Figure 1A ) Western blot analysis of Mpl in healthy donors ' ( HD ) platelets shows two distinct bands representing the mature glycoprotein ( top band ) and the ER ‐ core glycosylated form ( bottom band ) . The ratio is ~ 1 . B ) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F , alone or cotransfected with cDNA coding for Mpl . C ) Summary of the ratios of mature : immature Mpl for at least three independent ( 2 for the JAK2V617F transfection ) blotting experiments under the protocol shown in ( B ) . Based on two − tailed Student t − test statistical analysis , there is a significant difference ( p = 0 . 0001 ) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells . Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V / V617F by comparison to controls ( p = 0 . 0159 and p = 0 . 0009 , respectively ) . The mature : immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V / V617F ( p < 0 . 01 ) . D ) Western blot analysis of K562 lysates after transfection with cDNA coding for Mpl , alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F . E ) Summary of data in ( A ) , ( B ) and ( D ) , showing the relative band intensity of mature and immature Mpl and normalized for levels of β ‐ actin in each lane . F ) Immunoprecipitation of the Mpl ‐ Jak2 complex in different cell lines analyzed by immunoblotting with anti ‐ Mpl and anti ‐ JAK2 antibodies .

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Figure 1A) Western blot analysis of Mpl in healthy donors' (HD) platelets shows two distinct bands representing the mature glycoprotein (top band) and the ER‐core glycosylated form (bottom band). The ratio is ~ 1.B) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F, alone or cotransfected with cDNA coding for Mpl.C) Summary of the ratios of mature:immature Mpl for at least three independent (2 for the JAK2V617F transfection) blotting experiments under the protocol shown in (B). Based on two−tailed Student t−test statistical analysis, there is a significant difference (p = 0.0001) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells. Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V/V617F by comparison to controls (p = 0.0159 and p = 0.0009, respectively). The mature:immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V/V617F (p < 0.01).D) Western blot analysis of K562 lysates after transfection with cDNA coding for Mpl, alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F.E) Summary of data in (A), (B) and (D), showing the relative band intensity of mature and immature Mpl and normalized for levels of β‐actin in each lane.F) Immunoprecipitation of the Mpl‐Jak2 complex in different cell lines analyzed by immunoblotting with anti‐Mpl and anti‐JAK2 antibodies.


Figure 2Confocal fluorescent images of HEL cells ( JAK2V617F + / + ) after immuno ‐ fluorescence labeling of endogenous Mpl ( A ) , or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 ( B ) . C ) Confocal fluorescence image of K562 cells ( JAK2WT + / + ) transiently transfected with cDNA coding for MplmOrange2 . Red arrow in this image points to plasma membrane localization . D and E ) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno ‐ fluorescent labeling of endogenous JAK2 in K562 ( D ) or HEL ( E ) cells . Profiles of fluorescence ( across individual cells and indicated by a white arrow ) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane ( black arrow heads ) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL . F and G ) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine ( F ) or JAK2V617FmCitrine ( G ) in K562 cells . Profiles of fluorescence ( white arrows ) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine , especially at the plasma membrane ( black arrow heads ) , but a very poor colocalization with JAK2V617FmCitrine ( grey arrow heads ) . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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Figure 2Confocal fluorescent images of HEL cells (JAK2V617F+/+) after immuno‐fluorescence labeling of endogenous Mpl (A), or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 (B).C) Confocal fluorescence image of K562 cells (JAK2WT+/+) transiently transfected with cDNA coding for MplmOrange2. Red arrow in this image points to plasma membrane localization.D and E) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno‐fluorescent labeling of endogenous JAK2 in K562 (D) or HEL (E) cells. Profiles of fluorescence (across individual cells and indicated by a white arrow) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane (black arrow heads) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL.F and G) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine (F) or JAK2V617FmCitrine (G) in K562 cells. Profiles of fluorescence (white arrows) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine, especially at the plasma membrane (black arrow heads), but a very poor colocalization with JAK2V617FmCitrine (grey arrow heads). Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


Figure 3A ) K562 cells labeled live for 30 min with an anti ‐ CD235a ‐ Atto655 ( glycophorin A ) antibody before treatment with the Duolink reagents . B ) K562Mpl ‐ mKO2 cells non ‐ treated with the Duolink reagents . C and D ) HEL cells primed live for 30 min with an anti ‐ CD235a ‐ Atto655 antibody before treatment with the Duolink reagents . E and F ) K562Mpl ‐ mKO2 cells submitted to the Duolink reaction . White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane , while green arrow heads are pointing at positive Duolink reactions within the cytoplasm . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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Figure 3A) K562 cells labeled live for 30 min with an anti‐CD235a‐Atto655 (glycophorin A) antibody before treatment with the Duolink reagents.B) K562Mpl‐mKO2 cells non‐treated with the Duolink reagents.C and D) HEL cells primed live for 30 min with an anti‐CD235a‐Atto655 antibody before treatment with the Duolink reagents.E and F) K562Mpl‐mKO2 cells submitted to the Duolink reaction. White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane, while green arrow heads are pointing at positive Duolink reactions within the cytoplasm. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


Figure 4HEL ( A ) and K562 ( B ) cells treated with non ‐ targeting ( NT ) or anti ‐ JAK2 siRNA analyzed by western blot . C ) Confocal live cell ( upper panel ) , or fixed cells ( lower left panel ) , imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti ‐ JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells . After treatment with anti ‐ JAK2 siRNA , Mpl strongly colocalizes with the ER marker , calnexin . This colocalization in treated vs non ‐ treated cells is quantified and shown in Figure C lower right panel . Nuclei are labeled with Hoechst and scale bars = 5 µm .

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Figure 4HEL (A) and K562 (B) cells treated with non‐targeting (NT) or anti‐JAK2 siRNA analyzed by western blot.C) Confocal live cell (upper panel), or fixed cells (lower left panel), imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti‐JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells. After treatment with anti‐JAK2 siRNA, Mpl strongly colocalizes with the ER marker, calnexin. This colocalization in treated vs non‐treated cells is quantified and shown in Figure C lower right panel. Nuclei are labeled with Hoechst and scale bars = 5 µm.


Figure 5Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells . A - M ) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 ( A - F ) or HEL ( G - M ) cells . Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image . Black arrows in the plots point to overlap between MplmKO2 and either ER ‐ tracker ( C , I ) , or Sec31a ( B , J ) that marks ER exit sites . MplmOrange2 did not colocalize with markers for the Golgi ( Golgin ‐ 97 ) or early endosomes ( EEA1 ) in either cell line . However , MplmKO2 colocalized greatly with autophagic ( LC3 ) ( E , L ) and lysosomal ( LAMP1 ) ( F , M ) markers . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient . Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells . A - M ) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 ( A - F ) or HEL ( G - M ) cells . Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image . Black arrows in the plots point to overlap between MplmKO2 and either ER ‐ tracker ( C , I ) , or Sec31a ( B , J ) that marks ER exit sites . MplmOrange2 did not colocalize with markers for the Golgi ( Golgin ‐ 97 ) or early endosomes ( EEA1 ) in either cell line . However , MplmKO2 colocalized greatly with autophagic ( LC3 ) ( E , L ) and lysosomal ( LAMP1 ) ( F , M ) markers . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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Figure 5Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A-M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A-F) or HEL (G-M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A-M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A-F) or HEL (G-M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


Figure 6Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 ( A ) and HEL ( B ) cells . Electron micrographs of K562 ( C ) and HEL ( D ) cells transiently expressing MplmKO2 ‐ miniSOG . Dark precipitate indicates the presence of the miniSOG ‐ tagged Mpl protein after photo ‐ precipitation of DAB . White boxes indicate original location of higher magnification images with corresponding letters . E - G ) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C , showing MplmKO2 ‐ miniSOG dark precipitate into autophagic carrier vesicles ( black arrows , Figure E ) . Osmiophilic reaction products also heavily stain mature AP in these preparations ( black arrows , Figure G ) . H ) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D , cells transiently expressing MplmKO2 - miniSOG , showing different stage of formation of APs ( black arrows numbered from stages 1 - 4 ) . Scale bars = 5 µm ( A - D ) or 100 nm ( E - H ) .

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Figure 6Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 (A) and HEL (B) cells. Electron micrographs of K562 (C) and HEL (D) cells transiently expressing MplmKO2‐miniSOG. Dark precipitate indicates the presence of the miniSOG‐tagged Mpl protein after photo‐precipitation of DAB. White boxes indicate original location of higher magnification images with corresponding letters.E-G) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C, showing MplmKO2‐miniSOG dark precipitate into autophagic carrier vesicles (black arrows, Figure E). Osmiophilic reaction products also heavily stain mature AP in these preparations (black arrows, Figure G).H) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D, cells transiently expressing MplmKO2-miniSOG , showing different stage of formation of APs (black arrows numbered from stages 1-4). Scale bars = 5 µm (A-D) or 100 nm (E-H).


Figure 7A ) Western blot analysis and quantification of streptavidin ‐ immunoprecipitated surface ‐ biotinylated proteins , probed with an anti ‐ Mpl antibodyB and C ) Confocal images of K562 cells transiently expressing MplmOrange2 . Cells in ( B ) are untreated . Cells in ( C ) were stimulated 60 ' with Tpo , washed and chased for 15 min before imaging . D ) Quantification of plasma membrane fluorescence in resting and Tpo ‐ stimulated cells . Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background . A two ‐ tailed Student t ‐ test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal . Scale bars = 5 µm . E ) Western blot analysis of protein phosphorylation in HEL , K562Mpl ‐ mKO2 and K562Mpl ‐ mOrange2 cells after Tpo stimulation ( 50 ng / mL Tpo , 10 min ) .

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Figure 7A) Western blot analysis and quantification of streptavidin‐immunoprecipitated surface‐biotinylated proteins, probed with an anti‐Mpl antibodyB and C) Confocal images of K562 cells transiently expressing MplmOrange2. Cells in (B) are untreated. Cells in (C) were stimulated 60' with Tpo, washed and chased for 15 min before imaging.D) Quantification of plasma membrane fluorescence in resting and Tpo‐stimulated cells. Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background. A two‐tailed Student t‐test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal. Scale bars = 5 µm.E) Western blot analysis of protein phosphorylation in HEL, K562Mpl‐mKO2 and K562Mpl‐mOrange2 cells after Tpo stimulation (50 ng/mL Tpo, 10 min).


Figure 8A and B ) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl ‐ mKO2 cells after treatments for 60 min with Tpo , ionomycin ( I ) , PMA , Mon or A23187 . C ) Total lysate , surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl ‐ mKO2 cells treated with Mon or A23187 for 15 h . D ) Western blot analysis of HEL cells treated , or not , with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng / mL for 30 min . The quantification of the blot is shown . E ) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3 − MA ( 3 − Methyladenine ) or Rp . F ) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R ‐ V5 6 h post ‐ transfection . G ) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55 - V5 or HA - STX5 .

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Figure 8A and B) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl‐mKO2 cells after treatments for 60 min with Tpo, ionomycin (I), PMA, Mon or A23187.C) Total lysate, surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl‐mKO2 cells treated with Mon or A23187 for 15 h.D) Western blot analysis of HEL cells treated, or not, with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng/mL for 30 min. The quantification of the blot is shown.E) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3−MA (3−Methyladenine) or Rp.F) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R‐V5 6 h post‐transfection.G) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55-V5 or HA-STX5.


Figure 1B , HeLa cells were treated with DMSO or 2µM Mito - C for 15 min and immunostained with anti - TOM20 ( green ) antibody and DAPI ; cropped areas show the mitochondria morphology changes . C , Quantification of mitochondria morphology ( based on TOM20 immunostaining as shown in B ) from HeLa cells treated with DMSO or 2µM Mito - C for 15 min or 2h , expressed as ratio of fragmented mitochondria ( percentage of total ) . Errors bars show the standard error of the mean ( SEM ) ( n = 100 , technical replicates ) . E , Time lapse video - microscopy snapshot on HeLa cells transfected with NAF - 1 - mRFP ( red ) and treated with fluoMito - C ( green ) . The distance / intensity fluorescence quantification graph illustrates the codistribution of fluoMito - C and NAF - 1 - mRFP .

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Figure 1B, HeLa cells were treated with DMSO or 2µM Mito-C for 15 min and immunostained with anti-TOM20 (green) antibody and DAPI; cropped areas show the mitochondria morphology changes.C, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in B) from HeLa cells treated with DMSO or 2µM Mito-C for 15 min or 2h, expressed as ratio of fragmented mitochondria (percentage of total). Errors bars show the standard error of the mean (SEM) (n=100, technical replicates).E, Time lapse video-microscopy snapshot on HeLa cells transfected with NAF-1-mRFP (red) and treated with fluoMito-C (green). The distance/intensity fluorescence quantification graph illustrates the codistribution of fluoMito-C and NAF-1-mRFP.


Figure 2A , Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA ; Bar Chart ( right panel ) shows replicates quantification of MitoNEET expression . Errors bars show the standard deviation ( SD ) of 3 independent experiments . B , Western blot analysis of NAF - 1 expression in HeLa cells transfected with CISD2 siRNA ; graph ( right panel ) shows the replicates quantification of NAF - 1 expression . Errors bars show the standard deviation ( SD ) of 3 independent experiments . D , HeLa cells transfected with siRNA to reduce expression of MitoNEET ( CISD1 ) , NAF - 1 ( CISD2 ) or MiNT ( CISD3 ) and immunostained with anti TOM20 antibody . Arrowheads indicate fragmented mitochondria . E , Quantification of mitochondria morphology ( based on TOM20 immunostaining as shown in D ) from HeLa cells transfected with siRNA targeting mitoNEET ( siCISD1 ) , NAF1 ( siCISD2 ) or MiNT ( siCISD3 ) . Quantification is expressed as ratio of tubular and fragmented mitochondria . Errors bars show the standard error of the mean ( SEM ) . ( n = 125 - 130 cells ) . F , Electron microscopy pictures from 150 INS - 1 cells transfected as described in G . G , Western blot analysis of NAF - 1 expression in cells stably transfected with control shRNA , shRNA targeting reduction of NAF - 1 protein expression ( shCISD2 ) , or shRNA targeting reduction of NAF - 1 protein expression complemented with plasmid derived expression of NAF - 1 ( shCISD2 + CISD2 ) ; western blot quantification of NAF - 1 is shown on the accompanying bar chart . Errors bars show the standard deviation ( SD ) of 3 independent experiments . H , Quantification of mitochondrial length in EM images from cells transfected as described in G . Errors bars show the standard error of the mean ( SEM ) ( n = 260 - 270 , technical replicates ) .

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Figure 2A, Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA; Bar Chart (right panel) shows replicates quantification of MitoNEET expression. Errors bars show the standard deviation (SD) of 3 independent experiments.B, Western blot analysis of NAF-1 expression in HeLa cells transfected with CISD2 siRNA; graph (right panel) shows the replicates quantification of NAF-1 expression. Errors bars show the standard deviation (SD) of 3 independent experiments.D, HeLa cells transfected with siRNA to reduce expression of MitoNEET (CISD1), NAF-1 (CISD2) or MiNT (CISD3) and immunostained with anti TOM20 antibody. Arrowheads indicate fragmented mitochondria.E, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in D) from HeLa cells transfected with siRNA targeting mitoNEET (siCISD1), NAF1 (siCISD2) or MiNT (siCISD3). Quantification is expressed as ratio of tubular and fragmented mitochondria. Errors bars show the standard error of the mean (SEM). (n =125-130 cells).F, Electron microscopy pictures from 150 INS-1 cells transfected as described in G.G, Western blot analysis of NAF-1 expression in cells stably transfected with control shRNA, shRNA targeting reduction of NAF-1 protein expression (shCISD2), or shRNA targeting reduction of NAF-1 protein expression complemented with plasmid derived expression of NAF-1 (shCISD2+CISD2); western blot quantification of NAF-1 is shown on the accompanying bar chart. Errors bars show the standard deviation (SD) of 3 independent experiments.H, Quantification of mitochondrial length in EM images from cells transfected as described in G. Errors bars show the standard error of the mean (SEM) (n=260-270, technical replicates).


Figure 3A , HeLa cells treated or not , with 2µM Mito - C for 15 min were immunostained with anti - TOM20 ( red ) and anti - DRP1 ( green ) ; arrowheads in the far - right panel indicate recruitment of DRP1 onto the mitochondrial surface ( TOM20 ) . B , Quantification of DRP1 signal on TOM20 positive structures . Errors bars show the standard error of the mean ( SEM ) ( n = 45 , technical replicates ) . CC , Western blot analysis of DRP1 , GAPDH ( cytosolic marker ) and TOM20 ( mitochondrial marker ) protein in cytosolic and mitochondrial fractions treated or not , with 2µM Mito - C as indicated . D , Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions . Errors bars show the standard deviation ( SD ) of 5 independent experiments . EE , HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not , with Mito - C ( T for transfected , NT for not transfected ) . F , Quantification of the mitochondrial phenotypes observed in E . Errors bars show the standard error of the mean ( SEM ) . ( 30 - 35 images each with an average of 15 - 20 cells from triplicate independent experiments were analyzed ) . GG , Electron micrographs ( EM ) from HeLa cells treated with 2µM Mito - C or DMSO for 120 minutes . Empty arrowheads indicate ER - mitochondria contact sites . H , Quantification of ER - mitochondria membrane contact sites events ( reported in 50µm² section ; 50 images from an average of 30 cells from triplicate independent experiments were analyzed ) . Errors bars show the standard error of the mean ( SEM ) . I , Quantification of ER - mitochondria membrane contact sites density ( in µm ; 30 images from an average of 30 cells from triplicate independent experiments were analyzed ) . Errors bars show the standard error of the mean ( SEM ) .

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

Figure 3A, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20). B, Quantification of DRP1 signal on TOM20 positive structures. Errors bars show the standard error of the mean (SEM) (n=45, technical replicates). CC, Western blot analysis of DRP1, GAPDH (cytosolic marker) and TOM20 (mitochondrial marker) protein in cytosolic and mitochondrial fractions treated or not, with 2µM Mito-C as indicated. D, Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions. Errors bars show the standard deviation (SD) of 5 independent experiments. EE, HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not, with Mito-C (T for transfected, NT for not transfected). F, Quantification of the mitochondrial phenotypes observed in E. Errors bars show the standard error of the mean (SEM). (30-35 images each with an average of 15-20 cells from triplicate independent experiments were analyzed). GG, Electron micrographs (EM) from HeLa cells treated with 2µM Mito-C or DMSO for 120 minutes. Empty arrowheads indicate ER-mitochondria contact sites. H, Quantification of ER-mitochondria membrane contact sites events (reported in 50µm² section; 50 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). I, Quantification of ER-mitochondria membrane contact sites density (in µm; 30 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). Da


Figure 4B , Huh7 cells were treated with or without ( DMSO ) Mito - C , infected with dengue virus and fixed after 48h of infection . Cells were then immunostained for endogenous TOM20 ( green channel ) , viral non - structural protein NS5 ( red ) and DAPI ( Blue ) . Cropped areas illustrate the mitochondrial morphology in described conditions . C , Quantification of the mitochondrial phenotypes observed in b . Errors bars show the standard error of the mean ( SEM ) . ( 15 - 20 images each with an average of 20 - 25 cells from triplicate independent experiments were analyzed ) .

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

Figure 4B, Huh7 cells were treated with or without (DMSO) Mito-C, infected with dengue virus and fixed after 48h of infection. Cells were then immunostained for endogenous TOM20 (green channel), viral non-structural protein NS5 (red) and DAPI (Blue). Cropped areas illustrate the mitochondrial morphology in described conditions.C, Quantification of the mitochondrial phenotypes observed in b. Errors bars show the standard error of the mean (SEM). (15-20 images each with an average of 20-25 cells from triplicate independent experiments were analyzed).


Fig 1 ( B ) Gal4 DNA - binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins ( murine ) in Y2H . ( C ) HEK293 cells transfected with indicated MUP - V5 , myc - UBQLN1 ( human ) , or empty vector were immunoprecipitated ( IP ) with anti - myc or anti - UBQLN1 antibodies . Western blot ( WB ) of IP and lysate were probed as indicated . ( D - E ) WB of input lysate and IP from HEK293 cells expressing myc - UBQLN1 and Rv1926 - V5 ( D ) or Rv1566 - V5 ( E ) . IP was performed using antibodies directed against myc , V5 , or isotype control ( IgG ) .

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

Fig 1(B) Gal4 DNA-binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins (murine) in Y2H.(C) HEK293 cells transfected with indicated MUP-V5, myc-UBQLN1 (human), or empty vector were immunoprecipitated (IP) with anti-myc or anti-UBQLN1 antibodies. Western blot (WB) of IP and lysate were probed as indicated.(D-E) WB of input lysate and IP from HEK293 cells expressing myc-UBQLN1 and Rv1926-V5 (D) or Rv1566-V5 (E). IP was performed using antibodies directed against myc, V5, or isotype control (IgG).


Fig 2 ( B ) Mtb were incubated with lysate from HEK293 cells transfected with vector control , myc - UBQLN1 , GFP , or V5 - parkin . WB analysis of the input ( I ) , 5th wash ( W5 ) , and elution ( E ) , probed with indicated antibodies . Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet . ( C ) Mtb were incubated with lysate from HEK293 cells transfected with myc - UBQLN1 - ΔUBA , myc - UBQLN1 - ΔUBL , and myc - UBQLN1 - UBA . WB containing the input , 5th wash , and elution were incubated with anti - myc antibody . The predicted sizes of myc - UBQLN1 - ΔUBL , myc - UBQLN1 - ΔUBA , and myc - UBQLN1 - UBA are 55 kD , 50 kD , and 6 kD , respectively . ( D ) Mtb were incubated with recombinant GST - UBQLN1 , GST - UBQLN1 - ΔUBL , or GST - UBQLN1 - ΔUBA . WB containing the input and elution was probed with an anti - GST antibody .

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

Fig 2(B) Mtb were incubated with lysate from HEK293 cells transfected with vector control, myc-UBQLN1, GFP, or V5-parkin. WB analysis of the input (I), 5th wash (W5), and elution (E), probed with indicated antibodies. Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet.(C) Mtb were incubated with lysate from HEK293 cells transfected with myc-UBQLN1-ΔUBA, myc-UBQLN1-ΔUBL, and myc-UBQLN1-UBA. WB containing the input, 5th wash, and elution were incubated with anti-myc antibody. The predicted sizes of myc-UBQLN1-ΔUBL, myc-UBQLN1-ΔUBA, and myc-UBQLN1-UBA are 55 kD, 50 kD, and 6 kD, respectively.(D) Mtb were incubated with recombinant GST-UBQLN1, GST-UBQLN1-ΔUBL, or GST-UBQLN1-ΔUBA. WB containing the input and elution was probed with an anti-GST antibody.


Fig 3 ( A ) BMDMs were infected with GFP - expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody . IFN - γ was added 1d prior to infection as indicated . ( B ) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment ; * * * P < 0 . 001 , Fisher ' s exact test , comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples . ( C ) Cellular lysate from BMDMs that were treated with IFN - γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2 . ( D ) BMDMs were transfected with siRNAs targeting UBQLN1 or a non - targeting control ( siCON ) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2 . ( C - D ) Actin served as a loading control . ( E ) BMDMs were infected with Mtb live / dead strain and treated with AnTc 24h prior to fixation . ( F ) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live / dead Mtb strain . IFN - γ was added 1d prior to infection if indicated . % Viable bacteria were quantified 24 and 48 hpi . Data are from 2 independent experiments . * P < 0 . 05 ; * * * P < 0 . 001 , Fisher ' s exact test , comparing the proportion of viable and non - viable bacteria in indicated samples . ( G ) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN - γ one day prior to infection with H37Rv or ΔesxA . CFU were enumerated 4 and 96 hpi . * P < 0 . 05 , unpaired Student ' s t - test . Data are representative of three independent experiments . ( H ) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb ( H37Rv ) and IFN - γ was measured by ELISA ( I ) UBQLN1 - silenced BMDMs and controls were infected with H37Rv or ΔesxA . ( H - I ) 24 hpi infected BMDMs were co - cultured with P25TCR - Tg CD4 + T cells , and IFN - γ was measured by ELISA . Data is representative of 3 independent experiments . * P < 0 . 05 , * * P < 0 . 005 , unpaired Student ' s t - test . ( B , F , Γ , H ) Results are mean + / - SEM ; ns , not significant .

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

Fig 3(A) BMDMs were infected with GFP-expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody. IFN-γ was added 1d prior to infection as indicated. (B) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment; ***P< 0.001, Fisher's exact test, comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples.(C) Cellular lysate from BMDMs that were treated with IFN-γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2.(D) BMDMs were transfected with siRNAs targeting UBQLN1 or a non-targeting control (siCON) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2. (C-D) Actin served as a loading control.(E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation.(F) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live/dead Mtb strain. IFN-γ was added 1d prior to infection if indicated. %Viable bacteria were quantified 24 and 48 hpi. Data are from 2 independent experiments. *P<0.05; ***P<0.001, Fisher's exact test, comparing the proportion of viable and non-viable bacteria in indicated samples.(G) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN-γ one day prior to infection with H37Rv or ΔesxA. CFU were enumerated 4 and 96 hpi. *P< 0.05, unpaired Student's t-test. Data are representative of three independent experiments.(H) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb (H37Rv) and IFN-γ was measured by ELISA(I) UBQLN1-silenced BMDMs and controls were infected with H37Rv or ΔesxA. (H-I) 24 hpi infected BMDMs were co-cultured with P25TCR-Tg CD4+ T cells, and IFN-γ was measured by ELISA. Data is representative of 3 independent experiments. *P<0.05, **P<0.005, unpaired Student's t-test. (B, F, Γ, H) Results are mean +/- SEM; ns, not significant.


Fig 4 ( A ) BMDMs treated with IFN - γ and infected for 24h with GFP - Mtb were immunostained for UBQLN1 and either ubiquitin ( using the FK2 antibody ) or p62 . GFP - LC3 BMDMs infected with DsRed - Mtb were immunostained for UBQLN1 . ( B ) Quantification of those Mtb that co - localize with at least one of the cellular markers shown in A . More than 375 total bacteria were counted in two independent experiments , and the number co - localizing with at least one marker is indicated as " total " under each bar . ( C ) Wt macrophages ( Atg16L1flox / flox ; Cre - ) and autophagy - deficient macrophages ( Atg16L1flox / flox Lyz - cre ; Cre + ) were activated with IFN - γ or not , followed by infection with the live / dead Mtb reporter strain . % viable was quantified after addition of AnTc . * * P < 0 . 005 , * * * P < 0 . 0001 , Fisher ' s exact test ( in which the proportions of viable to non - viable Mtb was compared ) . ( D ) Atg16L1flox / flox Cre + and Cre - BMDMs treated with IFN - γ and infected for 24h with GFP - Mtb were immunostained for UBQLN1 and p62 . The number of Mtb co - localizating with the cellular markers is shown . ( C - D ) At least 100 bacteria were analyzed in three independent experiments . ( E ) Images from IFN - γ treated BMDMs of indicated genotype infected with GFP - Mtb and immunostained with UBQLN1 and p62 . Arrows indicates p62 + UBQLN1 + Mtb .

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

Fig 4(A) BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as "total" under each bar.(C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher's exact test (in which the proportions of viable to non-viable Mtb was compared).(D) Atg16L1flox/flox Cre+ and Cre- BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments.(E) Images from IFN-γ treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.


Fig 5 ( A - C ) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA . Images show IFN - γ treated macrophages infected with GFP - Mtb ( A - B ) or DsRed - Mtb ( C ) for 24h . Cells were immunostained for ubiquitin ( Ub ) with the FK2 antibody ( A ) or p62 ( B ) , which are shown in red . ( C ) GFP - LC3 BMDMs were infected with DsRed - Mtb . LC3 is shown in green , whereas Mtb are red . ( A - C ) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN - γ as indicated . Results are mean + / - SEM from two independent experiments , with at least 100 bacteria analyzed per experiment . * * * P < 0 . 001 , Fisher ' s exact test ( in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN - γ ) .

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

Fig 5(A-C) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA. Images show IFN-γ treated macrophages infected with GFP-Mtb (A-B) or DsRed-Mtb (C) for 24h. Cells were immunostained for ubiquitin (Ub) with the FK2 antibody (A) or p62 (B), which are shown in red. (C) GFP-LC3 BMDMs were infected with DsRed-Mtb. LC3 is shown in green, whereas Mtb are red. (A-C) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN-γ as indicated. Results are mean +/- SEM from two independent experiments, with at least 100 bacteria analyzed per experiment. ***P<0.001, Fisher's exact test (in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN-γ).


Fig 6 ( A ) WT and Park2 - / - BMDMs were pretreated with IFN - γ , infected with GFP - Mtb for 24h , and immunostained with the FK2 antibody . Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments . ( B ) Park2 - / - BMDMs pretreated or not treated with IFN - γ were infected with GFP - Mtb for 24h and immunostained with UBQLN1 specific antibody ; Arrows indicate Mtb co - localized with UBQLN1 . ns - not significant . ( C ) Park2 - / - BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection , treated with IFN - γ , and infected with GFP - Mtb for 24h prior to immunostaining for ubiquitin ( FK2 antibody ) . Ubiquitin ( FK2 + ) bacteria were quantified from two independent experiments . ( A , C ) * * P < 0 . 01 , Fisher ' s exact test ( comparing the proportion of FK2 positive bacteria in indicated samples ) . Results are mean + / - SEM . A minimum of 100 Mtb were counted in each experiment .

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Fig 6(A) WT and Park2-/- BMDMs were pretreated with IFN-γ, infected with GFP-Mtb for 24h, and immunostained with the FK2 antibody. Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments.(B) Park2-/- BMDMs pretreated or not treated with IFN-γ were infected with GFP-Mtb for 24h and immunostained with UBQLN1 specific antibody; Arrows indicate Mtb co-localized with UBQLN1. ns- not significant.(C) Park2-/- BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection, treated with IFN-γ, and infected with GFP-Mtb for 24h prior to immunostaining for ubiquitin (FK2 antibody). Ubiquitin (FK2+) bacteria were quantified from two independent experiments. (A, C) **P<0.01, Fisher's exact test (comparing the proportion of FK2 positive bacteria in indicated samples). Results are mean +/- SEM. A minimum of 100 Mtb were counted in each experiment.


Figure 1B . Vero E6 cells were transfected with 10 nM of siRNA before infection by SARS - CoV - 2 at a multiplicity of infection ( MOI ) of 0 . 1 after 24 h . The numbers of viral RNA copies were quantified with RT - qPCR . The negative control siRNA served as the control and is abbreviated as " Ctrl . " C1 - C11 represent the final candidate sequences after selection . The siRNAs capable of inhibiting up to 99 % of viral envelope gene expression with P values < 0 . 005 compared with Ctrl siRNA are marked with * . P value by Student t test . E . IC50 of C6 and the fully modified C6G25S . Vero E6 cells were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6 or C6G25S before virus infection at an MOI of 0 . 1 . The viral RNA was quantified by RT - qPCR at 24 h after infection . F . IC50 data for viral RdRp inhibition by C6G25S . Vero E6 cells were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6G25S before virus infection at an MOI of 0 . 1 . The viral RNA was quantified by RT - qPCR at 24 h after infection .

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Figure 1B. Vero E6 cells were transfected with 10 nM of siRNA before infection by SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 after 24 h. The numbers of viral RNA copies were quantified with RT-qPCR. The negative control siRNA served as the control and is abbreviated as "Ctrl." C1-C11 represent the final candidate sequences after selection. The siRNAs capable of inhibiting up to 99% of viral envelope gene expression with P values < 0.005 compared with Ctrl siRNA are marked with *. P value by Student t test.E. IC50 of C6 and the fully modified C6G25S. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6 or C6G25S before virus infection at an MOI of 0.1. The viral RNA was quantified by RT-qPCR at 24 h after infection. F. IC50 data for viral RdRp inhibition by C6G25S. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6G25S before virus infection at an MOI of 0.1. The viral RNA was quantified by RT-qPCR at 24 h after infection. D


Figure 2B . IC50 for C6G25S against different variants . Vero E6 cells were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6G25S before infection with different strains of SARS - CoV - 2 . The viral RNA was quantified by RT - qPCR at 24 h after infection . Data are presented as mean ± SD of three biological replicates .

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Figure 2B. IC50 for C6G25S against different variants. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6G25S before infection with different strains of SARS-CoV-2. The viral RNA was quantified by RT-qPCR at 24 h after infection. Data are presented as mean ± SD of three biological replicates.


Figure 3A , B , C . K18 - hACE2 - transgenic mice treated with C6G25S by 1 . 48 mg / L of AI for 30 min ( A ) , 50 μL of saline containing 50 μg of C6G25S by IN ( B ) , or PBS ( C ) ( n = 5 per group ) . C6G25S distribution in lungs was visualized by in situ hybridization ( ISH ) staining with C6G25S - specific probe ( red color ) . Bronchi ( i ) and bronchioles ( ii ) marked with the boxes are enlarged on the right . E , F . C57 / B6 mice ( n = 3 per group ) were treated by AI with C6G25S 1 . 48 mg / L for 30 min ( E ) or 50 μL of saline containing 50 μg of C6G25S by IN administration ( F ) . The C6G25S deposited in whole lungs and nasal cavities was quantified after whole tissue homogenization followed by stem - loop RT - qPCR . Quantification data represent mean ± SD . P value by Student t test .

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Figure 3A, B, C. K18-hACE2-transgenic mice treated with C6G25S by 1.48 mg/L of AI for 30 min (A), 50 μL of saline containing 50 μg of C6G25S by IN (B), or PBS (C) (n = 5 per group). C6G25S distribution in lungs was visualized by in situ hybridization (ISH) staining with C6G25S-specific probe (red color). Bronchi (i) and bronchioles (ii) marked with the boxes are enlarged on the right.E, F. C57/B6 mice (n = 3 per group) were treated by AI with C6G25S 1.48 mg/L for 30 min (E) or 50 μL of saline containing 50 μg of C6G25S by IN administration (F). The C6G25S deposited in whole lungs and nasal cavities was quantified after whole tissue homogenization followed by stem-loop RT-qPCR. Quantification data represent mean ± SD. P value by Student t test.


Figure 4A . K18 - hACE2 - transgenic mice ( Winkler et al . , 2020 ) were treated once daily for 3 days before intranasal challenge with 104 plaque - forming units ( PFU ) of the original virus . Prophylactic treatment consists of 30 min of AI ( 1 . 48 mg / l of C6G25S ) , followed by IN of 50 μg C6G25S . Mice receiving vehicle control ( saline ) for both AI and IN are annotated as control . Viral RNA ( left ) and infectious virions ( right ) in lungs were quantified with RT - qPCR and plaque forming assay , respectively , at 2 days post - infection ( dpi ) . B . Mice were challenged intranasally with 104 PFU of virus and co - treatment with 1 . 48 mg / L of C6G25S or vehicle control ( saline ) by AI for 30 min on day 0 ( right after infection ) and day 1 . Viral RNA and infectious virions were quantified at 2 dpi .

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Figure 4A. K18-hACE2-transgenic mice (Winkler et al., 2020) were treated once daily for 3 days before intranasal challenge with 104 plaque-forming units (PFU) of the original virus. Prophylactic treatment consists of 30 min of AI (1.48 mg/l of C6G25S), followed by IN of 50 μg C6G25S. Mice receiving vehicle control (saline) for both AI and IN are annotated as control. Viral RNA (left) and infectious virions (right) in lungs were quantified with RT-qPCR and plaque forming assay, respectively, at 2 days post-infection (dpi). B. Mice were challenged intranasally with 104 PFU of virus and co-treatment with 1.48 mg/L of C6G25S or vehicle control (saline) by AI for 30 min on day 0 (right after infection) and day 1. Viral RNA and infectious virions were quantified at 2 dpi. C


Figure 5A . Immunohistochemical ( IHC ) staining of viral spike proteins in lung sections from K18 - hACE2 mice ( Winkler et al . , 2020 ) at day 5 post - infection . Spike proteins were detected with anti - spike antibody and stained brown . Images of bronchial epithelium of vehicle control treated ( i and ii ) and C6G25S treated ( iii and iv ) , alveoli of vehicle control treated ( v and vi ) and C6G25S treated ( vii and viii ) , bronchiole and blood vessel of vehicle control treated ( ix and x ) and C6G25S treated ( xi and xii ) group were shown . Syncytial cell was indicated by green arrow in ( xi ) and thrombosis in ( x ) . B . ISH staining of viral RNA in lungs of vehicle control group ( left ) and C6G25S treated group ( right ) at day 2 post - infection . Viral RNA was stained brown as green arrows indicated . Images are representative of 5 animals for each group . C . Images of IHC staining of Ly6G + Cells ( Brown color , arrows indicated ) in lungs of vehicle control ( left ) and C6G25S treated group ( right ) at day 2 post - infection . D . Images of IHC staining of F4 / 80 + Cells ( Brown color ) in lungs of vehicle control ( left ) and C6G25S treated group ( right ) at day 2 post - infection . E . Images of IHC staining of CD3 + T Cells ( Brown color , arrows indicated ) in lungs of vehicle control ( left ) and C6G25S treated group ( right ) at day 2 post - infection .

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Figure 5A. Immunohistochemical (IHC) staining of viral spike proteins in lung sections from K18-hACE2 mice (Winkler et al., 2020) at day 5 post-infection. Spike proteins were detected with anti-spike antibody and stained brown. Images of bronchial epithelium of vehicle control treated (i and ii) and C6G25S treated (iii and iv), alveoli of vehicle control treated (v and vi) and C6G25S treated (vii and viii), bronchiole and blood vessel of vehicle control treated (ix and x) and C6G25S treated (xi and xii) group were shown. Syncytial cell was indicated by green arrow in (xi) and thrombosis in (x).B. ISH staining of viral RNA in lungs of vehicle control group (left) and C6G25S treated group (right) at day 2 post-infection. Viral RNA was stained brown as green arrows indicated. Images are representative of 5 animals for each group.C. Images of IHC staining of Ly6G+ Cells (Brown color, arrows indicated) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection.D. Images of IHC staining of F4/80+ Cells (Brown color) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection. E. Images of IHC staining of CD3 + T Cells (Brown color, arrows indicated) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection. F


Figure 1Representative WBs of RPE - 1 wild - type ( WT ) cells and the two selected KO clones for AKAP450 ( akap KO ) , PCNT ( pcnt KO ) and CDK5Rap2 ( c5rap2 KO ) probed with the indicated antibodies . GM130 was used as a loading controlConfocal IF images of RPE - 1 WT , single KO ( akap KO , pcnt KO and c5rap2 KO ) and double KO ( pc - ak - 2KO , left panels ; pc - c5 - 2KO , right panels ) cells stained with the indicated antibodies . The yellow dashed line indicates the nucleus contour ( N ) and the arrows point the centrosome ( CTR ) . GA marks the Golgi Apparatus . Scale bars : 5 μm . Representative WB of RPE - 1 WT cells and the two selected PCNT / AKAP450 ( pc - ak - 2KO ) and PCNT / CDK5Rap2 ( pc - c5 - 2KO ) double knockout mutated clones probed with the indicated antibodies . GM130 was used as a loading controlRepresentative WB of RPE - 1 WT cells and the indicated single or double KO cell lines probed with anti - AKAP450 , PCNT and CDK5Rap2 antibodies ( left ) . Quantification of signal intensity from three independent WB experiments is shown at right . # indicates KO proteins . The arrow points the dramatic decrease of CDK5Rap2 levels in pc - ak - 2KO cell line . Hsp70 was used as a loading

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Figure 1Representative WBs of RPE-1 wild-type (WT) cells and the two selected KO clones for AKAP450 (akap KO), PCNT (pcnt KO) and CDK5Rap2 (c5rap2 KO) probed with the indicated antibodies. GM130 was used as a loading controlConfocal IF images of RPE-1 WT, single KO (akap KO, pcnt KO and c5rap2 KO) and double KO (pc-ak-2KO, left panels; pc-c5-2KO, right panels) cells stained with the indicated antibodies. The yellow dashed line indicates the nucleus contour (N) and the arrows point the centrosome (CTR). GA marks the Golgi Apparatus. Scale bars: 5 μm.Representative WB of RPE-1 WT cells and the two selected PCNT/AKAP450 (pc-ak-2KO) and PCNT/CDK5Rap2 (pc-c5-2KO) double knockout mutated clones probed with the indicated antibodies. GM130 was used as a loading controlRepresentative WB of RPE-1 WT cells and the indicated single or double KO cell lines probed with anti-AKAP450, PCNT and CDK5Rap2 antibodies (left). Quantification of signal intensity from three independent WB experiments is shown at right. # indicates KO proteins. The arrow points the dramatic decrease of CDK5Rap2 levels in pc-ak-2KO cell line. Hsp70 was used as a loading control


Figure 2Confocal images of WT , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO and pc - c5 - 2KO cells stained for EB1 ( green ) and the centrosomal protein CAP350 ( red ) . High magnification single - channel images of selected areas are shown at right . Scatter plot showing quantification of EB1 comets from three independent experiments as that shown in A . A region of interest of 3 μm radius around the CAP350 signal was used to count the number of comets . Horizontal black lines represent the mean . MT regrowth experiments of WT cells mixed with either WT ( as a control ) , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO or pc - c5 - 2KO cells . Samples were fixed 3 min after NZ washout and stained for EB1 ( green ) and the indicated protein in the red channel . Quantification of EB1 signal intensity around the centrosomeWT and pc - ak - 2KO cells were transfected with a combination of three siRNAs against CEP192 , fixed and triple labelled for EB1 , CEP192 and CAP350 , as a centrosomal marker . Scramble siRNA was used as a control . Confocal images of WT , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO and pc - c5 - 2KO cells stained for EB1 ( green ) EB1 comets around the centrosomeThree min MT regrowth experiments in WT and pc - ak - 2KO cells transfected with either scramble or CEP192 siRNAs . Quantification of the EB1 signal intensity around the centrosomeMT regrowth experiments in WT and c5rap2 KO or pcnt KO cells . Cells were stained as indicated . Arrows point the centrosome . Quantification of EB1 signal intensity at Golgi membranes .

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Figure 2Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) and the centrosomal protein CAP350 (red). High magnification single-channel images of selected areas are shown at right. Scatter plot showing quantification of EB1 comets from three independent experiments as that shown in A. A region of interest of 3 μm radius around the CAP350 signal was used to count the number of comets. Horizontal black lines represent the mean.MT regrowth experiments of WT cells mixed with either WT (as a control), akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO or pc-c5-2KO cells. Samples were fixed 3 min after NZ washout and stained for EB1 (green) and the indicated protein in the red channel. Quantification of EB1 signal intensity around the centrosomeWT and pc-ak-2KO cells were transfected with a combination of three siRNAs against CEP192, fixed and triple labelled for EB1, CEP192 and CAP350, as a centrosomal marker. Scramble siRNA was used as a control.Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) EB1 comets around the centrosomeThree min MT regrowth experiments in WT and pc-ak-2KO cells transfected with either scramble or CEP192 siRNAs.Quantification of the EB1 signal intensity around the centrosomeMT regrowth experiments in WT and c5rap2 KO or pcnt KO cells. Cells were stained as indicated. Arrows point the centrosome.Quantification of EB1 signal intensity at Golgi membranes.


Figure 3WB of AKAP450 co - immunoprecipitation assays revealed with the indicated antibodies . Asterisks mark immunoglobulin heavy chains . Cntrl : non - specific Igs immunoprecipitates ; IP : specific Igs immunoprecipitates ; SP : supernatant . WB of AKAP450 and PCNT ( B ) co - immunoprecipitation assays revealed with the indicated antibodies . Asterisks mark immunoglobulin heavy chains . Cntrl : non - specific Igs immunoprecipitates ; IP : specific Igs immunoprecipitates ; SP : supernatant . Centrosomes from WT , single and double KO cells , as indicated , were labelled for AKAP450 and PCNT Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . Centrosomes from WT , single and double KO cells , as indicated , were labelled for AKAP450 and PCNT Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . Centrosomes from WT , single and double KO cells , as indicated , were labelled for AKAP450 and PCNT Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . Centrosomes from WT , single and double KO cells , as indicated , were labelled for CDK5Rap2 Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . Centrosomes from WT , single and double KO cells , as indicated , were labelled for γ - tubulin ( G ) . Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . WT and pc - ak - 2KO cells were transfected with either scramble or CEP192 siRNAs , fixed and stained for γ - tubulin . Quantification of the fluorescence intensity is show at right . IF images of WT and pcnt KO RPE - 1 cells treated with NZ for 3h and triple stained for AKAP450 ( green ) , CDK5Rap2 ( red ) and GMAP210 as a Golgi marker ( blue ) . At right , quantification of fluorescence co - localization of either AKAP450 or CDK5Rap2 with the Golgi marker GMAP210 in WT and pcnt KO cells .

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Figure 3WB of AKAP450 co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.WB of AKAP450 and PCNT (B) co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.Centrosomes from WT, single and double KO cells, as indicated, were labelled for CDK5Rap2 Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.Centrosomes from WT, single and double KO cells, as indicated, were labelled for γ-tubulin (G). Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.WT and pc-ak-2KO cells were transfected with either scramble or CEP192 siRNAs, fixed and stained for γ-tubulin. Quantification of the fluorescence intensity is show at right.IF images of WT and pcnt KO RPE-1 cells treated with NZ for 3h and triple stained for AKAP450 (green), CDK5Rap2 (red) and GMAP210 as a Golgi marker (blue). At right, quantification of fluorescence co-localization of either AKAP450 or CDK5Rap2 with the Golgi marker GMAP210 in WT and pcnt KO cells.


Figure 4Control and centrinone - treated cells were subjected to a 3 - min MT regrowth assay , fixed and labelled for EB1 ( green ) and giantin ( red ) . Insets show magnified images of boxed regions . Quantification of the total number of growing MTs per cell ( left ) , the number of MTs nucleated per Golgi fragment ( middle ) and the number of MT - nucleating Golgi fragments ( right ) in both control and centrosome - less RPE - 1 cells . Scatter plots show each individual data point . Horizontal black lines bars represent the mean . RPE - 1 cells were treated wi th centrinone for either 7 days ( left ) or for 5 days and then allowed to recover for 48h in the absence of the drug prior to fixation ( right ) . Representative images of a triple IF staining with EB1 ( green ) , γ - tubulin ( red ) and giantin ( blue ) as a Golgi marker are shown . Insets show MT nucleation from Golgi elements ( left ) or from multiple centrioles ( right ) and are enlarged at right . Arrowheads indicate the position of Golgi elements . Quantification of the percentage of cells exhibiting Golgi - nucleating activity in centrinone - treated cells 48h after the washout of the drug . Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63 . At least 100 cells from two independent experiments were analyzed in each condition .

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Figure 4Control and centrinone-treated cells were subjected to a 3-min MT regrowth assay, fixed and labelled for EB1 (green) and giantin (red). Insets show magnified images of boxed regions.Quantification of the total number of growing MTs per cell (left), the number of MTs nucleated per Golgi fragment (middle) and the number of MT-nucleating Golgi fragments (right) in both control and centrosome-less RPE-1 cells. Scatter plots show each individual data point. Horizontal black lines bars represent the mean.RPE-1 cells were treated with centrinone for either 7 days (left) or for 5 days and then allowed to recover for 48h in the absence of the drug prior to fixation (right). Representative images of a triple IF staining with EB1 (green), γ-tubulin (red) and giantin (blue) as a Golgi marker are shown. Insets show MT nucleation from Golgi elements (left) or from multiple centrioles (right) and are enlarged at right. Arrowheads indicate the position of Golgi elements.Quantification of the percentage of cells exhibiting Golgi-nucleating activity in centrinone-treated cells 48h after the washout of the drug. Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63. At least 100 cells from two independent experiments were analyzed in each condition.


Figure 5Control ( top ) and centrinone - treated ( bottom ) RPE - 1 cells double stained for either AKAP450 , CDK5Rap2 , PCNT or CEP192 ( all shown in green ) and giantin ( red ) as a Golgi marker . DNA was counterstained with DAPI and is shown in blue . Single channel ( left , green ) and merged images ( right ) are shown in each case . White arrows indicate the centrosome ( CTR ) . IF images of control and centrinone - treated RPE - 1 cells treated with NZ to induce GA fragmentation and double stained with either anti - AKAP450 , anti - CDK5Rap2 or anti - PCNT ( all shown in green ) and anti - giantin antibodies ( white ) . Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker . The yellow dashed line indicates the contour of the nucleus . Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces . Box - and - whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles , and whiskers represent 90th and 10th percentiles . The median is depicted with a solid line . Individual Golgi elements from at least 14 cells were delineated ( > 500 elements / experiment ) and the intensity of each protein spot associated with each Golgi element was measured . Data were collected from two independent experiments and normalized to WT mean . MT - regrowth experiment after NZ treatment in centrinone - treated cells . High magnification images at the bottom are representative of a triple staining with α - tubulin ( green ) , GM130 ( blue ) and the indicated protein ( red ) and show MTs growing from the surface of the GA . Double labelling without α - tubulin are also shown ( top ) to allow better visualization of PCM - protein spots on Golgi elements acting as MT nucleation sites ( arrowheads ) . Centrinone - treated pcnt KO , c5rap2 KO and akap KO cells , were incubated with NZ for 3h , fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated ( n = 3 ) . MT regrowth assay in centrinone - treated WT , pcnt KO and akap KO RPE - 1 cells stained with antibodies to EB1 and giantin . At right , quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone , as a measure of MT - nucleation from the GA . Data were collected from two independent experiments and normalized to WT mean .

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

Figure 5Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker. DNA was counterstained with DAPI and is shown in blue. Single channel (left, green) and merged images (right) are shown in each case. White arrows indicate the centrosome (CTR).IF images of control and centrinone-treated RPE-1 cells treated with NZ to induce GA fragmentation and double stained with either anti-AKAP450, anti-CDK5Rap2 or anti-PCNT (all shown in green) and anti-giantin antibodies (white). Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker. The yellow dashed line indicates the contour of the nucleus. Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces.Box-and-whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Individual Golgi elements from at least 14 cells were delineated (>500 elements/experiment) and the intensity of each protein spot associated with each Golgi element was measured. Data were collected from two independent experiments and normalized to WT mean.MT-regrowth experiment after NZ treatment in centrinone-treated cells. High magnification images at the bottom are representative of a triple staining with α-tubulin (green), GM130 (blue) and the indicated protein (red) and show MTs growing from the surface of the GA. Double labelling without α-tubulin are also shown (top) to allow better visualization of PCM-protein spots on Golgi elements acting as MT nucleation sites (arrowheads).Centrinone-treated pcnt KO, c5rap2 KO and akap KO cells, were incubated with NZ for 3h, fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated (n=3).MT regrowth assay in centrinone-treated WT, pcnt KO and akap KO RPE-1 cells stained with antibodies to EB1 and giantin. At right, quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone, as a measure of MT-nucleation from the GA. Data were collected from two independent experiments and normalized to WT mean.


Figure 6Representative MT network of a centrosome - less akap KO cell . MT - regrowth experiment after NZ treatment in centrinone - treated akap KO cells . A representative confocal image of a double labelling for EB1 ( green ) and giantin ( blue ) is shown . High magnification at right shows that growing MT asters are not associated to Golgi fragments . Centrinone - treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies . High magnification images of the boxed areas are shown as merge ( top ) or individual green or red labelling ( middle and bottom panels ) . Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 ( top panels ) or PCNT ( bottom panels ) . After 3 - min MT regrowth , cells were fixed and stained with the indicated antibodies . MT regrowth experiments in centrinone - treated akap KO cells transfected with either scramble or PCNT siRNAs . After a 3h NZ treatment , MTs were allowed to polymerize and , at the indicated time points after washout , cells were fixed and labelled for α - tubulin ( green ) , PCNT ( red ) and CAP350 ( blue ) . MT repolymerization experiments in centrinone - treated akap KO cells transfected with either scramble or PCNT siRNAs . After NZ treatment and removal , cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin . Cells were finally fixed and triple stained with EB1 ( green ) , PCNT ( red ) and CAP350 ( blue ) antibodies . Enlarged views of the boxed areas are presented at right . Scale bars , 5 μm .

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

Figure 6Representative MT network of a centrosome-less akap KO cell.MT-regrowth experiment after NZ treatment in centrinone-treated akap KO cells. A representative confocal image of a double labelling for EB1 (green) and giantin (blue) is shown. High magnification at right shows that growing MT asters are not associated to Golgi fragments.Centrinone-treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies. High magnification images of the boxed areas are shown as merge (top) or individual green or red labelling (middle and bottom panels).Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 (top panels) or PCNT (bottom panels). After 3-min MT regrowth, cells were fixed and stained with the indicated antibodies.MT regrowth experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After a 3h NZ treatment, MTs were allowed to polymerize and, at the indicated time points after washout, cells were fixed and labelled for α-tubulin (green), PCNT (red) and CAP350 (blue).MT repolymerization experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After NZ treatment and removal, cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin. Cells were finally fixed and triple stained with EB1 (green), PCNT (red) and CAP350 (blue) antibodies. Enlarged views of the boxed areas are presented at right. Scale bars, 5 μm.


Figure 7Confocal images showing MT network in WT , akap KO , c5rap2 KO , pcnt KO and pc - ak - 2KO RPE - 1 cells fixed and stained with antibodies against α - tubulin ( left ) or EB1 ( right ) . The red and yellow lines indicate the contour of cells . Confocal images showing MT network in centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO backgrounds . Cells containing extra centrosomes induced by centrinone washout were also quantified ( bottom panels ) . Quantification of α - tubulin fluorescence intensity ( as a measure of MT mass polymer , C ) , in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean . Quantification of number of EB1 comets per cell ( D ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean . Quantification of α , cell area ( E ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean . Quantification of MT density ( MT mass polymer / cell area ) ( F ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean .

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

Figure 7Confocal images showing MT network in WT, akap KO, c5rap2 KO, pcnt KO and pc-ak-2KO RPE-1 cells fixed and stained with antibodies against α-tubulin (left) or EB1 (right). The red and yellow lines indicate the contour of cells.Confocal images showing MT network in centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO backgrounds. Cells containing extra centrosomes induced by centrinone washout were also quantified (bottom panels).Quantification of α-tubulin fluorescence intensity (as a measure of MT mass polymer, C), in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.Quantification of number of EB1 comets per cell (D) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.Quantification of α , cell area (E) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.Quantification of MT density (MT mass polymer/cell area) (F) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


Figure 1A Bisulfite DNA methylation sequencing of the Athila6ATE transcriptional start site ( TSS ) in genotypes with silenced TEs ( left of the dashed line ) or transcriptionally active TEs ( right of the dashed line ) . Methylation in the CG ( red ) , CHG ( blue ) and CHH ( green ) sequence contexts are shown ( H = C , T , or A ) . Error bars represent Wilson score interval 95 % confidence limits . For multiple statistical tests , one ‐ way ANOVA was performed using Tukey ' s multiple comparison test . ns , not significant . B The reads ‐ per ‐ million ‐ ( RPM ) ‐ normalized accumulation of Athila6ATE siRNAs from TE ‐ silenced epigenomes ( left ) and ddm1TE ‐ active epigenomes ( right ) . Inset line graphs reflecting the same values are shown for select genotypes . All line graphs have the same y ‐ axis scale and are shown for increased resolution in genotypes with low siRNA levels .

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

Figure 1A Bisulfite DNA methylation sequencing of the Athila6ATE transcriptional start site (TSS) in genotypes with silenced TEs (left of the dashed line) or transcriptionally active TEs (right of the dashed line). Methylation in the CG (red), CHG (blue) and CHH (green) sequence contexts are shown (H = C, T, or A). Error bars represent Wilson score interval 95% confidence limits. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test. ns, not significant.B The reads‐per‐million‐(RPM)‐normalized accumulation of Athila6ATE siRNAs from TE‐silenced epigenomes (left) and ddm1TE‐active epigenomes (right). Inset line graphs reflecting the same values are shown for select genotypes. All line graphs have the same y‐axis scale and are shown for increased resolution in genotypes with low siRNA levels.


Figure 2AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . A A representative microRNA , miR158B . The location of miR158B from this locus is shown in orange . AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . B The SimpleHat2 non ‐ autonomous TE targeted by Pol IV ‐ RdDM . C The RDR6 ‐ RdDM target locus TAS3a . AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . D The Athila6A consensus element in the wt Col epigenome . E The same Athila6A consensus element as in ( D ) upon transcriptional activation in the ddm1 epigenome . AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . F The total small RNAs produced in the wt Col TE ‐ silenced epigenome . G The total small RNAs in ddm1 mutants . AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . H Bisulfite sequencing of different regions of the Athila6A element . Error bars represent Wilson score interval 95 % confidence limits . Differences in the methylation status were analyzed with a two ‐ tailed unpaired Student ' s t ‐ test . AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . I A second biological replicate of the FLAG ‐ AGO6 ‐ IP and no ‐ antigen control followed by deep sequencing shown for the Athila6A consensus element in ddm1 mutants . The transcriptional start sites of the LTR TSS ‐ and IR ‐ driven transcripts are shown . AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . J Analysis of the first base of AGO6 ‐ enriched siRNAs from the wt Col TE ‐ silenced epigenome ( left ) and the ddm1 TE ‐ transcriptionally active epigenome ( right ) .

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

Figure 2AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.A A representative microRNA, miR158B. The location of miR158B from this locus is shown in orange.AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.B The SimpleHat2 non‐autonomous TE targeted by Pol IV‐RdDM.C The RDR6‐RdDM target locus TAS3a.AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.D The Athila6A consensus element in the wt Col epigenome.E The same Athila6A consensus element as in (D) upon transcriptional activation in the ddm1 epigenome.AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.F The total small RNAs produced in the wt Col TE‐silenced epigenome.G The total small RNAs in ddm1 mutants.AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.H Bisulfite sequencing of different regions of the Athila6A element. Error bars represent Wilson score interval 95% confidence limits. Differences in the methylation status were analyzed with a two‐tailed unpaired Student's t‐test.AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.I A second biological replicate of the FLAG‐AGO6‐IP and no‐antigen control followed by deep sequencing shown for the Athila6A consensus element in ddm1 mutants. The transcriptional start sites of the LTR TSS‐ and IR‐driven transcripts are shown.AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.J Analysis of the first base of AGO6‐enriched siRNAs from the wt Col TE‐silenced epigenome (left) and the ddm1 TE‐transcriptionally active epigenome (right).