMCTX + LDMCPB vs vehicle).


B : HIF - 1α in HT29 liver metastatic nodules . Left : vehicle - treated controls . Middle / left : Doublet LDM cyclophosphamide + capecitabine ( LDMCTX + LDMCPB ) . Middle / right : LDM cyclophosphamide + MTD capecitabine ( LDMCTX + MTDCPB ) . Right : Automatic of effect of monotherapies or doublet regimens on HIF - 1α + areas in metastatic nodules . F ( 2 , 13 ) = 4 . 796 P = 0 . 028 for overall treatment by Brown - Forsythe ANOVA ; * , P = 0 . 038 LDMCTX + MTDCPB vs vehicle or P = 0 . 0123 LDMCTX + LDMCPB vs vehicle ; # , P = 0 . 0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini , Krieger Yekuteli post hoc .

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B: HIF-1α levels in HT29 liver metastatic nodules. Left: vehicle- treated controls. Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB). Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Right: Automatic quantification of the effect of monotherapies or doublet regimens on HIF-1α+ areas in individual metastatic nodules. F(2,13) = 4.796 and P = 0.028 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.038 LDMCTX + MTDCPB vs vehicle or P= 0.0123 LDMCTX + LDMCPB vs vehicle; #, P= 0.0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test.


C : Correlation between metastatic peri - metastatic HIF - 1α in liver . Left : Nodule immunostaining parenchymal HIF - 1α expression . Each point represents median values per nodule . Regression ( blue ) 95 % CI ( shaded blue ) are indicated . F ( 1 , 57 ) = 150 . 7 P < 0 . 0001 ; slope ≠ 0 by F - . Right : Correlation between intra - metastatic peri - metastatic parenchymatous ring in liver . Each point represents median values per nodule . Regression ( blue ) 95 % CI ( shaded blue ) are indicated . F ( 1 , 54 ) = 55 . 2 P < 0 . 0001 ; slope ≠ 0 by F - . .

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C: Correlation between metastatic and peri-metastatic HIF-1α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-1α expression. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,57) = 150.7 and P< 0.0001; slope ≠ 0 by F-test. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test..


D : Correlation between metastatic peri - metastatic HIF - 2α in liver . Left : Nodule immunostaining parenchymal HIF - 2α expression . Right : Correlation between intra - metastatic peri - metastatic parenchymatous ring in liver . Each point represents median values for each nodule . Regression ( blue ) 95 % CI ( shaded blue ) are indicated . F ( 1 , 54 ) = 55 . 2 P < 0 . 0001 ; slope ≠ 0 by F - . n , number of nodules .

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D: Correlation between metastatic and peri-metastatic HIF-2α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-2α expression. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values for each nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test. n, number of nodules.


A : EMT6 - CDDP lung metastatic nodule . Left : Histogram of cross - sectional metastatic diameter ( Ø ) . Middle : Metastatic diameter ( Ø ) by chemotherapy regimen . F ( 5 , 99 ) = 0 . 9310 P = 0 . 4644 ( not significant ) for overall treatment by Brown - Forsythe ANOVA . Right : Dichotomized metastatic at median diameter of vehicle - treated tumors . Lung nodule was classified as small ( blue ) or large ( red ) . nodule per group category ( small / large ) are plotted on right ordinate . χ2 ( df = 5 ) = 2 . 631 ; P = 0 . 7567 ( not significant ) for overall effects on .

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A: EMT6-CDDP lung metastatic nodule size. Left: Histogram of cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. F(5,99) = 0.9310 and P= 0.4644 (not significant) for overall treatment by Brown-Forsythe ANOVA. Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Lung nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 2.631; P= 0.7567 (not significant) for overall effects on size.


B : Hypoxic fraction in lung metastatic nodules . Pimonidazole adduct immunoreactivity was automatically quantified expressed as fractional positive areas per nodule ( rightmost panel ) . F ( 5 , 110 ) = 7 . 544 P < 0 . 0001 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 vs vehicle ; # , P = 0 . 0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini , Krieger Yekuteli post hoc .

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B: Hypoxic fraction in lung metastatic nodules. Pimonidazole adduct immunoreactivity was automatically quantified and expressed as fractional positive areas per nodule (rightmost panel). F(5,110) = 7.544 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicle; #, P= 0.0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini, Krieger and Yekuteli post hoc test.


C : HIF - 1α in lung metastatic nodules . Automatic of HIF - 1α measured as fractional positive areas per nodule ( rightmost panel ) . F ( 5 , 114 ) = 5 . 325 P = 0 . 0004 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 vs vehicleby Benjamini , Krieger Yekuteli post hoc .

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C: HIF-1α levels in lung metastatic nodules. Automatic quantification of HIF-1α levels measured as fractional positive areas per nodule (rightmost panel). F(5,114) = 5.325 and P= 0.0004 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicleby Benjamini, Krieger and Yekuteli post hoc test.


D : Microvessel density in lung metastatic nodules . Automatic of CD31 fractional areas per nodule ( rightmost panel ) . F ( 5 , 124 ) = 7 . 531 P < 0 . 0001 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 ( LDM capecitabine , cyclophosphamide , or doublet capecitabine + cyclophosphamide vs vehicle ) , # , P = 0 . 0417 ( doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine ) by Benjamini , Krieger Yekuteli post hoc .

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D: Microvessel density in lung metastatic nodules. Automatic quantification of CD31 fractional areas per nodule (rightmost panel). F(5,124) = 7.531 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 (LDM capecitabine, cyclophosphamide, or doublet capecitabine + cyclophosphamide vs vehicle), #, P= 0.0417 (doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine) by Benjamini, Krieger and Yekuteli post hoc test.


B : HIFα - inducible colon cancer signature ( HIFi - CCS ) . Left middle / left : List of HIFα targets overall survival in TCGA ( left ) or aggregated microarray data ( middle / left ) from colon adenocarcinomas . Cases are classified as low ( blue ) or high ( red ) HIFi - CCS according to their relationship to median . Kaplan - Meier followed by log - rank . Middle / right : HIFi - CCS lymph node ( LN ) invasion status . Data are standardized as z - scores . t ( 194 ) = 4 . 399 P < 0 . 0001 by Student t - with Welch ' s correction . Right : HIFi - CCS is associated with accelerated distant metastasis . Cases are classified as low ( blue ) or high ( red ) HIFi - CCS according to their relationship to median . Kaplan - Meier followed by log - rank .

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B: HIFα- inducible colon cancer signature (HIFi-CCS). Left and middle/left: List of HIFα targets and overall survival in TCGA (left) or aggregated microarray data (middle/left) from colon adenocarcinomas. Cases are classified as low (blue) or high (red) HIFi-CCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test. Middle/right: HIFi-CCS and lymph node (LN) invasion status. Data are standardized as z-scores. t(194) = 4.399 and P< 0.0001 by Student t-test with Welch's correction. Right: HIFi-CCS is associated with accelerated distant metastasis. Cases are classified as low (blue) or high (red) HIFi-CCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test.


C : HIFα - inducible breast cancer signature ( HIFi - BCS ) . Left middle / left : List of HIFα targets overall survival in METABRIC ( left ) or recurrence - free survival in microarray ( middle / left ) from breast adenocarcinomas . Cases are classified as low ( blue ) or high ( red ) HIFi - BCS according to their relationship to median . Kaplan - Meier followed by log - rank . Middle / right : HIFi - BCS estrogen receptor ( ER ) status . Data are standardized as z - scores . t ( 112 ) = 3 . 789 P = 0 . 0002 by Student t - with Welch ' s correction . Right : HIFi - BCS is associated with worsened distant metastasis - free survival . Cases are classified as low ( blue ) or high ( red ) HIFi - BCS whenever they are below or above distribution median . Kaplan - Meier followed by log - rank .

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C: HIFα- inducible breast cancer signature (HIFi-BCS). Left and middle/left: List of HIFα targets and overall survival in METABRIC (left) or recurrence- free survival in microarray (middle/left) from breast adenocarcinomas. Cases are classified as low (blue) or high (red) HIFi-BCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test. Middle/right: HIFi-BCS and estrogen receptor (ER) status. Data are standardized as z-scores. t(112) = 3.789 and P= 0.0002 by Student t-test with Welch's correction. Right: HIFi-BCS is associated with worsened distant metastasis-free survival. Cases are classified as low (blue) or high (red) HIFi-BCS whenever they are below or above the distribution median. Kaplan-Meier analysis followed by log-rank test.


D : Median RNAseq expression of HIFα targets as split by median HIFi - CCS ( left ) or HIFi - BCS ( right ) . Transcripts up / downregulated by ± 1 . 5 - fold are shown in blue or yellow , respectively . Error bars indicate 95 % CI of median . Log2 scale ; n = 209 ( colon , TCGA ) or n = 609 ( breast , METABRIC ) .

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D: Median RNAseq expression of individual HIFα targets as split by median HIFi-CCS (left) or HIFi-BCS (right). Transcripts up/downregulated by ±1.5-fold are shown in blue or yellow, respectively. Error bars indicate 95% CI of the median. Log2 scale; n= 209 (colon, TCGA) or n= 609 (breast, METABRIC).


A - D were infected at indicated MOI . Viral replication ( Left ) release ( Right ) were assessed by flow cytometry RT - qPCR . A ) Caco2 / TC7 ( MOI 0 . 01 ) B ) Calu - 3 ( MOI 0 . 001 ) C ) Vero ( MOI 0 , 01 ) D ) human airway epithelial ( HAEC ) release ( Right ) infectious release ( Left ) ( MOI 0 . 01 ) . Data are mean ± SD of at least 3 independent experiments . Statistical : Mixed - effect or Two ‐ way ANOVA compared to D614G reference , ns : non ‐ significant , * P < 0 . 05 , * * * * P < 0 . 0001 .

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A-D Cells were infected at the indicated MOI. Viral replication (Left) and release (Right) were assessed by flow cytometry and RT-qPCR. A) Caco2/TC7 cells (MOI 0.01) B) Calu-3 cells (MOI 0.001) C) Vero cells (MOI 0,01) D) primary human airway epithelial cells (HAEC) virus release (Right) and infectious virus release (Left) (MOI 0.01). Data are mean ± SD of at least 3 independent experiments. Statistical analysis: Mixed-effect analysis or Two‐way ANOVA compared to D614G reference, ns: non‐significant, *P < 0. 05, ****P < 0.0001.


( B ) Left Panel : Fusion was quantified by GFP / number of nuclei normalized to D614G for U2OS - ACE2 20h post infection at MOI 0 . 001 . Right Panel : Representative of U2OS - ACE2 20h post infection , GFP - Split ( Green ) Hoechst ( Blue ) . Top bottom are same with without Hoechst channel .

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(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for U2OS-ACE2 20h post infection at MOI 0.001. Right Panel: Representative images of U2OS-ACE2 20h post infection, GFP-Split (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel.


( C ) Left Panel : Quantified fusion of Vero infected at MOI 0 . 01 . Right Panel : Representative of Vero 48h post infection , GFP - Split ( Green ) Hoechst ( Blue ) .

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(C) Left Panel: Quantified fusion of Vero cells infected at MOI 0.01. Right Panel: Representative images of Vero cells 48h post infection, GFP-Split (Green) and Hoechst (Blue).


( B ) Left Panel : Fusion was quantified by GFP / number of nuclei normalized to D614G for each of transfected variant S . Right Panel : Representative of Vero GFP - split 18h post - transfection , GFP ( Green ) Hoechst ( Blue ) . Top bottom are same with without Hoechst channel . Scale bars : 200 µm .

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(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.


( C ) Left Panel : of variant S mediated fusion in Vero - GFP split by video microscopy . Results are mean ± SD from three fields per condition from one representative experiment . Right Panel : Fusion of at least 3 independent video microscopy experiments , 20h post transfection , normalized to D614G .

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(C) Left Panel: Quantification of variant S protein mediated fusion in Vero-GFP split cells by video microscopy. Results are mean ± SD from three fields per condition from one representative experiment. Right Panel: Fusion quantification of at least 3 independent video microscopy experiments, 20h post transfection, normalized to D614G.


( B ) Left Panel : Quantified fusion of Alpha + E484K variant S normalized to D614G S . Right Panel : Representative of fusion at 20h . Scale bar : 200 µm . Top bottom are same with without Hoechst channel .

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(B) Left Panel: Quantified fusion of the Alpha + E484K variant S protein normalized to D614G S. Right Panel: Representative images of fusion at 20h. Scale bar: 200 µm. Top and bottom are the same images with and without Hoechst channel.


( B ) Left Panel : EC50 values ( of ACE2 needed for 50 % binding ) for Alpha associated mutations . Colour code corresponds to location on S functional domains lower EC50 values signifies higher affinity to ACE2 binding . Right Panel : EC50 values for Beta associated mutations . Data set for N501Y D614G reference mutations are duplicated between left right panels as mutations are common to both variants .

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(B) Left Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha and associated mutations. Colour code corresponds to location on S protein functional domains and lower EC50 values signifies higher affinity to ACE2 binding. Right Panel: EC50 values for Beta and associated mutations. Data set for N501Y and D614G reference mutations are duplicated between left and right panels as mutations are common to both variants.


( A ) Vero GFP - split were transfected with variant S imaged 18h post - transfection . Left Panel : Fusion was quantified by GFP / number of nuclei normalized to D614G for each of transfected variant S . Right Panel : Representative of Vero GFP - split 18h post - transfection , GFP ( Green ) Hoechst ( Blue ) . Top bottom are same with without Hoechst channel . Scale bars : 200 µm .

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(A) Vero GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.


( D ) Caco2 GFP - split were transfected with variant S imaged 18h post - transfection . Left Panel : Fusion was quantified by GFP / number of nuclei normalized to D614G for each of transfected variant S . Right Panel : Representative of Caco2 GFP - split 18h post - transfection , GFP ( Green ) Hoechst ( Blue ) . Top bottom are same with without Hoechst channel . Scale bars : 200

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(D) Caco2 GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Caco2 GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm


( A ) Expression of rate - limiting glycolytic isoenzymes in R1 after knockdown of Stat3 using three independent shRNAs compared to an empty vector control shRNA ( sh - ctrl ) , as measured by Western blot . β - actin was employed as a loading control throughout . ( B - I ) Expression of pluripotency genes c - Myc , Oct - 4 , Nanog , Klf4 Sox - 2 in R1 after knockdown of Hk2 ( B ) , Pfkl ( C ) , Hk1 ( D ) , Hk3 ( E ) , Pfkm ( F ) , Pkm2 ( G ) , Pklr ( H ) Pfkp ( I ) glycolytic enzymes using two or three independent shRNAs , as measured by Western blot .

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(A) Expression of all rate-limiting glycolytic isoenzymes in R1 cells after knockdown of Stat3 using three independent shRNAs compared to an empty vector control shRNA (sh-ctrl), as measured by Western blot. β-actin was employed as a loading control throughout. (B-I) Expression of the pluripotency genes c-Myc, Oct-4, Nanog, Klf4 and Sox-2 in R1 cells after knockdown of Hk2 (B), Pfkl (C), Hk1 (D), Hk3 (E), Pfkm (F), Pkm2 (G), Pklr (H) and Pfkp (I) glycolytic enzymes using two or three independent shRNAs, as measured by Western blot. D


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 ,

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 (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 .

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

(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 .

O 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) 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 .

O O O O O O O O O O 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) 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 .

(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 O 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 O O O O 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.