( a ) Yolk granules flow in stereotyped patterns as epiboly progresses . PIV of time - lapse snapshots imaged by two - photon microscopy of a Tg ( β - actin : m - GFP ) embryo ( from Movie EV2 ) . Upon epiboly onset , yolk granules flow towards the animal pole displaying two internal vortices near the E - YSL . At 50 % , the movement of the yolk granules halts , but resumes afterwards until the end of epiboly . Animal ( A ) and vegetal ( V ) poles are indicated . Scale bar 100 μm . ( b ) Internal yolk content speed ( μm / min ) at the center of the embryo , over time ( min ) . Inset : Spherical coordinates . The main axis of the sphere is represented by z , from the animal to the vegetal pole . The polar angle is represented by φ and the radial coordinate is denoted ρ . ( c ) Vortex midpoints distance from the center ( ρ ) , over time ( min ) . ( d ) Vortex midpoints position in relation to the φ axis , over time ( min ) . Black lines represent individual measurements . Red lines represent averages . ( e ) Correlation between vortex displacement ( blue , average of n = 3 embryos ) vs . EVL margin displacement ( red , average of n = 4 embryos ) along the main axis ( z ) , over time ( min ) . Standard Errors are displayed . Arrow points to 50 % epiboly .

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(a) Yolk granules flow in stereotyped patterns as epiboly progresses. PIV of time-lapse snapshots imaged by two-photon microscopy of a Tg (β-actin:m-GFP) embryo (from Movie EV2). Upon epiboly onset, yolk granules flow towards the animal pole displaying two internal vortices near the E-YSL. At 50 %, the movement of the yolk granules halts, but resumes afterwards until the end of epiboly. Animal (A) and vegetal (V) poles are indicated. Scale bar 100 μm. (b) Internal yolk content speed (μm/min) at the center of the embryo, over time (min). Inset: Spherical coordinates. The main axis of the sphere is represented by z, from the animal to the vegetal pole. The polar angle is represented by φ and the radial coordinate is denoted ρ. (c) Vortex midpoints distance from the center (ρ), over time (min). (d) Vortex midpoints position in relation to the φ axis, over time (min). Black lines represent individual measurements. Red lines represent averages. (e) Correlation between vortex displacement (blue, average of n=3 embryos) vs. EVL margin displacement (red, average of n = 4 embryos) along the main axis (z), over time (min). Standard Errors are displayed. Arrow points to 50 % epiboly.


( a ) Yolk microrheology . Mean squared displacement ( MSD ) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafishembryos . Solid and dashed black lines are Mean ± Standard Error computed from 99 particles . Red line is the fit of the Stokes - Einstein equation . MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag , Δt , consistent with a Newtonian liquid behavior with a viscosity of 129 mPa · s . A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila ( He et al . , 2014 ) . Interestingly , the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C . elegansembryos ( Daniels et al . , 2006 ) and of the cytoplasm of several cell types ( Wirtz , 2009 ) .

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(a) Yolk microrheology. Mean squared displacement (MSD) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafishembryos. Solid and dashed black lines are Mean ± Standard Error computed from 99 particles. Red line is the fit of the Stokes-Einstein equation. MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag, Δt, consistent with a Newtonian liquid behavior with a viscosity of 129 mPa·s. A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila (He et al., 2014). Interestingly, the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C. elegansembryos (Daniels et al., 2006) and of the cytoplasm of several cell types (Wirtz, 2009).


( b ) Viscoelasticity of the yolk cortex . Effective complex modulus ( g * ) measured by indenting the surface of zebrafishembryos with AFM . Red symbols are the elastic modulus ( g ' ) and yellow symbols are the viscous modulus ( g ' ' ) , respectively . Standard Errors are displayed . In all the measured frequencies cortex rheology is dominated by a solid - like behavior with the viscous modulus 5 times lower that the elastic modulus . No significant frequency dependence was found in the elastic ( ANOVA , p = 0 . 123 ) and viscous modulus ( ANOVA , p = 0 . 719 ) .

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(b) Viscoelasticity of the yolk cortex. Effective complex modulus (g*) measured by indenting the surface of zebrafishembryos with AFM. Red symbols are the elastic modulus (g') and yellow symbols are the viscous modulus (g''), respectively. Standard Errors are displayed. In all the measured frequencies cortex rheology is dominated by a solid-like behavior with the viscous modulus 5 times lower that the elastic modulus. No significant frequency dependence was found in the elastic (ANOVA, p = 0.123) and viscous modulus (ANOVA, p = 0.719).


( c ) Flow lines from snapshots of the PIV measurements ( at 40 , 60 and 80 % epiboly ) of a two - photon video time - lapse ( from Movie EV4 ) of a Tg ( β - actin : m - GFP ) embryo ( top ) . Fitted flow lines using HR ar shown at the bottom . The relative mean square errors ( RMSE ) of the fitting are displayed as a percentage at the bottom left of each panel . Scale bar 100 μm .

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(c) Flow lines from snapshots of the PIV measurements (at 40, 60 and 80 % epiboly) of a two-photon video time-lapse (from Movie EV4) of a Tg (β-actin:m-GFP) embryo (top). Fitted flow lines using HR ar shown at the bottom. The relative mean square errors (RMSE) of the fitting are displayed as a percentage at the bottom left of each panel. Scale bar 100 μm.


( d ) Mechanical power maps over time obtained by HR of the same embryos as in ( c ) ( from Movie EV5 ) . The RMSE of the power is shown as a percentage at the bottom left of each panel . The mechanical work to produce doming ( 40 % epiboly ) arises mainly at the blastoderm ( left ) ; at 60 % epiboly ( middle ) DCs undergo ingression ( red ) while the E - YSL resists constriction ( blue ) . At 80 % epiboly ( right ) , the active work leading to the active movement of the EVL / yolk margin mainly maps at the E - YSL ( red ) while the blastoderm undergoes elastic resistance ( blue ) . White arrows point to the main observed events . Scale bar 100 μm .

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(d) Mechanical power maps over time obtained by HR of the same embryos as in (c) (from Movie EV5). The RMSE of the power is shown as a percentage at the bottom left of each panel. The mechanical work to produce doming (40 % epiboly) arises mainly at the blastoderm (left); at 60 % epiboly (middle) DCs undergo ingression (red) while the E-YSL resists constriction (blue). At 80 % epiboly (right), the active work leading to the active movement of the EVL / yolk margin mainly maps at the E-YSL (red) while the blastoderm undergoes elastic resistance (blue). White arrows point to the main observed events. Scale bar 100 μm.


( e ) AV ( red ) and CC ( green ) stresses and their differences ( blue ) normalized values in arbitrary units from the regression analysis ( Movie EV6 ) were plotted as a function of the φ angle ( 0 - 1 ) along the cortex from animal to vegetal . The equator - dotted yellow line - , yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed ( 40 % ; 60 % ; 80 % epiboly ) . The CC stress steeps up from animal to vegetal and the AV shows a more complex profile . Their difference changes senses around the EVL / yolk margin .

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(e) AV (red) and CC (green) stresses and their differences (blue) normalized values in arbitrary units from the regression analysis (Movie EV6) were plotted as a function of the φ angle (0-1) along the cortex from animal to vegetal. The equator - dotted yellow line -, yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed (40 %; 60 %; 80 % epiboly). The CC stress steeps up from animal to vegetal and the AV shows a more complex profile. Their difference changes senses around the EVL / yolk margin.


( a ) Correlation between the differential tension ( cyan ) represented by the recoil velocities differences ( m / s ) between CC and AV laser cuts and their difference ( blue ) calculated by regression analysis at the E - YSL during epiboly ( % ) . The difference values in normalized relative units were fitted applying a constant adjustment ratio at all time points .

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(a) Correlation between the differential tension (cyan) represented by the recoil velocities differences (m/s) between CC and AV laser cuts and their difference (blue) calculated by regression analysis at the E-YSL during epiboly (%). The difference values in normalized relative units were fitted applying a constant adjustment ratio at all time points.


( b ) AV tension ( averaged recoil velocity in m / s ) in the yolk cortex at different distances from the EVL ( 0 , 10 , 20 , 40 and 60 m ) and at different epiboly stages ( 50 % , blue ; 60 - 70 % , red ; 80 % epiboly , green ) . Significance changes are highlighted and Standard Errors are displayed .

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(b) AV tension (averaged recoil velocity in m/s) in the yolk cortex at different distances from the EVL (0, 10, 20, 40 and 60 m) and at different epiboly stages (50 %, blue; 60-70 %, red; 80 % epiboly, green). Significance changes are highlighted and Standard Errors are displayed.


( c ) Probing mechanical parameters of living embryos with AFM . Force - indentation curves recorded at cantilever velocity of 10 m / s ( blue ) fitted with a " liquid balloon " model ( red ) . Inset : AFM force - indentation description .

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(c) Probing mechanical parameters of living embryos with AFM. Force-indentation curves recorded at cantilever velocity of 10 m/s (blue) fitted with a "liquid balloon" model (red). Inset: AFM force-indentation description.


( d ) Averaged values of mean surface tension ( pN / μm ) on the yolk obtained with AFM at different epiboly stages ( left ) , and the significant difference of tension at 60 - 70 % epiboly ( p = 0 . 0245 ) between the E - YSL and the vegetal pole ( right ) . Standard Errors are displayed .

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(d) Averaged values of mean surface tension (pN / μm) on the yolk obtained with AFM at different epiboly stages (left), and the significant difference of tension at 60-70 % epiboly (p = 0.0245) between the E-YSL and the vegetal pole (right). Standard Errors are displayed.


( a ) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut ( red line time point ) in the E - YSL of a Myosin - GFP transgenic embryo [ Tg ( β - actin : myl 12 . 1 - e - GFP ) ] . After a rapid opening , an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched ( elastic ) than the vegetal yolk cellmembrane / cortex . When the gap is closed , the cut center returns to its initial position and the elastic equilibrium is restored . Scale bar 10 μm .

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(a) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut (red line time point) in the E-YSL of a Myosin-GFP transgenic embryo [Tg (β-actin:myl 12.1-e-GFP)]. After a rapid opening, an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched (elastic) than the vegetal yolk cellmembrane/cortex. When the gap is closed, the cut center returns to its initial position and the elastic equilibrium is restored. Scale bar 10 μm.


( d ) Dynamic simulation of AV and CC stresses normalized values in arbitrary units ( from Movie EV9 - 40 % ; 65 % ; 80 % epiboly ) represented as in Figure 1a . The AV and CC stresses are equal at the poles , as the epiboly simulation progresses , the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin .

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(d) Dynamic simulation of AV and CC stresses normalized values in arbitrary units (from Movie EV9 - 40 %; 65 %; 80 % epiboly) represented as in Figure 1a. The AV and CC stresses are equal at the poles, as the epiboly simulation progresses, the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin.


( a ) Speed of the EVL advancing front at different epiboly stages ( φ angle ) for two distinct simulated conditions : 1 ) a difference of elastic tension between the EVL and the yolk associated to a two - phase reduction of the contractile E - YSL width ( from 20º to 5º ) as epiboly advances ( from 50º to 155º ) ( wild type - blue ) . The contractile E - YSL width decreases at a slower rate until 80º and shrinks afterwards at a rate 2 . 25 times faster , and 2 ) Same assumptions as in 1 but with no shrinkage of the E - YSL ( constant 20º ) ( contractileless - red ) .

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(a) Speed of the EVL advancing front at different epiboly stages (φ angle) for two distinct simulated conditions: 1) a difference of elastic tension between the EVL and the yolk associated to a two-phase reduction of the contractile E-YSL width (from 20º to 5º) as epiboly advances (from 50º to 155º) (wild type - blue). The contractile E-YSL width decreases at a slower rate until 80º and shrinks afterwards at a rate 2.25 times faster, and 2) Same assumptions as in 1 but with no shrinkage of the E-YSL (constant 20º) (contractileless - red).


( b ) Experimental values for the EVL advancing front speeds at different epiboly stages ( φ angle - top ) and at different times ( bottom ) for wild type controls ( blue ) and Msn1yolk morphants with reduced E - YSL contractility ( red ) . Experimental profiles closely approach to those inferred in the simulations ( compare to ( a ) ) .

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(b) Experimental values for the EVL advancing front speeds at different epiboly stages (φ angle - top) and at different times (bottom) for wild type controls (blue) and Msn1yolk morphants with reduced E-YSL contractility (red). Experimental profiles closely approach to those inferred in the simulations (compare to (a)).


( c ) Timeline showing medial sections and power isolines for wild type ( top ) and Msn1yolk morphants ( bottom ) along epiboly . Mechanical parameters were inferred by HR from experimental velocity fields ( Movie EV10 ) . The RMSE of power is shown as a percentage at each time point . DCs in the Msn1yolk morphants undergo ingression on schedule at 50 % epiboly ( yellow arrow ) . Importantly , as in wild type controls , the active work in morphants is detected at the E - YSL ( red arrow ) . This power supply fades away as epiboly slows down and eventually fails . Scale bar 100 μm .

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(c) Timeline showing medial sections and power isolines for wild type (top) and Msn1yolk morphants (bottom) along epiboly. Mechanical parameters were inferred by HR from experimental velocity fields (Movie EV10). The RMSE of power is shown as a percentage at each time point. DCs in the Msn1yolk morphants undergo ingression on schedule at 50 % epiboly (yellow arrow). Importantly, as in wild type controls, the active work in morphants is detected at the E-YSL (red arrow). This power supply fades away as epiboly slows down and eventually fails. Scale bar 100 μm.


( d ) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly and its temporal equivalent ( 9 hours after egg laying ) for Msn1yolk morphants . The characteristic profiles of longitudinal and latitudinal stresses and the animal to vegetal stress gradient that are built up during epiboly are never properly developed in morphants .

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(d) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly and its temporal equivalent (9 hours after egg laying) for Msn1yolk morphants. The characteristic profiles of longitudinal and latitudinal stresses and the animal to vegetal stress gradient that are built up during epiboly are never properly developed in morphants.


D - E ) Immunofluorescence against CITRINE ( electroporated cells ) , SOX10 ( Schwann cell precursors and Schwann cells ) and ISL1 ( sensory and sympathetic neurons ) on embryos electroporated with either control CRISPR plasmid ( D ) or a CRISPR plasmid containing a guide - RNA against Sox8 ( E ) . CITRINE + cells populate the developing dorsal root ganglia ( DRG ) and peripheral nerves . Examples of CITRINE + / SOX10 + cells shown by arrowheads . Asterisks show ventral boundary cap glia . Scale bar is 50 µm in overviews and 10 µm in insets . F ) Quantification of the fate distribution of CITRINE + cells as a % between glial ( SOX10 + ) cells , sensory neurons ( ISL1 + ) or neither ( SOX10 - / ISL1 - ) in the DRG of control and Sox8KD chick embryos . Biological replicates - N = 3 embryos per condition ( wild type versus Sox8 KD ) . Data represented as mean ± SEM . Statistical significance determined using the Holm - Sidak method ( alpha = 0 . 05 ) ( multiple t - tests , unpaired ) . SOX10 + cells : p = 0 . 7901 , ISL1 + cells : p = 0 . 9206 , SOX10 - / ISL1 - cells : p = 0 . 9206 . For statistical significance : non - significant - p - value ≥ 0 . 05 . G ) Quantification of ( left ) the % of SOX10 + cells in the peripheral nerves that are CITRINE + in control and Sox8KD chick embryos and ( right ) the % of PH3 + CITRINE + cells corresponding to proliferative cells . Biological replicates - N = 4 embryos per condition for SOX10 + distribution and N = 2 for PH3 quantification ( wild type versus Sox8 KD ) . Data represented as mean ± SEM . Statistical significance determined unpaired t - test with two - tailed p value . SOX10 + cells : p = 0 . 0044 , PH3 + cells : p = 0 . 4929 . For statistical significance : non - significant - p - value ≥ 0 . 05 , * * - p - value < 0 . 01 . Data information : In total , 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean ± SEM per embryo ( corresponding to biological replicates ) : 3 - 4 electroporated embryos were analyzed per condition ( control and SOX8 knock down ) with 4 - 5 sections stained and analyzed per embryo per region of interest ( DRG , DRG : dorsal root ganglia , sg : sympathetic

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D-E) Immunofluorescence against CITRINE (electroporated cells), SOX10 (Schwann cell precursors and Schwann cells) and ISL1 (sensory and sympathetic neurons) on embryos electroporated with either control CRISPR plasmid (D) or a CRISPR plasmid containing a guide-RNA against Sox8 (E). CITRINE+ cells populate the developing dorsal root ganglia (DRG) and peripheral nerves. Examples of CITRINE+/SOX10+ cells shown by arrowheads. Asterisks show ventral boundary cap glia. Scale bar is 50 µm in overviews and 10 µm in insets. F) Quantification of the fate distribution of CITRINE+ cells as a % between glial (SOX10+) cells, sensory neurons (ISL1+) or neither (SOX10-/ISL1-) in the DRG of control and Sox8KD chick embryos. Biological replicates - N=3 embryos per condition (wild type versus Sox8 KD). Data represented as mean±SEM. Statistical significance determined using the Holm-Sidak method (alpha = 0.05) (multiple t-tests, unpaired). SOX10+ cells: p=0.7901, ISL1+ cells: p=0.9206, SOX10-/ISL1- cells: p=0.9206. For statistical significance: non-significant - p-value≥0.05. G) Quantification of (left) the % of SOX10+ cells in the peripheral nerves that are CITRINE+ in control and Sox8KD chick embryos and (right) the % of PH3+ CITRINE+ cells corresponding to proliferative cells. Biological replicates - N=4 embryos per condition for SOX10+ distribution and N=2 for PH3 quantification (wild type versus Sox8 KD). Data represented as mean±SEM. Statistical significance determined unpaired t-test with two-tailed p value. SOX10+ cells: p=0.0044, PH3+ cells: p=0.4929. For statistical significance: non-significant - p-value≥0.05, ** - p-value<0.01. Data information: In total, 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean±SEM per embryo (corresponding to biological replicates): 3-4 electroporated embryos were analyzed per condition (control and SOX8 knock down) with 4-5 sections stained and analyzed per embryo per region of interest (DRG, DRG: dorsal root ganglia, sg: sympathetic ganglion


I ) Immunofluorescence against CITRINE ( electroporated cells ) , SOX10 ( Schwann cell precursors and Schwann cells ) and TH ( sympathetic neurons and chromaffin cells ) of the sympathoadrenal domain on control and Sox8KD embryos . CITRINE + / SOX10 + cells shown by empty arrowheads while CITRINE + / TH + cells are shown by filled arrowheads . Scale bar = 50 µm . J ) Quantification of the fate distribution of CITRINE + cells as a % between glial ( SOX10 + ) cells , chromaffin cells ( TH + ) or neither ( SOX10 - / TH - ) in the proximity of the dorsal aorta and visceral nerve of wild type and Sox8 KD chick embryos . Biological replicates - N = 4 embryos per condition . Data represented as mean ± SEM . Statistical significance determined using the Holm - Sidak method ( alpha = 0 . 05 ) ( multiple t - tests , unpaired ) . SOX10 + cells : p = 0 . 0067 , TH + cells : p = 0 . 0067 , SOX10 - / TH - cells : p = 0 . 8819 . For statistical significance : non - significant - p - value ≥ 0 . 05 , * - p - value < 0 . 05 . Data information : In total , 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean ± SEM per embryo ( corresponding to biological replicates ) : 3 - 4 electroporated embryos were analyzed per condition ( control and SOX8 knock down ) with 4 - 5 sections stained and analyzed per embryo per region of interest Sympathoadrenal domain ) .

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I) Immunofluorescence against CITRINE (electroporated cells), SOX10 (Schwann cell precursors and Schwann cells) and TH (sympathetic neurons and chromaffin cells) of the sympathoadrenal domain on control and Sox8KD embryos. CITRINE+/SOX10+ cells shown by empty arrowheads while CITRINE+/TH+ cells are shown by filled arrowheads. Scale bar = 50 µm. J) Quantification of the fate distribution of CITRINE+ cells as a % between glial (SOX10+) cells, chromaffin cells (TH+) or neither (SOX10-/TH-) in the proximity of the dorsal aorta and visceral nerve of wild type and Sox8 KD chick embryos. Biological replicates - N=4 embryos per condition. Data represented as mean±SEM. Statistical significance determined using the Holm-Sidak method (alpha = 0.05) (multiple t-tests, unpaired). SOX10+ cells: p=0.0067, TH+ cells: p=0.0067, SOX10-/TH- cells: p=0.8819. For statistical significance: non-significant - p-value≥0.05, * - p-value<0.05. Data information: In total, 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean±SEM per embryo (corresponding to biological replicates): 3-4 electroporated embryos were analyzed per condition (control and SOX8 knock down) with 4-5 sections stained and analyzed per embryo per region of interest Sympathoadrenal domain).


RNA - Seq analysis of : different time points of primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected Expression levels were normalized as Log2 fold change in infected vs matched mock - infected controls For ( A ) data were analyzed by Fisher test ( number of donors = 3 biological replicates ) . Boxplots in ( A ) depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . For each time point , dots illustrate the pathway enrichment analysis of genes up - regulated in infected vs matched mock - infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of differentially expressed genes ( DEGs ) in the pathway ; grey dots are not statistically

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RNA-Seq analysis of: different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected Expression levels were normalized as Log2 fold change in infected vs matched mock-infected controls For (A) data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in (A) depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes (DEGs) in the pathway; grey dots are not statistically significant


RNA - Seq analysis of PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART Expression levels were normalized as Log2 fold change or as enrichment score in ART - treated vs ART - naïve animals ( B ) . ( B ) data were analyzed by Gene Set Enrichment Analysis ( GSEA , number of animals = 8 ) . Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART - naïve and ART - treated time points . Red color indicates leading edge genes . All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress ( 184 genes ; GO : 0034599 ) .

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RNA-Seq analysis of PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART Expression levels were normalized as Log2 fold change or as enrichment score in ART-treated vs ART-naïve animals (B). (B) data were analyzed by Gene Set Enrichment Analysis (GSEA, number of animals =8). Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART-naïve and ART-treated time points. Red color indicates leading edge genes. All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress (184 genes; GO:0034599).


( A ) Total to oxidized glutathione ratio ( GSH / GSSG ) in mock infected or HIV - 1 infected CD4 + T - cells ( 7 dpi , n = 4 biological replicates ; mean ± SEM ) .

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(A) Total to oxidized glutathione ratio (GSH/GSSG) in mock infected or HIV-1 infected CD4+ T-cells (7 dpi, n=4 biological replicates; mean±SEM).


representative picture ( B ) of ROS content in HIV - 1 infected or mock infected primary CD4 + T - cells at 7dpi ( productive infection ) and 14 dpi ( latent infection ) . ROS content was measured by immunofluorescence , HIV - 1 DNA + cells were identified by

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representative picture (B) of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH


quantification of ROS content in HIV - 1 infected or mock infected primary CD4 + T - cells at 7dpi ( productive infection ) and 14 dpi ( latent infection ) . ROS content was measured by immunofluorescence , HIV - 1 DNA + cells were identified by FISH ( D ) . The black dash in ( C ) indicates the median . Box plots in ( D ) depict median and 25 - 75 percentile , while whiskers extend from min to max . n = number of cells . Scale bar = 2μm .

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quantification of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH (D). The black dash in (C) indicates the median. Box plots in (D) depict median and 25-75 percentile, while whiskers extend from min to max. n= number of cells. Scale bar = 2μm.


( E ) Subcellular localization of Nrf2 in CD4 + T - cells infected with HIV - 1 or mock infected ( 3dpi ) as analyzed by biochemical fractionation and western blot ; Whole Cell Extract ( WCE ) , Cytoplasm ( Cyt ) , Nucleoplasm ( Nuc ) .

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(E) Subcellular localization of Nrf2 in CD4+ T-cells infected with HIV-1 or mock infected (3dpi) as analyzed by biochemical fractionation and western blot; Whole Cell Extract (WCE), Cytoplasm (Cyt), Nucleoplasm (Nuc).


( F ) Nuclear localization ( left ) and content ( right ) of Nrf2 in HIV - 1 RNA + and HIV - 1 RNA - cells ( 7dpi ) as measured by combining immunofluorescence and HIV - 1 RNA FISH . Black dash indicates median , n = number of cells . Scale bar =

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(F) Nuclear localization (left) and content (right) of Nrf2 in HIV-1 RNA+ and HIV-1 RNA- cells (7dpi) as measured by combining immunofluorescence and HIV-1 RNA FISH. Black dash indicates median, n= number of cells. Scale bar = 2μm


Time course of the mRNA levels of main downstream antioxidant targets of Nrf2 during the transition from productive ( 3 - 9 dpi ) to latent ( 14 dpi ) infection , as measured by qPCR Data in ( G ) are depicted as mean ± SEM of 3 biological replicates . raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in infected vs mock infected cells Trx = thioredoxin ; NQO1 = NAD ( P ) H [ quinone ] dehydrogenase 1 ; HMOX - 1 = heme oxygenase 1 ; G6PD = glucose - 6 - phosphate dehydrogenase ; GCLC = glutamate - cysteine ligase ; TrxR1 = thioredoxin reductase 1 .

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Time course of the mRNA levels of main downstream antioxidant targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection, as measured by qPCR Data in (G) are depicted as mean±SEM of 3 biological replicates. raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in infected vs mock infected cells Trx= thioredoxin; NQO1= NAD(P)H [quinone] dehydrogenase 1; HMOX-1= heme oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC= glutamate-cysteine ligase ; TrxR1= thioredoxin reductase 1.


Time course of the protein levels of main downstream antioxidant targets of Nrf2 during the transition from productive ( 3 - 9 dpi ) to latent ( 14 dpi ) infection , as measured by western blot GCLC and HMOX - 1 in ( H ) were probed upon membrane stripping .

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Time course of the protein levels of main downstream antioxidant targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection, as measured by western blot GCLC and HMOX-1 in (H) were probed upon membrane stripping.


( I ) Relative mRNA expression of Nrf2 downstream antioxidant targets in J - Lat 9 . 2 cells left untreated ( latent ) or treated for 24h with 10µM TPA ( reactivated ) . Data are depicted as mean ± SEM of 3 biological replicates . raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in TPA - reactivated vs latent cells Trx = thioredoxin ; NQO1 = NAD ( P ) H [ quinone ] dehydrogenase 1 ; HMOX - 1 = heme oxygenase 1 ; G6PD = glucose - 6 - phosphate dehydrogenase ; GCLC =

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(I) Relative mRNA expression of Nrf2 downstream antioxidant targets in J-Lat 9.2 cells left untreated (latent) or treated for 24h with 10µM TPA (reactivated). Data are depicted as mean±SEM of 3 biological replicates. raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in TPA-reactivated vs latent cells Trx= thioredoxin; NQO1= NAD(P)H [quinone] dehydrogenase 1; HMOX-1= heme oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC=


( A ) gag mRNA expression ( left ) and relative viability ( right ) of HIV - 1 infected Jurkat - TAg cells transfected with non targeting siRNA ( NC ) or siRNAs targeting the HMOX - 1 gene . Cells were infected 24hr post - transfection and assayed for mRNA expression ( by qPCR ) or viability ( by MTT assay ) 48hr post - infection . Data ( mean ± SEM ; n = 3 technical replicates ) were normalized using the NC control and analyzed by unpaired t - test . P < 0 . 05 ; * * P < 0 . 01 .

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(A) gag mRNA expression (left) and relative viability (right) of HIV-1 infected Jurkat-TAg cells transfected with non targeting siRNA (NC) or siRNAs targeting the HMOX-1 gene. Cells were infected 24hr post-transfection and assayed for mRNA expression (by qPCR) or viability (by MTT assay) 48hr post-infection. Data (mean±SEM; n=3 technical replicates) were normalized using the NC control and analyzed by unpaired t-test. P<0.05; **P<0.01.


( B RNA - Seq analysis of different time points of primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected . The gene set used for the analyses is GO iron ion import ( 12 genes , GO : 0097286 ) . Data in ( B ) were analyzed by Fisher test . Boxplots depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . Dots below boxplots illustrate , for each time point , the pathway enrichment analysis of genes up - regulated in infected vs matched mock - infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of DEGs in the pathway ; grey dots are not statistically significant .

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(B RNA-Seq analysis of different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. The gene set used for the analyses is GO iron ion import (12 genes, GO:0097286). Data in (B) were analyzed by Fisher test. Boxplots depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. Dots below boxplots illustrate, for each time point, the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of DEGs in the pathway; grey dots are not statistically significant.


( C ) Heatmaps of the standardized expression of the GO iron ion import pathway genes in HIV - 1 infected and mock infected samples over time . Expression levels were standardized [ ( mean gene expression - SD ) / SD ] for each gene in each time point . The TFRC ( TfR1 ) gene , which was considered for further analysis , is highlighted .

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(C) Heatmaps of the standardized expression of the GO iron ion import pathway genes in HIV-1 infected and mock infected samples over time. Expression levels were standardized [(mean gene expression - SD)/SD] for each gene in each time point. The TFRC (TfR1) gene, which was considered for further analysis, is highlighted.


( D ) TfR1 expression in HIV - 1 infected or mock infected CD4 + T - cells as visualized by confocal microscopy . Fluorescent values shown were calculated as described in ( McCloy et al , 2014 ) . Data were analyzed by one - way ANOVA followed by Sidak ´ s post - test to compare corresponding mock infected vs infected pairs . Black dash indicates median , n = number of cells . Scale bar = 2μm .

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(D) TfR1 expression in HIV-1 infected or mock infected CD4+ T-cells as visualized by confocal microscopy. Fluorescent values shown were calculated as described in (McCloy et al, 2014). Data were analyzed by one-way ANOVA followed by Sidak´s post-test to compare corresponding mock infected vs infected pairs. Black dash indicates median, n= number of cells. Scale bar = 2μm.


( E ) Western blot ( upper panel ) and relative expression ( lower panel ) of FTH - 1 protein in HIV - 1 infected vs mock infected CD4 + T - cells over time . Western blot quantification was performed with Fiji - Image J ( Schindelin et al , 2012 ) , normalized to the housekeeping protein beta - actin and expressed as Log2 fold change in HIV - 1 infected vs mock infected cells ( mean ± SEM ; n = 3 biological replicates ) . P < 0 . 05 ;

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(E) Western blot (upper panel) and relative expression (lower panel) of FTH-1 protein in HIV-1 infected vs mock infected CD4+ T-cells over time. Western blot quantification was performed with Fiji-Image J (Schindelin et al, 2012), normalized to the housekeeping protein beta-actin and expressed as Log2 fold change in HIV-1 infected vs mock infected cells (mean±SEM; n=3 biological replicates). P<0.05;


( F ) Percentage of intracellular p24 + gag CD4 + T - cells infected with HIV - 1 and left untreated or treated for 48hr with the iron donor Fe - NTA at 150μM concentration ( n = 3 ; mean ± range of three biological replicates ) .

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(F) Percentage of intracellular p24+ gag CD4+ T-cells infected with HIV-1 and left untreated or treated for 48hr with the iron donor Fe-NTA at 150μM concentration (n=3; mean ± range of three biological replicates).


( G ) Percentage of GFP + J - Lat cells ( clones 9 . 2 and 15 . 4 ) left untreated or treated for 48hr with TPA ( 10μM ) , the iron donor FeNT ( 150μM ) or a combination of the two . Data are expressed as mean ± SEM of four technical replicates for each cell line and were analyzed by one - way ANOVA followed by Tukey ´ s post - test . * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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(G) Percentage of GFP+ J-Lat cells (clones 9.2 and 15.4) left untreated or treated for 48hr with TPA (10μM), the iron donor FeNT (150μM) or a combination of the two. Data are expressed as mean±SEM of four technical replicates for each cell line and were analyzed by one-way ANOVA followed by Tukey´s post-test. *P<0.05; ***P<0.001; ****P<0.0001.


( A RNA - Seq analyses of different time points in primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected . Data were analyzed by Fisher test ( number of donors = 3 biological replicates ) . Boxplots in depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . For each time point , dots below boxplots illustrate the pathway enrichment analysis of genes up - regulated in infected vs matched mock infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of differentially expressed genes in the pathway ; grey dots are not statistically significant . The gene set considered for the analyses is Biocarta _ PML _ pathway ( 17 genes , M4891 ) .

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(A RNA-Seq analyses of different time points in primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. Data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots below boxplots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes in the pathway; grey dots are not statistically significant. The gene set considered for the analyses is Biocarta_PML_pathway (17 genes, M4891).


B ) proteomic analyses of different time points in primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected . Data were analyzed by Fisher test ( number of donors = 3 biological replicates ) . Boxplots in depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . For each time point , dots below boxplots illustrate the pathway enrichment analysis of proteins up - regulated in infected vs matched mock infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of differentially expressed proteins in the pathway ; grey dots are not statistically significant .

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B) proteomic analyses of different time points in primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. Data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots below boxplots illustrate the pathway enrichment analysis of proteins up-regulated in infected vs matched mock infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed proteins in the pathway; grey dots are not statistically significant.


( C Western blot of PML protein in HIV - 1 infected vs mock infected CD4 + T - cells over time . Raw data were normalized using actin ( protein ) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells .

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(C Western blot of PML protein in HIV-1 infected vs mock infected CD4+ T-cells over time. Raw data were normalized using actin (protein) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells.


D ) relative expression PML mRNA in HIV - 1 infected vs mock infected CD4 + T - cells over time . Raw data were normalized using GAPDH or 18S ( mRNA ) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells . Data were analyzed by two - way ANOVA followed by Tukey ´ s post - test ( mean ± SEM of 3 and 4 biological replicates for PML

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D) relative expression PML mRNA in HIV-1 infected vs mock infected CD4+ T-cells over time. Raw data were normalized using GAPDH or 18S (mRNA) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells. Data were analyzed by two-way ANOVA followed by Tukey´s post-test (mean±SEM of 3 and 4 biological replicates for PML mRNA


( E ) Western blot and quantification of PML protein in two different clones of J - Lat cells left untreated or treated with TPA for 24h . Data were analyzed by unpaired t - test ( mean ± SEM ; n = 3 technical replicates for each cell line ) .

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(E) Western blot and quantification of PML protein in two different clones of J-Lat cells left untreated or treated with TPA for 24h. Data were analyzed by unpaired t-test (mean±SEM; n=3 technical replicates for each cell line).


( F PML protein expression in CD4 + T - cells infected with HIV - 1 or mock infected at 7dpi as measured by western blot . Cells were left untreated or treated with the antioxidant compound NAC ( 10mM , F )

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(F PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the antioxidant compound NAC (10mM, F)


G ) PML protein expression in CD4 + T - cells infected with HIV - 1 or mock infected at 7dpi as measured by western blot . Cells were left untreated or treated with the iron chelator deferiprone ( L1 , 50μM , G ) .

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G) PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the iron chelator deferiprone (L1, 50μM, G).


( A Representative image and quantification of the number of PML NBs in HIV - 1 DNA + vs mock infected CD4 + T - cells . Scale bars = 2μm . Infected cells were identified with DNA FISH and PML NBs were stained by IF .

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(A Representative image and quantification of the number of PML NBs in HIV-1 DNA+ vs mock infected CD4+ T-cells. Scale bars = 2μm. Infected cells were identified with DNA FISH and PML NBs were stained by IF.


B ) Representative image and quantification of the number of PML NBs in HIV - 1 RNA + vs HIV - 1 RNA - CD4 + T - cells . Scale bars = 2μm . Infected cells were identified with RNA FISH and PML NBs were stained by IF .

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B) Representative image and quantification of the number of PML NBs in HIV-1 RNA+ vs HIV-1 RNA- CD4+ T-cells. Scale bars = 2μm. Infected cells were identified with RNA FISH and PML NBs were stained by IF.


Representative immunofluorescence images and quantifications of PML NBs ( green , maximal projection ) and mab414 / nuclear pore ( red , one Z stack ) in J - Lat 9 . 2 cells . Cells were left latent or reactivated for 24 hrs with 10μM TPA . Proteasome function was blocked for 4 hrs in both latent and TPA reactivated cells using MG132 ( 10μM ) or Bortezomib ( 100nM )

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Representative immunofluorescence images and quantifications of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Proteasome function was blocked for 4 hrs in both latent and TPA reactivated cells using MG132 (10μM) or Bortezomib (100nM)


Representative immunofluorescence images and quantifications of PML NBs ( green , maximal projection ) and mab414 / nuclear pore ( red , one Z stack ) in J - Lat 9 . 2 cells . Cells were left latent or reactivated for 24 hrs with 10μM TPA . Alternatively , ubiquitination was blocked for 4 hrs using TAK243 ( 10μM )

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Representative immunofluorescence images and quantifications of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Alternatively, ubiquitination was blocked for 4 hrs using TAK243 (10μM)


( E , F ) Representative immunofluorescence images ( E ) and quantification ( F ) of PML NBs ( green ) and SUMO 2 / 3 ( red ) in J - Lat 9 . 2 cells left latent or reactivated for 24 hours with 10μM TPA . Cells were imaged by STED microscopy , scale bar 2 μm .

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(E,F) Representative immunofluorescence images (E) and quantification (F) of PML NBs (green) and SUMO 2/3 (red) in J-Lat 9.2 cells left latent or reactivated for 24 hours with 10μM TPA. Cells were imaged by STED microscopy, scale bar 2 μm.


( G , H ) Representative immunofluorescence images ( G ) and quantification ( H ) of PML NBs ( green , maximal projection ) and SUMO 2 / 3 ( red , maximal projection ) during mock and productive HIV - 1 infection of primary CD4 + T - cells at 7dpi . Scale bar 10μm .

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(G,H) Representative immunofluorescence images (G) and quantification (H) of PML NBs (green, maximal projection) and SUMO 2/3 (red, maximal projection) during mock and productive HIV-1 infection of primary CD4+ T-cells at 7dpi. Scale bar 10μm.


A S . cerevisiae wt or hsp42Δ cells expressing mCherry ‐ VHL ( red ) and GFP ‐ luciferase ‐ DM ‐ NLS ( green ) were grown at 30 ° C and shifted to 37 ° C for 90 min . MG132 was added prior to temperature upshift . Changes in protein localizations were recorded . DNA was stained by DAPI ( blue ) .

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A S. cerevisiae wt or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐luciferase‐DM‐NLS (green) were grown at 30°C and shifted to 37°C for 90 min. MG132 was added prior to temperature upshift. Changes in protein localizations were recorded. DNA was stained by DAPI (blue).


B S . cerevisiae wt cells or hsp42Δ cells expressing GFP ‐ VHL ( green ) were treated as in ( A ) . Nuclear membranes were visualized by Nsp1immunofluorescence microscopy labeling ( red ) .

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B S. cerevisiae wt cells or hsp42Δ cells expressing GFP‐VHL (green) were treated as in (A). Nuclear membranes were visualized by Nsp1immunofluorescence microscopy labeling (red).


C S . cerevisiae wt cells or hsp42Δ cells expressing mCherry ‐ VHL ( red ) and GFP ‐ Nup49 ( green ) were treated as in ( A ) . Three ‐ dimensional reconstructions of respective cells are given .

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C S. cerevisiae wt cells or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐Nup49 (green) were treated as in (A). Three‐dimensional reconstructions of respective cells are given.


D Time ‐ lapse microscopy of wt cells expressing mCherry ‐ VHL ( green ) and GFP ‐ Nup49 ( red ) upon temperature upshift from 30 ° C to 37 ° C . * indicates collision of peripheral foci leading to fusion , and an arrow indicates apparent encounters of peripheral foci and nuclear INQ , followed by foci separation .

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D Time‐lapse microscopy of wt cells expressing mCherry‐VHL (green) and GFP‐Nup49 (red) upon temperature upshift from 30°C to 37°C. * indicates collision of peripheral foci leading to fusion, and an arrow indicates apparent encounters of peripheral foci and nuclear INQ, followed by foci separation.


A - D Cryo ‐ sections of ( A ) S . cerevisiae wt cells expressing GFP ‐ luciferase ‐ DM ‐ NLS , ( B ) hsp42Δ cells expressing GFP ‐ VHL . Sections were immunogold ‐ labeled with GFP ‐ specific antibodies . Gold particles are marked ( black arrows ) . Electron ‐ dense regions represent protein aggregates ( A ) . Locations of cytosol ( C ) , nuclear envelope ( NE , orange arrows ) , and nucleus ( N ) are given . Scale bars , 200 nm . E , F Average numbers of gold particles associated with nuclear and cytosolic protein aggregates or distributed throughout the cytosol or nucleus were determined .

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A-D Cryo‐sections of (A) S. cerevisiae wt cells expressing GFP‐luciferase‐DM‐NLS, (B) hsp42Δ cells expressing GFP‐VHL. Sections were immunogold‐labeled with GFP‐specific antibodies. Gold particles are marked (black arrows). Electron‐dense regions represent protein aggregates (A). Locations of cytosol (C), nuclear envelope (NE, orange arrows), and nucleus (N) are given. Scale bars, 200 nm.E, F Average numbers of gold particles associated with nuclear and cytosolic protein aggregates or distributed throughout the cytosol or nucleus were determined.


A - D Cryo ‐ sections of S . cerevisiae ( C , D ) wt or hsp42Δ cells expressing Hsp104 ‐ GFP . Sections were immunogold ‐ labeled with GFP ‐ specific antibodies . Gold particles are marked ( black arrows ) . Electron ‐ dense regions represent protein aggregates ( A ) . Locations of cytosol ( C ) , nuclear envelope ( NE , orange arrows ) , and nucleus ( N ) are given . Scale bars , 200 nm . E , F Average numbers of gold particles associated with nuclear and cytosolicprotein aggregates or distributed throughout the cytosol or nucleus were determined .

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A-D Cryo‐sections of S. cerevisiae (C, D) wt or hsp42Δ cells expressing Hsp104‐GFP. Sections were immunogold‐labeled with GFP‐specific antibodies. Gold particles are marked (black arrows). Electron‐dense regions represent protein aggregates (A). Locations of cytosol (C), nuclear envelope (NE, orange arrows), and nucleus (N) are given. Scale bars, 200 nm.E, F Average numbers of gold particles associated with nuclear and cytosolicprotein aggregates or distributed throughout the cytosol or nucleus were determined.


A S . cerevisiae wt cells expressing GFP ‐ VHL ( green ) were grown at 30 ° C and shifted to 37 ° C for 90 min in the presence of MG132 . Ubiquitin ( red ) was stained by immunofluorescence microscopy using specific antibodies . DNA was stained by DAPI ( blue ) . Changes in protein localizations were recorded . Scale bars , 2 μm .

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A S. cerevisiae wt cells expressing GFP‐VHL (green) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132. Ubiquitin (red) was stained by immunofluorescence microscopy using specific antibodies. DNA was stained by DAPI (blue). Changes in protein localizations were recorded. Scale bars, 2 μm.


B tGnd1 ‐ GFP is stabilized in ubr1Δ san1Δ cells . S . cerevisiae wt and ubr1Δ san1Δ mutant cells expressing tGnd1 ‐ GFP were grown at 30 ° C . Cycloheximide was added , and protein levels were determined at the indicated time points by Western blot using GFP ‐ specific antibodies . Zwf1 levels are given as a loading control .

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B tGnd1‐GFP is stabilized in ubr1Δ san1Δ cells. S. cerevisiae wt and ubr1Δ san1Δ mutant cells expressing tGnd1‐GFP were grown at 30°C. Cycloheximide was added, and protein levels were determined at the indicated time points by Western blot using GFP‐specific antibodies. Zwf1 levels are given as a loading control.


C Ubiquitination status of tGnd1 ‐ GFP expressed in S . cerevisiae wt and ubr1Δ san1Δ cells was determined by GFPpull ‐ down and Western blot analysis using ubiquitin ‐ specific antibodies . Levels of isolated tGnd1 ‐ GFP after GFPpull ‐ down were determined by Western blot using GFP ‐ specific antibodies and are given as a control .

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C Ubiquitination status of tGnd1‐GFP expressed in S. cerevisiae wt and ubr1Δ san1Δ cells was determined by GFPpull‐down and Western blot analysis using ubiquitin‐specific antibodies. Levels of isolated tGnd1‐GFP after GFPpull‐down were determined by Western blot using GFP‐specific antibodies and are given as a control.


D S . cerevisisae wt , ubr1Δ san1Δ , and ubr1Δ san1Δ btn2Δ cells expressing tGnd1 ‐ GFP ( green ) were grown at 30 ° C . DNA and nuclear envelopes were visualized by coexpressing Htb1 ‐ mCherry or Nic96 ‐ mCherry , respectively ( red ) . The fraction of cells showing specific tGnd1 ‐ GFP foci numbers per cell was determined . INQ formation in cells containing tGnd1 ‐ GFP foci was determined ( % cells with INQ ) based on close vicinity of tGnd1 ‐ GFP foci to Htb1 ‐ mCherry or localization inside Nic96 ‐ mCherry signals , yielding comparable results . Scale bars , 2 μm .

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D S. cerevisisae wt, ubr1Δ san1Δ, and ubr1Δ san1Δ btn2Δ cells expressing tGnd1‐GFP (green) were grown at 30°C. DNA and nuclear envelopes were visualized by coexpressing Htb1‐mCherry or Nic96‐mCherry, respectively (red). The fraction of cells showing specific tGnd1‐GFP foci numbers per cell was determined. INQ formation in cells containing tGnd1‐GFP foci was determined (% cells with INQ) based on close vicinity of tGnd1‐GFP foci to Htb1‐mCherry or localization inside Nic96‐mCherry signals, yielding comparable results. Scale bars, 2 μm.


A , B S . cerevisiae hsp42Δ and hsp42Δ nup42Δ cells expressing mCherry ‐ VHL ( red ) were grown at 30 ° C and shifted to 37 ° C in the presence of MG132 . Changes in protein localization were monitored . Scale bars , 2 μm . The total number of mCherry ‐ VHL foci per cell was determined .

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A, B S. cerevisiae hsp42Δ and hsp42Δ nup42Δ cells expressing mCherry‐VHL (red) were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. Scale bars, 2 μm. The total number of mCherry‐VHL foci per cell was determined.


C Dynamic exchange of a misfolded model protein between cytosol and nucleus . FLIP measurements of mCherry ‐ VHL were performed in S . cerevisiae wild ‐ type cells at 30 ° C . Nuclear or cytosolic areas were bleached , and loss of fluorescence intensities in non ‐ bleached compartments was determined . Bleaching and acquisition controls are given . Error bars : SEM .

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C Dynamic exchange of a misfolded model protein between cytosol and nucleus. FLIP measurements of mCherry‐VHL were performed in S. cerevisiae wild‐type cells at 30°C. Nuclear or cytosolic areas were bleached, and loss of fluorescence intensities in non‐bleached compartments was determined. Bleaching and acquisition controls are given. Error bars: SEM.


S . cerevisiae hsp42Δ and hsp42Δ tet ‐ off sis1 cells expressing GFP ‐ VHL were grown for 20 h in the absence ( − Dox ) or presence ( + Dox ) of doxycycline at 30 ° C and shifted for 90 min to 37 ° C in the presence of MG132 . Changes in protein localization were monitored . DNA was stained by DAPI ( blue ) . The total number of GFP ‐ VHL foci per cell and frequencies ( % ) of INQ formation were determined . Scale bars , 2 μm .

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S. cerevisiae hsp42Δ and hsp42Δ tet‐off sis1 cells expressing GFP‐VHL were grown for 20 h in the absence (−Dox) or presence (+Dox) of doxycycline at 30°C and shifted for 90 min to 37°C in the presence of MG132. Changes in protein localization were monitored. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.


B S . cerevisiae wt , hsp42Δ , sti1Δ , and hsp42Δ sti1Δ cells expressing GFP ‐ VHL were grown at 30 ° C and shifted to 37 ° C in the presence of MG132 . Changes in protein localization were monitored . The total number of GFP ‐ VHL foci per cell and frequencies ( % ) of INQ formation were determined . Scale bars , 2 μm .

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B S. cerevisiae wt, hsp42Δ, sti1Δ, and hsp42Δ sti1Δ cells expressing GFP‐VHL were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.


A , B S . cerevisiae wt , hsp42Δ , btn2Δ , and hsp42Δ btn2Δ cells expressing GFP ‐ VHL ( green , A ) or GFP ‐ luciferase ‐ DM ‐ NLS ( green , B ) were grown at 30 ° C and shifted to 37 ° C for 90 min in the presence of MG132 , and protein localizations were recorded . DNA was stained by DAPI ( blue ) , and the nuclear envelope was stained by Nsp1immunofluorescence microscopy ( red ) . Solubilities of GFP ‐ VHL and GFP ‐ luciferase ‐ DM ‐ NLS were determined after stress application by Western blot using GFP ‐ specific antibodies . Solubility of actin was determined as a control . T , total fraction ; S , soluble fraction ; P , pellet fraction . Scale bars , 2 μm .

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A, B S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing GFP‐VHL (green, A) or GFP‐luciferase‐DM‐NLS (green, B) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132, and protein localizations were recorded. DNA was stained by DAPI (blue), and the nuclear envelope was stained by Nsp1immunofluorescence microscopy (red). Solubilities of GFP‐VHL and GFP‐luciferase‐DM‐NLS were determined after stress application by Western blot using GFP‐specific antibodies. Solubility of actin was determined as a control. T, total fraction; S, soluble fraction; P, pellet fraction. Scale bars, 2 μm.


C S . cerevisiae wt , hsp42Δ , btn2Δ , and hsp42Δ btn2Δ cells expressing mCherry ‐ VHL were heat ‐ shocked to 38 ° C . Upon return to 30 ° C , protein synthesis was stopped by cycloheximide ( CHX ) addition and degradation of mCherry ‐ VHL was monitored by Western blot analysis . The stability of GFP ‐ luciferase ‐ DM ‐ NLS expressed in S . cerevisiae wt and btn2Δ cells was determined accordingly . Ccs1 or Zwf1 levels are given as loading controls .

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C S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing mCherry‐VHL were heat‐shocked to 38°C. Upon return to 30°C, protein synthesis was stopped by cycloheximide (CHX) addition and degradation of mCherry‐VHL was monitored by Western blot analysis. The stability of GFP‐luciferase‐DM‐NLS expressed in S. cerevisiae wt and btn2Δ cells was determined accordingly. Ccs1 or Zwf1 levels are given as loading controls.


D S . cerevisiae hsp42Δ btn2Δ cells expressing either GFP or LuciDM ‐ GFP and Htb1 ‐ mCherry were grown at 30 ° C and heat ‐ shocked to 37 ° C for 30 min . Line intensity plots of luciferase ‐ DM ‐ GFP and Htb1 ‐ mCherry before and after heat shock are given . The ratio of nuclear and cytosolic luciferase ‐ DM ‐ GFP fluorescence intensity was determined at 30 ° C and 37 ° C ( n > 25 ) . Scale bars , 2 μm .

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D S. cerevisiae hsp42Δ btn2Δ cells expressing either GFP or LuciDM‐GFP and Htb1‐mCherry were grown at 30°C and heat‐shocked to 37°C for 30 min. Line intensity plots of luciferase‐DM‐GFP and Htb1‐mCherry before and after heat shock are given. The ratio of nuclear and cytosolic luciferase‐DM‐GFP fluorescence intensity was determined at 30°C and 37°C (n > 25). Scale bars, 2 μm.


A , B Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence microscopy . DNA was stained by either Htb1 ‐ mCherry or DAPI . Scale bars , 2 μm . In case of Hsp42 immunofluorescence , a line intensity plot of a deconvoluted widefield image is given .

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A, B Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence microscopy. DNA was stained by either Htb1‐mCherry or DAPI. Scale bars, 2 μm. In case of Hsp42 immunofluorescence, a line intensity plot of a deconvoluted widefield image is given.


C S . cerevisiae btn2Δ hsp42Δ Hsp42 ‐ NLS cells expressing GFP ‐ VHL ( green ) were grown at 30 ° C and heat ‐ shocked to 37 ° C for 30 min in the presence of MG132 . Changes in protein localizations were recorded . DNA was stained by DAPI ( blue ) . The total number of GFP ‐ VHL foci per cell and frequencies ( % ) of INQ formation were determined . Scale bars , 2 μm .

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C S. cerevisiae btn2Δ hsp42Δ Hsp42‐NLS cells expressing GFP‐VHL (green) were grown at 30°C and heat‐shocked to 37°C for 30 min in the presence of MG132. Changes in protein localizations were recorded. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.


D S . cerevisiaebtn2Δ hsp42Δ Hsp42 ‐ NLS cells expressing mCherry ‐ VHL ( red ) were treated as described in ( C ) . The nuclear membrane was visualized by Nsp1 immunofluorescence ( green ) and colocalization of mCherry ‐ VHL foci with Hsp42 was probed by Hsp42 ( green ) immunofluorescence microscopy . DNA was stained with DAPI ( blue ) . Scale bars , 2 μm .

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D S. cerevisiaebtn2Δ hsp42Δ Hsp42‐NLS cells expressing mCherry‐VHL (red) were treated as described in (C). The nuclear membrane was visualized by Nsp1 immunofluorescence (green) and colocalization of mCherry‐VHL foci with Hsp42 was probed by Hsp42 (green) immunofluorescence microscopy. DNA was stained with DAPI (blue). Scale bars, 2 μm.


E Hsp42 and Btn2 levels were determined prior and post ‐ heat shock at the indicated time points by Western blot . The numbers of molecules / cell were calculated using purified proteins as standard .

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E Hsp42 and Btn2 levels were determined prior and post‐heat shock at the indicated time points by Western blot. The numbers of molecules/cell were calculated using purified proteins as standard.


A S . cerevisiae wt cells were treated with MMS and Btn2 levels were determined at the indicated time points . Zwf1 levels are given as a loading control .

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A S. cerevisiae wt cells were treated with MMS and Btn2 levels were determined at the indicated time points. Zwf1 levels are given as a loading control.


B , C S . cerevisiae wt and btn2Δ cells expressing Hos2 ‐ GFP ( green , B ) or GFP ‐ VHL ( green , C ) were grown at 30 ° C and treated with MMS . Changes in protein localizations were recorded at the indicated time points . DNA was stained by DAPI ( blue ) . The total number of GFP ‐ VHL foci per cell and frequencies ( % ) of INQ formation were determined .

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B, C S. cerevisiae wt and btn2Δ cells expressing Hos2‐GFP (green, B) or GFP‐VHL (green, C) were grown at 30°C and treated with MMS. Changes in protein localizations were recorded at the indicated time points. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined.


( a ) RT - PCR expression and quantification of IGF1 , IGF1Rβ and IGF2 levels in untreated C2C12 and shCD20 C2C12 myoblasts and 10 nM IGF1 - treated C2C12 and over - expressing CD20 C2C12 myoblasts . Each experiment was replicated independently four times .

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(a) RT-PCR expression and quantification of IGF1, IGF1Rβ and IGF2 levels in untreated C2C12 and shCD20 C2C12 myoblasts and 10 nM IGF1-treated C2C12 and over-expressing CD20 C2C12 myoblasts. Each experiment was replicated independently four times.


( b ) Immunofluorescence for IGF2 ( in green ) in untreated and 10 nM IGF1 - treated C2C12 myoblasts . Scale bars = 75 μm .

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(b) Immunofluorescence for IGF2 (in green) in untreated and 10 nM IGF1-treated C2C12 myoblasts. Scale bars = 75 μm.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( c ) C2C12 cells treated with IGF1 for two hours ( 2 h ) or overnight ( ON ) as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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Representative CD20 immunoprecipitation using anti-pSer+pThr in (c) C2C12 cells treated with IGF1 for two hours (2 h) or overnight (ON) as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( d ) sh - empty ( shCTR ) - and shCD20 - treated C2C12 cells treated with anti - Flag and 10 nM anti - IGF2R , as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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Representative CD20 immunoprecipitation using anti-pSer+pThr in (d) sh-empty (shCTR)- and shCD20-treated C2C12 cells treated with anti-Flag and 10 nM anti-IGF2R, as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( e ) C2C12 cells treated with IGF1 and IGF2 , as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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Representative CD20 immunoprecipitation using anti-pSer+pThr in (e) C2C12 cells treated with IGF1 and IGF2, as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


( f ) Representative WB of IGF2R and phosphorylated IGF2R ( pIGF2R ) in untreated , shCD20 - treated and anti - IGF2R - treated C2C12 cells . Densitometric analysis of data are expressed as the ratio of IGF2R / actin or pIGF2R / IGF2R in arbitrary units in the lower panels . One - way ANOVA . * p < 0 . 05 ; Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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(f) Representative WB of IGF2R and phosphorylated IGF2R (pIGF2R) in untreated, shCD20-treated and anti-IGF2R-treated C2C12 cells. Densitometric analysis of data are expressed as the ratio of IGF2R/actin or pIGF2R/IGF2R in arbitrary units in the lower panels. One-way ANOVA. *p<0.05; Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


( g ) IGF2R immunoprecipitation products were immunoblotted for Gαi2 and WB expression of IGF2R in untreated , IGF2 - treated , anti - IGF2R - treated and IGF2 + anti - IGF2R treated C2C12 cells . Densitometric analysis of data are expressed as the ratio of Gαi2 / IGF2R in arbitrary units in the lower panel .

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(g) IGF2R immunoprecipitation products were immunoblotted for Gαi2 and WB expression of IGF2R in untreated, IGF2-treated, anti-IGF2R-treated and IGF2+anti-IGF2R treated C2C12 cells. Densitometric analysis of data are expressed as the ratio of Gαi2/IGF2R in arbitrary units in the lower panel.


( h ) Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for p - Tyr in untreated , IGF2 - treated and IGF2 + anti - IGF2R - treated ( 1 : 500 , 1 : 1000 and 1 : 2000 dilutions of anti - IGF2R ) C2C12 cells ( cells were treated for 2 and 24 h ) . Densitometric analysis of data are expressed as the ratio of pTYR / IGF1Rβ in arbitrary units in the lower panel . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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(h) Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for p-Tyr in untreated, IGF2-treated and IGF2+anti-IGF2R-treated (1:500, 1:1000 and 1:2000 dilutions of anti-IGF2R) C2C12 cells (cells were treated for 2 and 24 h). Densitometric analysis of data are expressed as the ratio of pTYR/IGF1Rβ in arbitrary units in the lower panel. Each experiment was performed in triplicate wells. All values are expressed as the mean±SEM.


( d ) Over - expressing CD20 in C2C12 myoblasts that co - expressed CD20 ( in green ) and IGF2R ( in red ) . DAPI - labelled nuclei are shown in blue . Scale bars = 75 μm .

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(d) Over-expressing CD20 in C2C12 myoblasts that co-expressed CD20 (in green) and IGF2R (in red). DAPI-labelled nuclei are shown in blue. Scale bars=75 μm.


( e ) Representative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20 , respectively , in UNTREATED and shCD20 - treated C2C12 cell membranes and whole lysates of proteins . The immunoprecipitation output is shown as IP neg .

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(e) Representative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20, respectively, in UNTREATED and shCD20-treated C2C12 cell membranes and whole lysates of proteins. The immunoprecipitation output is shown as IP neg.


( f ) Myotubes immunofluorescence of cells in proliferation medium ( P . M . ) and after 2 , 4 and 6 days of myogenic differentiation in serum free medium . Control ( untreated ) , shCD20 - treated C2C12 cells , C2C12 and HSkM myoblasts pre - treated with anti - IGF2R for 24 hours were stained . Desmin - positive myotubes are shown in green . Scale bars = 75 μm .

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(f) Myotubes immunofluorescence of cells in proliferation medium (P.M.) and after 2, 4 and 6 days of myogenic differentiation in serum free medium. Control (untreated), shCD20-treated C2C12 cells, C2C12 and HSkM myoblasts pre-treated with anti-IGF2R for 24 hours were stained. Desmin-positive myotubes are shown in green. Scale bars=75 μm.


( g ) Fusion index quantification after 6 days of differentiation . One - way ANOVA . * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as mean ± SEM .

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(g) Fusion index quantification after 6 days of differentiation. One-way ANOVA. **p<0.01; ***p<0.001; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as mean±SEM.


( h ) Representative WB of anti - myogenin , anti - Myf5 , anti - MyHC and anti - β - actin in total protein lysates obtained from untreated , shCD20 - treated C2C12 cells , and C2C12 and HSkM myoblasts pre - treated with anti - IGF2R for 24 hours ; cells were collected under P . M . and after 2 , 4 and 6 days of myogenic differentiation . Densitometric analysis of WB data expressed as the ratio of the indicated antibody / β - actin in arbitrary units . Two - way ANOVA test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 in comparison to the results obtained in untreated cells at the corresponding time point . # # p < 0 . 01 ; # # # p < 0 . 001 ; # # # # p < 0 . 0001 in comparison to the results obtained in shCD20 - treated C2C12 cells at the corresponding time point . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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(h) Representative WB of anti-myogenin, anti-Myf5, anti-MyHC and anti-β-actin in total protein lysates obtained from untreated, shCD20-treated C2C12 cells, and C2C12 and HSkM myoblasts pre-treated with anti-IGF2R for 24 hours; cells were collected under P.M. and after 2, 4 and 6 days of myogenic differentiation. Densitometric analysis of WB data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. Two-way ANOVA test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 in comparison to the results obtained in untreated cells at the corresponding time point. ##p<0.01; ###p<0.001; ####p<0.0001 in comparison to the results obtained in shCD20-treated C2C12 cells at the corresponding time point. Each experiment was performed in triplicate wells. All values are expressed as the mean±SEM.


Immunofluorescence analysis of CD20 and IGF2R expression ( in red ) in the TA muscles of three - month - old C57Bl6 / J and mdx mice . Scale bars = 75μm . ( a ) .

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Immunofluorescence analysis of CD20 and IGF2R expression (in red) in the TA muscles of three-month-old C57Bl6/J and mdx mice. Scale bars=75μm. (a).


IGF2R ( green ) and sarcomeric actin ( magenta ) expression in the VM muscles of two DMD patients . Scale bars = 25μm . ( b ) .

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IGF2R (green) and sarcomeric actin (magenta) expression in the VM muscles of two DMD patients. Scale bars=25μm. (b).


Representative WB analysis of CD20 and β - actin ( c ) expression in the TA and VM muscles of C57Bl6 / J and mdx mice ( n = 10 mice per group ) . Densitometry analysis of WB data are expressed as the CD20 / β - actin ratio , in arbitrary units in the lower panels One - way ANOVA . * * * * p < 0 . 0001 .

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Representative WB analysis of CD20 and β-actin (c) expression in the TA and VM muscles of C57Bl6/J and mdx mice (n=10 mice per group). Densitometry analysis of WB data are expressed as the CD20/β-actin ratio, in arbitrary units in the lower panels One-way ANOVA. ****p<0.0001.


Representative WB analysis of pSer + pThr and GADPH ( d ) expression in the TA and VM muscles of C57Bl6 / J and mdx mice ( n = 10 mice per group ) . Densitometry analysis of WB data are expressed as the pSer + pThr / CD20 ratio in arbitrary units in the lower panels One - way ANOVA . * * * * p < 0 . 0001 .

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Representative WB analysis of pSer+pThr and GADPH (d) expression in the TA and VM muscles of C57Bl6/J and mdx mice (n=10 mice per group). Densitometry analysis of WB data are expressed as the pSer+pThr/CD20 ratio in arbitrary units in the lower panels One-way ANOVA. ****p<0.0001.


( e ) Representative WB analysis of IGF2R and GAPDH expression in total protein lysates of the human ( healthy and DMD ) and murine ( C57Bl6 / J and mdx ) TA and VM muscles ( n = 2 per healthy and DMD ; n = 10 mice per group ) ; quantifications are expressed as the IGF2R / GAPDH ratio in arbitrary units .

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(e) Representative WB analysis of IGF2R and GAPDH expression in total protein lysates of the human (healthy and DMD) and murine (C57Bl6/J and mdx) TA and VM muscles (n=2 per healthy and DMD; n=10 mice per group); quantifications are expressed as the IGF2R/GAPDH ratio in arbitrary units.


( f ) Representative WB analysis of IGF2R expression in sarcolemma protein lysates of the TA and VM muscles of healthy , DMD , C57Bl6 / J and mdx ( n = 2 per healthy and DMD ; n = 10 mice per group ) . Densitometry analysis of IGF2R expression , results are shown as a fold increase , as indicated in the lower panel in arbitrary units . One - way ANOVA . * p < 0 . 05 ; * * * * p < 0 . 0001 . All values are expressed as the mean ± SEM .

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(f) Representative WB analysis of IGF2R expression in sarcolemma protein lysates of the TA and VM muscles of healthy, DMD, C57Bl6/J and mdx (n=2 per healthy and DMD; n=10 mice per group). Densitometry analysis of IGF2R expression, results are shown as a fold increase, as indicated in the lower panel in arbitrary units. One-way ANOVA. *p<0.05; ****p<0.0001. All values are expressed as the mean±SEM.


Quantification of the relative frequency of myofibre cross - sectional areas ( CSAs ) expressed as a frequency distribution in the TA and VM muscles of untreated mdx mice or mdx mice treated with a low or high dose of anti - IGF2R for 4 and 9 weeks ( a ) .

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Quantification of the relative frequency of myofibre cross-sectional areas (CSAs) expressed as a frequency distribution in the TA and VM muscles of untreated mdx mice or mdx mice treated with a low or high dose of anti-IGF2R for 4 and 9 weeks (a).


( b ) Quantification of regenerating myofibres as a proportion of centrally nucleated fibres in the total number of fibres after anti - IGF2R administration . The quantification of regenerating and M - cadherin - positive satellite cells per section is shown . One - way ANOVA test , * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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(b) Quantification of regenerating myofibres as a proportion of centrally nucleated fibres in the total number of fibres after anti-IGF2R administration. The quantification of regenerating and M-cadherin-positive satellite cells per section is shown. One-way ANOVA test, **p<0.01; ***p<0.001; ****p<0.0001.