Kaplan - Meier analysis of mice survival relative to the two experimental groups . Long - rank test , p < 0 . 0001 . The median survival time of mice injected with UOK257 / shLuc is 219 . 5 days in ( c )

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Kaplan-Meier analysis of mice survival relative to the two experimental groups. Long-rank test, p < 0.0001. The median survival time of mice injected with UOK257/shLuc is 219.5 days in (c)


Plots show tumor volumes in mice injected with UOK257 / shLuc ( n = 8 ) or UOK257 / shTFE3 ( n = 9 ) ( e ) * p = 0 . 023 for plot in ( e )

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Plots show tumor volumes in mice injected with UOK257/shLuc (n=8) or UOK257/shTFE3 (n=9) (e) *p= 0.023 for plot in (e)


Kaplan - Meier analysis of mice survival relative to the two experimental groups . The median survival time of mice injected with UOK257 / shLuc is 244 . 5 days in ( f ) .

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Kaplan-Meier analysis of mice survival relative to the two experimental groups. The median survival time of mice injected with UOK257/shLuc is 244.5 days in (f).


E WT and C2βD1212A / D1212A mice were subjected to thromboembolic stroke model . Representatives T2 - weighted MRI of coronal section taken 24 hours after the onset of in situ clot formation by α - thrombin were shown . Graph quantification of infarct volume measurements . Data represent mean ± SEM ( n = 23 - 25 mice per group ; * * p < 0 . 01 , Unpaired t - test ) .

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E WT and C2βD1212A/D1212A mice were subjected to thromboembolic stroke model. Representatives T2-weighted MRI of coronal section taken 24 hours after the onset of in situ clot formation by α-thrombin were shown. Graph quantification of infarct volume measurements. Data represent mean ± SEM (n=23-25 mice per group; **p<0.01, Unpaired t-test).


WT and C2βD1212A / D1212A mice were subjected to tMCAO followed by 24h of reperfusion . ( B ) Protein expression of TNFα , IL - 1β and IL - 6 . Data represent mean ± SEM ( n = 4 mice per group ; * p = 0 . 05 ; Mann - Whitney test ) . Data represent mean ± SEM ( n = 5 - 7 mice per group ; * * p < 0 . 01 ; Mann - Whitney test ) .

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WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (B) Protein expression of TNFα, IL-1β and IL-6. Data represent mean ± SEM (n=4 mice per group; *p=0.05; Mann-Whitney test). Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test).


A Gene expression of TNFα ( n = 8 - 10 mice for WT and n = 6 mice for C2βD1212A / D1212A ) , IL - 1β ( n = 8 mice for WT and n = 10 - 12 mice for C2βD1212A / D1212A ) , IL - 6 ( n = 8 mice for WT and n = 10 - 12 mice for C2βD1212A / D1212A ) and VCAM - 1 ( n = 8 - 10 mice for WT and n = 10 mice for C2βD1212A / D1212A ) after 1 hour of ischemia without reperfusion ( right panel ) . Gene expression of IL - 6 ( n = 10 - 12 mice for WT and n = 8 mice for C2βD1212A / D1212A ) and VCAM - 1 ( n = 12 mice for WT and n = 10mice for C2βD1212A / D1212A ) after 1 hour of ischemia followed by 4 hours of reperfusion ( bottom , left panel ) on WT and C2βD1212A / D1212A mice . The mRNA levels are given as the fold increase normalized to PGK1 relative to the healthy contralateral hemisphere of WT mice . Data represent mean ± SEM ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , Mann - Whitney test or unpaired t - test , as accordance ) .

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A Gene expression of TNFα (n=8-10 mice for WT and n=6 mice for C2βD1212A/D1212A), IL-1β (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A), IL-6 (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A) and VCAM-1 (n=8-10 mice for WT and n=10 mice for C2βD1212A/D1212A) after 1 hour of ischemia without reperfusion (right panel). Gene expression of IL-6 (n=10-12 mice for WT and n=8 mice for C2βD1212A/D1212A) and VCAM-1 (n=12 mice for WT and n=10mice for C2βD1212A/D1212A) after 1 hour of ischemia followed by 4 hours of reperfusion (bottom, left panel) on WT and C2βD1212A/D1212A mice. The mRNA levels are given as the fold increase normalized to PGK1 relative to the healthy contralateral hemisphere of WT mice. Data represent mean ± SEM (***p<0.001, **p<0.01, *p<0.05, Mann-Whitney test or unpaired t-test, as accordance).


Representative photographs of coronal brain sections and graph quantification of infarct volume ( dashed white line ) measurements in bone marrow ( BM ) chimeric mice 24 hours after tMCAO . WT > WT : transplantation of WT BM into WT hosts ; C2βD1212A > WT : transplantation of C2βD1212A / D1212A BM into WT hosts ; C2βD1212A > C2βD1212A : transplantation of C2βD1212A / D1212A BM into C2βD1212A / D1212A hosts ; WT > C2βD1212A transplantation of WT BM into kinase - dead hosts . Data represent mean ± SEM ; n = 13 - 16 mice per group ; * p < 0 . 05 * * p < 0 . 01 ; * * * p < 0 . 001 vs WT > WT , Mann - Whitney test or unpaired t - test , as accordance .

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Representative photographs of coronal brain sections and graph quantification of infarct volume (dashed white line) measurements in bone marrow (BM) chimeric mice 24 hours after tMCAO. WT>WT: transplantation of WT BM into WT hosts; C2βD1212A>WT: transplantation of C2βD1212A/D1212A BM into WT hosts; C2βD1212A>C2βD1212A: transplantation of C2βD1212A/D1212A BM into C2βD1212A/D1212A hosts; WT> C2βD1212A transplantation of WT BM into kinase-dead hosts. Data represent mean ± SEM; n=13-16 mice per group; *p<0.05 **p<0.01; ***p<0.001 vs WT>WT, Mann-Whitney test or unpaired t-test, as accordance.


B Analysis of APPL1 , EEA1 , LAMP1 and Rab11a staining in sh - Control and sh - PI3KC2β hCMEC / D3 cells . White dotted lines delineate cells ; Scale bar , 10µm ; nuclei are shown in blue ( DAPI ) .

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B Analysis of APPL1, EEA1, LAMP1 and Rab11a staining in sh-Control and sh-PI3KC2β hCMEC/D3 cells. White dotted lines delineate cells; Scale bar, 10µm; nuclei are shown in blue (DAPI).


Quantification was performed to determine number of positive vesicles per cell ( ImageJ software ) . Nuclei are shown in blue ( DAPI ) . Data represent mean ± SEM of cell fields randomly selected ( n = 13 - 20 for ( C ) and n = 11 - 20 for ( D ) ) . Data are collected from at least 3 - 4 independent experiments . For violin plots ( D ) , thick lines represent median and thin lines interquartile ; * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 , unpaired t - test or Mann - Whitney , as accordance .

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Quantification was performed to determine number of positive vesicles per cell (ImageJ software). Nuclei are shown in blue (DAPI). Data represent mean ± SEM of cell fields randomly selected (n=13-20 for (C) and n=11-20 for (D)). Data are collected from at least 3-4 independent experiments. For violin plots (D), thick lines represent median and thin lines interquartile; ***p<0.001; **p<0.01; *p<0.05, unpaired t-test or Mann-Whitney, as accordance.


( A ) Representative VE - cadherin staining and graph quantification of fluorescence intensity normalized to total number of cells per field ( DAPI labelling ) in sh - Control and sh - PI3KC2β hCMEC / D3 in resting condition and after 24 hours activation with 25 ng / ml of TNFα . Control normalized to 100 % . Scale bar , 15 µm . Data represent mean ± SEM of cell fields randomly selected ( dots n = 14 - 19 ) . Data are collected from 4 - 5 independent experiments ; * * * p < 0 . 001 , unpaired t - test .

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(A) Representative VE-cadherin staining and graph quantification of fluorescence intensity normalized to total number of cells per field (DAPI labelling) in sh-Control and sh-PI3KC2β hCMEC/D3 in resting condition and after 24 hours activation with 25 ng/ml of TNFα. Control normalized to 100%. Scale bar, 15 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=14-19). Data are collected from 4-5 independent experiments; ***p<0.001, unpaired t-test.


( B ) Graph quantification of fluorescence intensity of VE - Cadherin normalized to total cell number per field ( DAPI labelling ) in control condition and after 5 hours in oxygen - glucose deprivation ( OGD ) condition . Control normalized to 100 % . Data represent mean ± SEM of cell fields randomly selected ( dots n = 16 ) . Data were collected from 5 independent experiments ) ; * * * p < 0 . 001 ; * * p < 0 . 01 , Mann - Whitney test .

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(B) Graph quantification of fluorescence intensity of VE-Cadherin normalized to total cell number per field (DAPI labelling) in control condition and after 5 hours in oxygen-glucose deprivation (OGD) condition. Control normalized to 100%. Data represent mean ± SEM of cell fields randomly selected (dots n=16). Data were collected from 5 independent experiments);***p<0.001; **p<0.01, Mann-Whitney test.


A Representative images of PI3P and PI ( 3 , 4 ) P2 labeling and quantification using mCherry - FYVEHRS and GFP - PH ( TAPP1 ) probes respectively in sh - Control or sh - PI3KC2β hCMEC / D3 . Scale bar , 10 µm . Nuclei are shown in blue ( DAPI ) . Staining intensity was quantified by ImageJ software and normalized to total number of cells ( DAPI labelling ) per field . Data represent mean ± SEM of cell fields randomly selected for PI3P ( dots n = 21 ) and for PI ( 3 , 4 ) P2 ( dots n = 14 - 16 ) . Data were collected from at least 5 independent experiments ; * * * p < 0 . 001 , unpaired t - test with Welch ' s correction .

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

A Representative images of PI3P and PI(3,4)P2 labeling and quantification using mCherry-FYVEHRS and GFP-PH(TAPP1) probes respectively in sh-Control or sh-PI3KC2β hCMEC/D3. Scale bar, 10 µm. Nuclei are shown in blue (DAPI). Staining intensity was quantified by ImageJ software and normalized to total number of cells (DAPI labelling) per field. Data represent mean ± SEM of cell fields randomly selected for PI3P (dots n=21) and for PI(3,4)P2 (dots n=14-16). Data were collected from at least 5 independent experiments; ***p<0.001, unpaired t-test with Welch's correction.


C Graph quantification of APPL1 - , EEA1 - and Rab11 - endosomes colocalized with mCitrin - VE - cadherin in sh - Control and sh - PI3KC2β hCMEC / D3 cells . Data are shown as percentage of total count endosomes per cell . Data are expressed as mean ± SEM ( n = 33 - 35 cells for APPL1 and EEA1 endosomes and n = 16 - 18 cells for Rab11 endosomes ; 3 independent experiments ) ; * * p < 0 . 001 , Mann - Whitney test or unpaired t - test , as accordance .

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C Graph quantification of APPL1-, EEA1- and Rab11- endosomes colocalized with mCitrin-VE-cadherin in sh-Control and sh-PI3KC2β hCMEC/D3 cells. Data are shown as percentage of total count endosomes per cell. Data are expressed as mean ± SEM (n=33-35 cells for APPL1 and EEA1 endosomes and n=16-18 cells for Rab11 endosomes; 3 independent experiments); **p<0.001, Mann-Whitney test or unpaired t-test, as accordance.


D Representative confocal microscopic images of VE - cadherin immunostaining in sh - Control cells , sh - PI3KC2β cells or sh - PI3KC2β cells transduced with Rab11S25N ( Rab11 - DN ) . Graph quantification of VE - cadherin ; control normalized to 100 % . Scale bar , 10 µm . Data represent mean ± SEM of cell fields randomly selected ( dots n = 6 - 9 ) . Data are collected from at least 3 independent experiments ; * * p < 0 . 01 Mann - Whitney test .

D Representative confocal microscopic images of VE-cadherin immunostaining in sh-Control cells, sh-PI3KC2β cells or sh-PI3KC2β cells transduced with Rab11S25N (Rab11-DN). Graph quantification of VE-cadherin; control normalized to 100%. Scale bar, 10 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=6-9). Data are collected from at least 3 independent experiments; **p<0.01 Mann-Whitney test.


( a ) Ten - month - old adult mice and 20 - month - old aged mice were treated with an intraperitoneal injection of 5 mg kg - 1 CCCP and their hearts were collected 12 h later . Representative electron micrographs of hearts from CCCP - treated adult and aged mice ; original magnification × 5 , 000 ; scale bar , 2 μm . The magnified photograph represents an autophagic vacuole containing mitochondria ; original magnification × 12 , 000 ; scale bar , 500 nm . Mitochondria incorporated into an autophagic vacuole in baseline and CCCP - treated conditions were quantified blindly from 8 - 10 images from different fields ( original magnification × 5 , 000 ) ( n = 3 per group ) .

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(a) Ten-month-old adult mice and 20-month-old aged mice were treated with an intraperitoneal injection of 5 mg kg-1 CCCP and their hearts were collected 12 h later. Representative electron micrographs of hearts from CCCP-treated adult and aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents an autophagic vacuole containing mitochondria; original magnification × 12,000; scale bar, 500 nm. Mitochondria incorporated into an autophagic vacuole in baseline and CCCP-treated conditions were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).


( b , c ) CCCP - induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria - rich fraction in adult and aged mice ( b ) , and DOX - treated mice ( c ) . Representative immunoblots are shown from four independent experiments .

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(b,c) CCCP-induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria-rich fraction in adult and aged mice (b), and DOX-treated mice (c). Representative immunoblots are shown from four independent experiments.


( d ) Parkin was overexpressed in young and senescent MEFs using adenovirus - mediated transduction for the in vitro cell - based bioassay to assess the ability to clear damaged mitochondria . Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM . Results from four independent experiments performed in duplicate are shown . MEFs at passage 3 and passage 9 were used as young and senescent MEFs , respectively .

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(d) Parkin was overexpressed in young and senescent MEFs using adenovirus-mediated transduction for the in vitro cell-based bioassay to assess the ability to clear damaged mitochondria. Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM. Results from four independent experiments performed in duplicate are shown. MEFs at passage 3 and passage 9 were used as young and senescent MEFs, respectively.


( e ) Mitochondrial DNA content was assessed by real - time PCR . Results are shown from six - independent experiments .

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(e) Mitochondrial DNA content was assessed by real-time PCR. Results are shown from six-independent experiments.


( f ) Mitochondrial content was assessed by immunoblotting for the mitochondrial chaperone , GRP75 ( matrix protein ) and complex I subunit , NDUFA9 ( inner membrane protein ) . MEFs were treated with 100 nM bafilomycin - A1 , ( Baf - A1 , an inhibitor of the vacuolar - type proton ATPase ) to inhibit autophagy . Representative immunoblots are shown from three independent experiments .

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(f) Mitochondrial content was assessed by immunoblotting for the mitochondrial chaperone, GRP75 (matrix protein) and complex I subunit, NDUFA9 (inner membrane protein). MEFs were treated with 100 nM bafilomycin-A1, (Baf-A1, an inhibitor of the vacuolar-type proton ATPase) to inhibit autophagy. Representative immunoblots are shown from three independent experiments.


( g ) Representative images of GFP - LC3 expressing MEFs treated with 20 μM CCCP for 10 h before the immunostaining of mitochondria with anti - TOM20 ( red ) ; original magnification , × 400 and × 1 , 000 ; scale bar , 100 μm and scale bar , 15 μm . Line scans below the images indicate colocalization between LC3 ( green ) and mitochondria ( red ) and correlate to the arrows drawn in the images . WHL indicates whole - heart lysate . Data are shown as the means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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(g) Representative images of GFP-LC3 expressing MEFs treated with 20 μM CCCP for 10 h before the immunostaining of mitochondria with anti-TOM20 (red); original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm. Line scans below the images indicate colocalization between LC3 (green) and mitochondria (red) and correlate to the arrows drawn in the images. WHL indicates whole-heart lysate. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


( a ) The senescence - associated molecules p53 , p21 , p16 , H - Ras 12V and Rb were overexpressed in MEFs by retrovirus - mediated transduction . After puromycin selection , mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus - mediated Parkin expression . Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM . Results are shown from four independent experiments performed in duplicate .

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(a) The senescence-associated molecules p53, p21, p16, H-Ras 12V and Rb were overexpressed in MEFs by retrovirus-mediated transduction. After puromycin selection, mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus-mediated Parkin expression. Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM. Results are shown from four independent experiments performed in duplicate.


( b ) Representative images of MEFs infected with the GFP - LC3 adenovirus and stained with anti - TOM20 ( red ) to visualize mitochondria 10 h after mitophagy induction ; original magnification , × 400 and × 1 , 000 ; scale bar , 100 μm and scale bar , 15 μm .

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(b) Representative images of MEFs infected with the GFP-LC3 adenovirus and stained with anti-TOM20 (red) to visualize mitochondria 10 h after mitophagy induction; original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm.


( c ) Mitochondrial content of H - Ras 12 V expressing WT , p53 − / − and p21 − / − MEFs was assessed by flow cytometry 24 h after mitophagy induction . Results are shown from four independent experiments performed in duplicate .

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(c) Mitochondrial content of H-Ras 12 V expressing WT, p53−/− and p21−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. Results are shown from four independent experiments performed in duplicate.


( d ) Mitochondrial content of WT and p53 − / − MEFs was assessed by flow cytometry 24 h after mitophagy induction . WT and p53 − / − MEFs were transfected with control or p53 - specific siRNAs or cultured in complete medium for 24 h in the presence or absence of 20 μM pifithrin - α before mitophagy induction . Results are shown from four independent experiments performed in duplicate .

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(d) Mitochondrial content of WT and p53−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. WT and p53−/− MEFs were transfected with control or p53-specific siRNAs or cultured in complete medium for 24 h in the presence or absence of 20 μM pifithrin-α before mitophagy induction. Results are shown from four independent experiments performed in duplicate.


( e ) Murine HL - 1 cardiac myocytes were infected with retrovirus vectors encoding p53 , p21 or p16 and after puromycin selection , underwent mitophagy induction . Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown .

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(e) Murine HL-1 cardiac myocytes were infected with retrovirus vectors encoding p53, p21 or p16 and after puromycin selection, underwent mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown.


( f ) HL - 1 cells were transfected with control , p53 , p21 or p16 - specific siRNAs , followed by treatment with 0 . 02 μM DOX and mitophagy induction . Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate .

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(f) HL-1 cells were transfected with control, p53, p21 or p16-specific siRNAs, followed by treatment with 0.02 μM DOX and mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate.


( g ) CCCP - induced recruitment of Parkin and expression of Mfn1 and Drp1 in DOX - treated WT , p53 − / − and p21 − / − mice were determined by immunoblotting of the heartmitochondria - rich fraction . Representative immunoblots are shown from four independent experiments . Data are shown as the means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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(g) CCCP-induced recruitment of Parkin and expression of Mfn1 and Drp1 in DOX-treated WT, p53−/− and p21−/− mice were determined by immunoblotting of the heartmitochondria-rich fraction. Representative immunoblots are shown from four independent experiments. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


( a ) Whole - cell lysates of HL - 1 cells treated with 0 . 02 μM DOX and transfected with FLAG - tagged Parkin or the FLAG - empty vector were immunoprecipitated with the anti - Flag - M2 antibody and blotted with anti - p53 and Flag antibodies .

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(a) Whole-cell lysates of HL-1 cells treated with 0.02 μM DOX and transfected with FLAG-tagged Parkin or the FLAG-empty vector were immunoprecipitated with the anti-Flag-M2 antibody and blotted with anti-p53 and Flag antibodies.


( b ) Whole - cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti - Parkin antibody .

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(b) Whole-cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti-Parkin antibody.


( c ) Cytosolic heart lysates of adult and aged mice were immunoprecipitated with anti - Parkin , p53 and control IgG antibodies .

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(c) Cytosolic heart lysates of adult and aged mice were immunoprecipitated with anti-Parkin, p53 and control IgG antibodies.


( d ) Schematic representation of N - terminally FLAG - tagged WT Parkin and various mutants of Parkin . ( e ) The association of endogenous p53 with various Parkin mutants in DOX - treated HeLa cells . The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53 .

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(d) Schematic representation of N-terminally FLAG-tagged WT Parkin and various mutants of Parkin. (e) The association of endogenous p53 with various Parkin mutants in DOX-treated HeLa cells. The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53.


( f ) Schematic representation of the GST - p53 fusion proteins . ( g ) GST pull - down assays using GST - p53 fusion proteins and in vitro transcribed / translated FLAG - Parkin . Residues 81 - 160 of p53 were sufficient to bind Parkin .

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(f) Schematic representation of the GST-p53 fusion proteins. (g) GST pull-down assays using GST-p53 fusion proteins and in vitro transcribed/translated FLAG-Parkin. Residues 81-160 of p53 were sufficient to bind Parkin.


( a ) HL - 1 cells transfected with control or p53 - specific siRNAs no . 1 and cultured in complete medium for 24 h in the presence or absence of 0 . 02 μM DOX . After treatment with 20 μM CCCP for 6 h , the fractionation experiment was performed . Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting . Representative immunoblots are shown from three independent experiments .

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(a) HL-1 cells transfected with control or p53-specific siRNAs no.1 and cultured in complete medium for 24 h in the presence or absence of 0.02 μM DOX. After treatment with 20 μM CCCP for 6 h, the fractionation experiment was performed. Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting. Representative immunoblots are shown from three independent experiments.


( b ) These samples were subjected to immunoprecipitation with the anti - VDAC antibody to evaluate ubiquitination . Representative immunoblots are shown from three independent experiments .

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(b) These samples were subjected to immunoprecipitation with the anti-VDAC antibody to evaluate ubiquitination. Representative immunoblots are shown from three independent experiments.


( c , d ) Representative images of YFP - Parkin overexpressing p53 − / − HCT116 cells re - transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8h ( c ) and 36 h ( d ) before the immunostaining of mitochondria with anti - TOM20 ( red ) and endogenous poly - ubiquitin with a specific antibody FK - 2 ( white ) ; original magnification , × 630 ; scale bar , 20 μm .

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(c,d) Representative images of YFP-Parkin overexpressing p53−/− HCT116 cells re-transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8h (c) and 36 h (d) before the immunostaining of mitochondria with anti-TOM20 (red) and endogenous poly-ubiquitin with a specific antibody FK-2 (white); original magnification, × 630; scale bar, 20 μm.


( e - g ) Parkin mitochondrial translocation ( e ) and ubiquitination ( f ) 8 h after CCCP treatment and mitochondrial clearance ( g ) 36 h after CCCP treatment were quantified . A minimum of 300 - 400 YFP - positive cells were scored in three independent experiments . The single - letter amino - acid code is used . NES indicates nuclear export signal ; NLS , nuclear localization signal ; ER , endoplasmic reticulum ; Ub , ubiquitin . Data are shown as the means ± s . d . * P0 . 05 , compared with the corresponding control ( two - tailed unpaired Student ' s t - test ) .

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(e-g) Parkin mitochondrial translocation (e) and ubiquitination (f) 8 h after CCCP treatment and mitochondrial clearance (g) 36 h after CCCP treatment were quantified. A minimum of 300-400 YFP-positive cells were scored in three independent experiments. The single-letter amino-acid code is used. NES indicates nuclear export signal; NLS, nuclear localization signal; ER, endoplasmic reticulum; Ub, ubiquitin. Data are shown as the means±s.d. *P0.05, compared with the corresponding control (two-tailed unpaired Student's t-test).


( a ) Representative electron micrographs of DOX - treated hearts . Arrows indicate abnormal mitochondria defined by marked swelling , vacuolation ( loss of electron density ) or disrupted cristae ; original magnification × 5 , 000 ; scale bar , 2 μm . The magnified photograph represents abnormal mitochondria ( blue line ) and an autophagic vacuole containing mitochondria ( red line ) ; original magnification × 12 , 000 ; scale bar , 500 nm . ( b , c ) Mitochondria incorporated into an autophagic vacuole ( b ) and abnormal mitochondria ( c ) were quantified blindly from 8 - 10 images from different fields ( original magnification × 5 , 000 ) ( n = 3 per group ) .

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(a) Representative electron micrographs of DOX-treated hearts. Arrows indicate abnormal mitochondria defined by marked swelling, vacuolation (loss of electron density) or disrupted cristae; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents abnormal mitochondria (blue line) and an autophagic vacuole containing mitochondria (red line); original magnification × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).


( d ) The 8 - hydroxy - 2 ′ - deoxyguanosine ( 8 - OHdG ) , popular marker for oxidative DNA damage , in mitochondria DNA from DOX - treated hearts . Results are shown from 6 - 8 hearts analysed in duplicate .

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(d) The 8-hydroxy-2′-deoxyguanosine (8-OHdG), popular marker for oxidative DNA damage, in mitochondria DNA from DOX-treated hearts. Results are shown from 6-8 hearts analysed in duplicate.


( e ) Maximum dP / dt was examined using cardiac catheterization under graded dobutamine infusion in PBS - treated ( control ) and DOX - treated mice ( n = 6 - 8 per group ) . DKO indicates double knockout . Data are shown as the means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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(e) Maximum dP/dt was examined using cardiac catheterization under graded dobutamine infusion in PBS-treated (control) and DOX-treated mice (n=6-8 per group). DKO indicates double knockout. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


We used 20 - month - old heterozygous littermates surviving grossly tumor free . ( a , b ) The sequential state III oxygen consumption rate of heart isolated mitochondria ( 300 μg ) was monitored with a fibre - optic system . Substrates and inhibitors were added sequentially at the final concentrations indicated as follow . FCCP , 1 μM carbonylcyanide - p - trifluoromethoxyphenylhydrazone ; M / P , 5 mM malate + 5 mM pyruvate ; Rot , 100 nM rotenone ; Suc , 5 mM succinate ; AA , 50 nM antimycin A ; TMPD / Asc , 0 . 4 mM N , N , N ′ , N ′ - tetramethyl - p - phenylenediamine + 1 mM ascorbate ; KCN , 5 mM potassium cyanide . Representative results are shown ( a ) and oxygen consumption by each complex was calculated ( n = 5 - 8 per group , b ) .

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We used 20-month-old heterozygous littermates surviving grossly tumor free. (a,b) The sequential state III oxygen consumption rate of heart isolated mitochondria (300 μg) was monitored with a fibre-optic system. Substrates and inhibitors were added sequentially at the final concentrations indicated as follow. FCCP, 1 μM carbonylcyanide-p-trifluoromethoxyphenylhydrazone; M/P, 5 mM malate+5 mM pyruvate; Rot, 100 nM rotenone; Suc, 5 mM succinate; AA, 50 nM antimycin A; TMPD/Asc, 0.4 mM N,N,N′,N′-tetramethyl-p-phenylenediamine +1 mM ascorbate; KCN, 5 mM potassium cyanide. Representative results are shown (a) and oxygen consumption by each complex was calculated (n=5-8 per group, b).


( c , d ) Mitochondrial particle size ( c ) and membrane potential ( d ) were determined using flow cytometry for TMRE in cardiac mitochondria preparations . Results are shown from 4 - 8 hearts analysed in duplicate .

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(c,d) Mitochondrial particle size (c) and membrane potential (d) were determined using flow cytometry for TMRE in cardiac mitochondria preparations. Results are shown from 4-8 hearts analysed in duplicate.


( e ) H2O2 production with 0 . 5 mM NADH , complex I - linked substrate , was determined in the absence or presence of 2 μM complex I inhibitor rotenone using Amplex Red ( n = 5 - 8 per group ) .

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(e) H2O2 production with 0.5 mM NADH, complex I-linked substrate, was determined in the absence or presence of 2 μM complex I inhibitor rotenone using Amplex Red (n=5-8 per group).


( f ) Myocardial high - energy phosphate metabolism under 8γ dobutamine loading was measured biochemically with HPLC ( n = 5 per group ) .

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(f) Myocardial high-energy phosphate metabolism under 8γ dobutamine loading was measured biochemically with HPLC (n=5 per group).


( g ) Maximum dP / dt was examined using cardiac catheterization with graded a dobutamine infusion ( n = 5 per group ) .

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(g) Maximum dP/dt was examined using cardiac catheterization with graded a dobutamine infusion (n=5 per group).


( h ) Photographs showing heart sections after SA - β - gal staining ; original magnification , × 200 ; scale bar , 40 μm . SA - β - gal - positive cells were quantified ( n = 4 per group ) . Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section . HT indicates heterozygous ; DHT double heterozygous . Data are shown as the means ± s . d . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 ( two - tailed unpaired Student ' s t - test )

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(h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section. HT indicates heterozygous; DHT double heterozygous. Data are shown as the means±s.d. *P0.05; **P0.01; ***P0.001 (two-tailed unpaired Student's t-test)


( a ) Representative electron micrographs of the hearts of 20 - month - old aged mice ; original magnification × 5 , 000 ; scale bar , 2 μm . The magnified photograph shows an autophagic vacuole containing mitochondria ; original magnification , × 12 , 000 ; scale bar , 500 nm . ( b , c ) Mitochondria incorporated into an autophagic vacuole ( b ) and abnormal mitochondria ( c ) were quantified blindly from 8 - 10 images from different fields ( original magnification × 5 , 000 ) ( n = 3 per group ) .

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(a) Representative electron micrographs of the hearts of 20-month-old aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph shows an autophagic vacuole containing mitochondria; original magnification, × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).


( d , e ) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser . With the initial presence of 5 μg mitochondria per well , 0 . 5 mM NADH and 4 μM FCCP , injections were performed as follows : port A , 50 μl of 20 μM rotenone ( 2 μM final ) ; port B , 55 μl of 100 mM succinate ( 10 mM final ) ; port C , 60 μl of 40 μM antimycin A ( 4 μM final ) ; port D , 65 μl of 100 mM ascorbate plus 1 mM TMPD ( 10 μM and 100 μM final , respectively ) . Representative results of aged littermates are shown ( d ) and oxygen consumption by each complex was calculated from four independent experiments , five replicates per sample , and four samples per experiment ( n = 8 per group , e ) .

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(d,e) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser. With the initial presence of 5 μg mitochondria per well, 0.5 mM NADH and 4 μM FCCP, injections were performed as follows: port A, 50 μl of 20 μM rotenone (2 μM final); port B, 55 μl of 100 mM succinate (10 mM final); port C, 60 μl of 40 μM antimycin A (4 μM final); port D, 65 μl of 100 mM ascorbate plus 1 mM TMPD (10 μM and 100 μM final, respectively). Representative results of aged littermates are shown (d) and oxygen consumption by each complex was calculated from four independent experiments, five replicates per sample, and four samples per experiment (n=8 per group, e).


( f ) NADH - driven H2O2 production was determined using Amplex Red . Results are shown from five hearts analysed in duplicate .

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(f) NADH-driven H2O2 production was determined using Amplex Red. Results are shown from five hearts analysed in duplicate.


( g ) Maximum dP / dt was examined using cardiac catheterization with graded dobutamine infusion in WT and Parkin Tg aged littermates ( n = 8 per group ) .

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(g) Maximum dP/dt was examined using cardiac catheterization with graded dobutamine infusion in WT and Parkin Tg aged littermates (n=8 per group).


( h ) Photographs showing heart sections after SA - β - gal staining ; original magnification , × 200 ; scale bar , 40 μm . SA - β - gal - positive cells were quantified ( n = 4 per group ) . Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section .

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(h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section.


( i ) Real - time PCR assessing the expression of cell cycle inhibitors and proinflammatory cytokines ( n = 8 per group ) .

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(i) Real-time PCR assessing the expression of cell cycle inhibitors and proinflammatory cytokines (n=8 per group).


( j ) Representative immunoblots assessing the expression of cell cycle inhibitors are shown ; Data are shown as the means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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(j) Representative immunoblots assessing the expression of cell cycle inhibitors are shown; Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


( a ) Electron micrographs of the IM ( black arrowheads ) associated with the ER ( black arrows ) from Atg4BC74A - expressing NIH 3T3 cells . The IM appears to expand between the two ER cisternae and forms an autophagosome . The white arrowhead indicates vesicular clusters visible in the vicinity of the IM . The white arrow indicates the autophagosome .

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(a) Electron micrographs of the IM (black arrowheads) associated with the ER (black arrows) from Atg4BC74A-expressing NIH 3T3 cells. The IM appears to expand between the two ER cisternae and forms an autophagosome. The white arrowhead indicates vesicular clusters visible in the vicinity of the IM. The white arrow indicates the autophagosome.


( b ) The ratio of ER - associated autophagic structures ( ER - IM complexes ) to total autophagic structures . Data are expressed as the mean ± s . d . of triplicates from representative experiments .

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(b) The ratio of ER-associated autophagic structures (ER-IM complexes) to total autophagic structures. Data are expressed as the mean ± s.d. of triplicates from representative experiments.


( c ) Localization of GFP - DFCP1 with Atg16L1 in both control and Atg4BC74A - expressing cells . Insets show enlarged images of boxed regions .

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(c) Localization of GFP-DFCP1 with Atg16L1 in both control and Atg4BC74A-expressing cells. Insets show enlarged images of boxed regions.


( d - g ) Gold - enhanced immunogold electron microscopy revealed that GFP - DFCP1 localized to ER - IM complexes in both control ( d , e ) and Atg4BC74A - expressing cells ( f ) . Immunogold particles are indicated by small arrows . No specific labelling was observed on ER - IM complexes in cells expressing GFP ( g ) . Large arrows and arrowheads indicate the ER and the IM , respectively . Scale bars , 200 nm in a and d - g , an d 10 μm in c .

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(d-g) Gold-enhanced immunogold electron microscopy revealed that GFP-DFCP1 localized to ER-IM complexes in both control (d, e) and Atg4BC74A-expressing cells (f). Immunogold particles are indicated by small arrows. No specific labelling was observed on ER-IM complexes in cells expressing GFP (g). Large arrows and arrowheads indicate the ER and the IM, respectively. Scale bars, 200 nm in a and d-g, an d 10 μm in c.


Atg4BC74A - expressing cells ( for Atg16L1 ) or control cells ( for PDI , COP II and β - COP ) were labelled with specific antibodies against the indicated markers . The left and right anti - Atg16L1 and anti - PDI images are from two different ER - IM complexes . Arrows , arrowheads and open arrowheads indicate the ER , IMs and VTCs , respectively . Small arrows in the image from the anti - COP II experiment indicate immunogold particles . Scale bars , 200 nm .

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Atg4BC74A-expressing cells (for Atg16L1) or control cells (for PDI, COP II and β-COP) were labelled with specific antibodies against the indicated markers. The left and right anti-Atg16L1 and anti-PDI images are from two different ER-IM complexes. Arrows, arrowheads and open arrowheads indicate the ER, IMs and VTCs, respectively. Small arrows in the image from the anti-COP II experiment indicate immunogold particles. Scale bars, 200 nm.


( e ) Images show 1 . 5 - nm tomographic slices of the boxed region in c , and depict the IM ( black arrow ) , whose edge ( red arrowhead ) is connected to the associated ER ( black arrowhead ) . Numbers indicate the depth of the tomographic slices . Scale bars , 200 nm in a , 100 nm in b and c , and 50 nm in d and e .

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(e) Images show 1.5-nm tomographic slices of the boxed region in c, and depict the IM (black arrow), whose edge (red arrowhead) is connected to the associated ER (black arrowhead). Numbers indicate the depth of the tomographic slices. Scale bars, 200 nm in a, 100 nm in b and c, and 50 nm in d and e.


A 2 - nm tomographic slice of each ER - IM complex , reconstructed from a 200 - nm thick section , and their 3D models are depicted in the left panels . Red arrows indicate sites at which an edge of the IM is connected to the ER . Boxed regions are enlarged in the right panels . 3D models of the boxed regions , shown in a different orientation , depict the connection between the IM and ER ( red arrows ) . Tomographic slices of the boxed regions show the edge of the IM ( red arrowheads ) is connected to the ER . Numbers indicate the depth of the tomographic slices . Scale bars , 100 nm .

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A 2-nm tomographic slice of each ER-IM complex, reconstructed from a 200-nm thick section, and their 3D models are depicted in the left panels. Red arrows indicate sites at which an edge of the IM is connected to the ER. Boxed regions are enlarged in the right panels. 3D models of the boxed regions, shown in a different orientation, depict the connection between the IM and ER (red arrows). Tomographic slices of the boxed regions show the edge of the IM (red arrowheads) is connected to the ER. Numbers indicate the depth of the tomographic slices. Scale bars, 100 nm.


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 .

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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 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 .

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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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 + 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


( G ) Size - exclusion chromatography profiles and corresponding SDS - PAGE of the indicated samples . Two mutant RZZ complexes containing mutations in ROD L110F - L119F - L120K or I191M are indicated respectively as FFK and I / M . Note that in the bottom SDS - PAGE the first and second lane were deliberately inverted and contain the first eluted fraction and the molecular weight marker . For all other shown SDS - PAGE gels , the marker precedes the first fraction .

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

(G) Size-exclusion chromatography profiles and corresponding SDS-PAGE of the indicated samples. Two mutant RZZ complexes containing mutations in ROD L110F-L119F-L120K or I191M are indicated respectively as FFK and I/M. Note that in the bottom SDS-PAGE the first and second lane were deliberately inverted and contain the first eluted fraction and the molecular weight marker. For all other shown SDS-PAGE gels, the marker precedes the first fraction.


( B ) Coomassie and ProQ Diamond - stained gels on mChRZZSF ( 4 µM ) treated with the indicated kinases ( 1 µM ) for 15 hours at 20ºC . Rev = reversine , used at 10 µM .

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

(B) Coomassie and ProQ Diamond-stained gels on mChRZZSF (4 µM) treated with the indicated kinases (1 µM) for 15 hours at 20ºC. Rev = reversine, used at 10 µM.


( D ) Levels of Zwilch at kinetochores of HeLa cells that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis . Subsequently , cells were immediately treated with 500 nM reversine , 3 . 3 µM nocodazole , and 10 µM MG132 for 1 hour and imaged while in mitosis . CREST serum was used to visualize kinetochores and DAPI to stain DNA . Scale bar : 10 µm . Three biological replicates were performed . ( E ) Cells treated as for panel D were treated for visualization of Spindly . Scale bar : 10 µm .

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

(D) Levels of Zwilch at kinetochores of HeLa cells that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 500 nM reversine, 3.3 µM nocodazole, and 10 µM MG132 for 1 hour and imaged while in mitosis. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed. (E) Cells treated as for panel D were treated for visualization of Spindly. Scale bar: 10 µm.


( F ) Scatter dot plots representing normalized total area of the Zwilch and Spindly signals , normalized to the reversine - untreated control , in the indicated number of cells from the experiment shown in panels C - D . Red lines indicate mean and standard deviation . Statistical analysis was performed with a nonparametric t - test comparing two unpaired groups ( Mann - Whitney test ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

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

(F) Scatter dot plots representing normalized total area of the Zwilch and Spindly signals, normalized to the reversine-untreated control, in the indicated number of cells from the experiment shown in panels C-D. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.


( G ) Representative images showing the effects of a knockdown of the endogenous Spindly in HeLa cells . RNAi treatment was performed for 48 h with 50 nM siRNA . Before fixation , cells were synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis . Subsequently , cells were immediately treated with 3 . 3 μM nocodazole for an additional hour . CREST serum was used to visualize kinetochores and DAPI to stain DNA . Scale bar : 10 µm . Three biological replicates were performed .

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

(G) Representative images showing the effects of a knockdown of the endogenous Spindly in HeLa cells. RNAi treatment was performed for 48 h with 50 nM siRNA. Before fixation, cells were synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM nocodazole for an additional hour. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed.


( H ) Scatter dot plots representing normalized intensity ratios of Spindly over CREST for individual kinetochores of cells from the experiment shown in panel G . Red lines indicate median with interquartile range .

O O O O O O O O O O O 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) Scatter dot plots representing normalized intensity ratios of Spindly over CREST for individual kinetochores of cells from the experiment shown in panel G. Red lines indicate median with interquartile range.


( I ) Levels of CENP - E and Zwilch were assessed in control cells and in cells treated as in panel G to knockdown Spindly . Three biological replicates were performed . ( J ) Scatter dot plots representing normalized total area of the CENP - E and Zwilch signals , normalized to the RNAi negative control , in the indicated number of cells from the experiment shown in panel I . Red lines indicate mean and standard deviation . Statistical analysis was performed with a nonparametric t - test comparing two unpaired groups ( Mann - Whitney test ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

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

(I) Levels of CENP-E and Zwilch were assessed in control cells and in cells treated as in panel G to knockdown Spindly. Three biological replicates were performed. (J) Scatter dot plots representing normalized total area of the CENP-E and Zwilch signals, normalized to the RNAi negative control, in the indicated number of cells from the experiment shown in panel I. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.


( E ) The phosphorylation state of RZZ and SpindlyF was monitored by ProQ Diamond after SDS - PAGE separation of reactions . Data information : Two technical replicates for panels ,

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(E) The phosphorylation state of RZZ and SpindlyF was monitored by ProQ Diamond after SDS-PAGE separation of reactions. Data information: Two technical replicates for panels , E


( D ) Heat - induced mChRZZGFPSF and MPS1 - induced rings of mChRZZmChSF have similar diameters . As shown in panel B , heat - and MPS1 - induced rings of mChRZZmChSF also have very similar dimensions . Data information : Experiments in D were carried out once .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heat-induced mChRZZGFPSF and MPS1-induced rings of mChRZZmChSF have similar diameters. As shown in panel B, heat- and MPS1-induced rings of mChRZZmChSF also have very similar dimensions. Data information: Experiments in D were carried out once.


( A ) Size - exclusion chromatography and SDS - PAGE of elution fractions of the indicated samples . Each Spindly construct was incubated with RZZ in absence ( dotted lines ) or in presence ( continuous lines ) of farnesyl transferase and farnesyl pyrophosphate . Elution shifts of SpindlyF and of the resulting RZZSF complexes is indicative of successful interaction . Two technical replicates were performed .

O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(A) Size-exclusion chromatography and SDS-PAGE of elution fractions of the indicated samples. Each Spindly construct was incubated with RZZ in absence (dotted lines) or in presence (continuous lines) of farnesyl transferase and farnesyl pyrophosphate. Elution shifts of SpindlyF and of the resulting RZZSF complexes is indicative of successful interaction. Two technical replicates were performed.


( D ) Representative images of fixed HeLa cells electroporated with full - length mChSpindly and mChSpindly354 - 605 constructs in cells depleted of endogenous Spindly by RNAi . Spindly localization was detected with an antibody against the C - terminal region of Spindly . Corona expansion or lack thereof were monitored through CENP - E . CREST serum was used to visualize kinetochores , DAPI to stain for DNA . Scale bar : 10 µm . Experiments were replicated three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of fixed HeLa cells electroporated with full-length mChSpindly and mChSpindly354-605 constructs in cells depleted of endogenous Spindly by RNAi. Spindly localization was detected with an antibody against the C-terminal region of Spindly. Corona expansion or lack thereof were monitored through CENP-E. CREST serum was used to visualize kinetochores, DAPI to stain for DNA. Scale bar: 10 µm. Experiments were replicated three times.


( E ) Scatter dot plots representing normalized intensity ratios of the indicated Spindly constructs over CREST for individual kinetochores of cells from the experiment shown in panel D . Red lines indicate median with interquartile range . Statistical analysis was performed with a nonparametric t - test comparing two unpaired groups ( Mann - Whitney test ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 . ( G ) Kinetochore intensities were quantified like in panel E . Red lines indicate median with interquartile range . Statistical analysis was performed with a nonparametric t - test comparing two unpaired groups ( Mann - Whitney test ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

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(E) Scatter dot plots representing normalized intensity ratios of the indicated Spindly constructs over CREST for individual kinetochores of cells from the experiment shown in panel D. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000. (G) Kinetochore intensities were quantified like in panel E. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.


( D ) ACE2 levels were characterized using RNAscope . A representative 3D view of a 232 x 232 μm2 field of view of the AT layer of an uninfected control at 3 days at air - liquid interface ( 3C ) is shown . The nuclear labelling is indicated in electric indigo and ACE2 mRNA is labelled in spring green LUT . ( G ) Same as in ( D ) with infected LoC at 3 dpi ( G ) shown .

(D) ACE2 levels were characterized using RNAscope. A representative 3D view of a 232 x 232 μm2 field of view of the AT layer of an uninfected control at 3 days at air-liquid interface (3C) is shown. The nuclear labelling is indicated in electric indigo and ACE2 mRNA is labelled in spring green LUT. (G) Same as in (D) with infected LoC at 3 dpi (G) shown.


( I , J ) A plot of the fold change in expression of host proteins required for viral entry in the AT and endothelial layer of LoCs at 1 day - post infection ( n = 4 biological replicates ) and 3 days - post infection ( n = 3 biological replicates ) vs . uninfected control LoCs ( n = 3 biological replicates ) . Data information : The bars represent the mean value , the solid line represents the median value , and the error bars represent the standard deviation . P - values are calculated using a Kruskal - Wallis one - way ANOVA test , * represents p < = 0 . 05 , * * represents p < = 0 . 01 , * * represents p < = 0 . 001 . Scale bar = 20 μm .

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(I, J) A plot of the fold change in expression of host proteins required for viral entry in the AT and endothelial layer of LoCs at 1 day-post infection (n=4 biological replicates) and 3 days-post infection (n=3 biological replicates) vs. uninfected control LoCs (n=3 biological replicates). Data information: The bars represent the mean value, the solid line represents the median value, and the error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis one-way ANOVA test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001. Scale bar = 20 μm.


( A The kinetics of release of SARS - CoV - 2 progeny was assessed from samples obtained from a wash of the apical face of the chip and the vascular effluent ( A ) collected at each day - post infection . ( A ) The number of viral genomes in each sample was quantified using a one - step qRT - PCR assay for the SARS - CoV - 2 N gene . The starting inoculum introduced to the apical side ( labelled ' Inn ' ) corresponded to 200 - 300 PFU . Lines join datapoints from the same LoC , reconstituted with ( green squares ) and without ( dark blue circles ) macrophages . Viral genome numbers within the apical wash showed a declining trend over 3 days of infection . Viral genomes are not detected ( ' nd ' ) in the vascular effluent for all the chips shown . Data information : In all plots , the bars represent the mean value , and the error bars represent the standard deviation , the solid black line in ( A ) represents the median .

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(A The kinetics of release of SARS-CoV-2 progeny was assessed from samples obtained from a wash of the apical face of the chip and the vascular effluent (A) collected at each day-post infection. (A) The number of viral genomes in each sample was quantified using a one-step qRT-PCR assay for the SARS-CoV-2 N gene. The starting inoculum introduced to the apical side (labelled 'Inn') corresponded to 200-300 PFU. Lines join datapoints from the same LoC, reconstituted with (green squares) and without (dark blue circles) macrophages. Viral genome numbers within the apical wash showed a declining trend over 3 days of infection. Viral genomes are not detected ('nd') in the vascular effluent for all the chips shown. Data information: In all plots, the bars represent the mean value, and the error bars represent the standard deviation, the solid black line in (A) represents the median.


( A - D ) 3D views of representative 232 x 232 μm2 ( A , B ) and 155 x 155 μm2 ( C , D ) fields of view from confocal imaging of an uninfected control ( A ) , and infected chips without macrophages at days 2 ( B ) and 3 ( C , D ) post - infection , respectively . Actin and nuclear labelling are shown in azure and indigo , respectively . ( B ) At 2 dpi , adjacent areas of endothelial cell clusters identified by a stronger nuclear stain and brighter actin staining ( yellow arrows ) and cells with increased permeability and loss of confluence ( white arrows ) are indicated . ( C ) At 3 dpi , a significant loss of barrier integrity is observed in the endothelial layer , whereas the AT layer directly above this field of view ( D ) is relatively intact . A small area of missing cells in the AT layer is indicated with a white arrow in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) 3D views of representative 232 x 232 μm2 (A, B) and 155 x 155 μm2 (C, D) fields of view from confocal imaging of an uninfected control (A), and infected chips without macrophages at days 2 (B) and 3 (C, D) post-infection, respectively. Actin and nuclear labelling are shown in azure and indigo, respectively. (B) At 2 dpi, adjacent areas of endothelial cell clusters identified by a stronger nuclear stain and brighter actin staining (yellow arrows) and cells with increased permeability and loss of confluence (white arrows) are indicated. (C) At 3 dpi, a significant loss of barrier integrity is observed in the endothelial layer, whereas the AT layer directly above this field of view (D) is relatively intact. A small area of missing cells in the AT layer is indicated with a white arrow in (D).


( G - J ) The fields of view in ( C , D ) are shown also in ( G , H ) respectively , SARS - CoV - 2 spike ( S ) proteins labelled via immunofluorescence are false - colored in amber . Zooms in ( I ) and ( J ) show clear evidence of intracellular spike protein within both endothelial and ATs , respectively .

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(G-J) The fields of view in (C, D) are shown also in (G, H) respectively, SARS-CoV-2 spike (S) proteins labelled via immunofluorescence are false-colored in amber. Zooms in (I) and (J) show clear evidence of intracellular spike protein within both endothelial and ATs, respectively.


( A - D ) Maximum intensity projects of 232 x 232 μm2 fields of view from the AT ( A , B ) and endothelial layers of an uninfected control LoC . ATs show strong actin ( A ) and TJP1 expression ( B , identified via antibody staining ) at the cell junction boundaries . Endothelial cells also show high levels of TJP - 1 expression ( C ) together with CD31 expression at cell junctions ( D , identified via antibody labelling ) . Actin , TJP - 1 , CD31 , and nuclear labelling are indicated with the azure , bright pink , spring green and electric indigo LUTs , respectively .

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(A-D) Maximum intensity projects of 232 x 232 μm2 fields of view from the AT (A, B) and endothelial layers of an uninfected control LoC. ATs show strong actin (A) and TJP1 expression (B, identified via antibody staining) at the cell junction boundaries. Endothelial cells also show high levels of TJP-1 expression (C) together with CD31 expression at cell junctions (D, identified via antibody labelling). Actin, TJP-1, CD31, and nuclear labelling are indicated with the azure, bright pink, spring green and electric indigo LUTs, respectively.


( E - H ) 3D views of one 232 x 232 μm2 field of view of the AT layer ( E ) and ( F - H ) three 232 x 232 μm2 fields of view of the endothelial layer of an infected LoC at 1 dpi . Areas with lower TJP1 expression in the AT layer in ( E ) are indicated by white arrows . ( F - H ) Areas with loss of tight junctions between cells on the endothelial surface is indicated by white arrows . The proportion of the surface area covered by confluent cells decreases sequentially from ( F ) to ( H ) . Endothelial cell clusters with low CD31 expression are clearly visible , and the proportion of the surface area covered by these clusters increases sequentially from ( F ) to ( H ) . In ( F - H ) , spike protein is identified via an anti - SARS - spike antibody and shown in amber LUT , and von Willebrand Factor identified by antibody labelling is shown in bring pink LUT .

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(E-H) 3D views of one 232 x 232 μm2 field of view of the AT layer (E) and (F-H) three 232 x 232 μm2 fields of view of the endothelial layer of an infected LoC at 1 dpi. Areas with lower TJP1 expression in the AT layer in (E) are indicated by white arrows. (F-H) Areas with loss of tight junctions between cells on the endothelial surface is indicated by white arrows. The proportion of the surface area covered by confluent cells decreases sequentially from (F) to (H). Endothelial cell clusters with low CD31 expression are clearly visible, and the proportion of the surface area covered by these clusters increases sequentially from (F) to (H). In (F-H), spike protein is identified via an anti-SARS-spike antibody and shown in amber LUT, and von Willebrand Factor identified by antibody labelling is shown in bring pink LUT.


( I - L ) Zooms of two endothelial cell clusters ( I , J and K , L ) in the endothelial layer of another infected LoC at 1 dpi confirm that little or no CD31 expression is detected ( I , K ) despite high levels of actin and TJP - 1 protein expression in these cells . ( J , L ) .

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(I-L) Zooms of two endothelial cell clusters (I, J and K, L) in the endothelial layer of another infected LoC at 1 dpi confirm that little or no CD31 expression is detected (I, K) despite high levels of actin and TJP-1 protein expression in these cells. (J, L).