Figure 3 ( 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 . ( 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 . ( 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 ) . ( 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 .

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 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 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 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 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 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

Figure 3(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.(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.(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).(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.


Figure 4 ( 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 . ( 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 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 ) . ( 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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Figure 4(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.(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 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).(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).


Figure 5 ( 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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Figure 5(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.


Figure 6Representative 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 ) ( 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 .

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 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

Figure 6Representative 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)(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.


Figure 1A 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 ) . 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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Figure 1A 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).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.


Figure 2A - 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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Figure 2A-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.


Figure 3A 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 . 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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Figure 3A 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.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.


Figure 4A , 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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Figure 4A, 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.


Figure 5S . 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 .

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 O O O O O O O O

Figure 5S. 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.


Figure 6A , 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 . 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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Figure 6A, 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.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.


Figure 7A , 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 . 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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Figure 7A, 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.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.


Figure 8A 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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Figure 8A 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.


Figure 1 ( 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 . 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 . 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 . ( 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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Figure 1(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.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.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.(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.


Figure 2 ( 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 . ( 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 . ( 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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Figure 2(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.(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.(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.


Figure 3Immunofluorescence 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 ) . 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 . ( 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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Figure 3Immunofluorescence 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).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.(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.


Figure 4Quantification 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 . ( c ) Representative WB analysis of neonatal myosin heavy chain ( neonatal MHC ) expression and quantification expressed as the ratio of neonatal MHC / β - actin in untreated mdx mice and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * * p < 0 . 0001 ( n = 10 mice per group ) . ( d ) Specific force ( maximal tetanic force normalized to cross - sectional area , Po / CSA ) from TA and VM muscles in C57Bl6 / J mice , untreated mdx mice , and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( n = 10 mice per group ) . SERCA activity quantified as ATPase hydrolysis activity vs . min ( e ) in C57Bl6 / J mice , untreated mdx mice , and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( n = 10 mice per group ) .

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Figure 4Quantification 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.(c) Representative WB analysis of neonatal myosin heavy chain (neonatal MHC) expression and quantification expressed as the ratio of neonatal MHC/β-actin in untreated mdx mice and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; ***p<0.0001 (n=10 mice per group).(d) Specific force (maximal tetanic force normalized to cross-sectional area, Po/CSA) from TA and VM muscles in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).SERCA activity quantified as ATPase hydrolysis activity vs. min (e) in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).


Figure 5 ( a ) Representative WB analysis of active 48 kDa and inactive 60 kDa calcineurin A and β - actin expression in the TA and VM muscles in untreated C57BL mice , untreated mdx mice and mdx treated with low and high doses of anti - IGF2R for 4 and 9 weeks . Densitometry analysis of data expressed as ratios of 48 kDa and 60 kDa calcineurin A / β - actin in arbitrary units . Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ( n = 10 mice per group ) . ( b ) Representative WB analysis of CAMKII , p - CAMKII and GAPDH expression quantified and shown as CAMKII / GAPDH and p - CAMKII / CAMKII ratios in arbitrary units . Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ( n = 10 mice per group ) . ( c ) Representative WB analysis of PKCα , p - PKCα and β - actin expression levels quantified and expressed as the PKCα / β - actin and p - PKCα / PKCα ratios in arbitrary units ( n = 10 mice per group ) . All values are expressed as the mean ± SEM .

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Figure 5(a) Representative WB analysis of active 48 kDa and inactive 60 kDa calcineurin A and β-actin expression in the TA and VM muscles in untreated C57BL mice, untreated mdx mice and mdx treated with low and high doses of anti-IGF2R for 4 and 9 weeks. Densitometry analysis of data expressed as ratios of 48 kDa and 60 kDa calcineurin A/β-actin in arbitrary units. Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001 (n=10 mice per group).(b) Representative WB analysis of CAMKII, p-CAMKII and GAPDH expression quantified and shown as CAMKII/GAPDH and p-CAMKII/CAMKII ratios in arbitrary units. Two-way ANOVA. *p<0.05; **p<0.01 (n=10 mice per group).(c) Representative WB analysis of PKCα, p-PKCα and β-actin expression levels quantified and expressed as the PKCα/β-actin and p-PKCα/PKCα ratios in arbitrary units (n=10 mice per group). All values are expressed as the mean±SEM.


Figure 6WB analysis performed in TA and VM muscles of mdx mice treated with anti - IGF2R using antibodies against CD20 , pSer + pThr and vinculin . Densitometry analysis of data expressed as the pSer + pThr / CD20 and CD20 / vinculin ratio in arbitrary units . One - way ANOVA * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) ( a ) . Representative WB analysis performed on TA and VM muscles of mdx mice treated with anti - IGF2R using anti - IGF1R , anti - pospho - IGF1R and GAPDH . Densitometry analysis of data expressed as the pIGF1R / IGF1R ratio normalized to GAPDH content in arbitrary units . One - way ANOVA . * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) ( b ) . All values are expressed as the mean ± SEM .

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Figure 6WB analysis performed in TA and VM muscles of mdx mice treated with anti-IGF2R using antibodies against CD20, pSer+pThr and vinculin. Densitometry analysis of data expressed as the pSer+pThr/CD20 and CD20/vinculin ratio in arbitrary units. One-way ANOVA *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group)(a).Representative WB analysis performed on TA and VM muscles of mdx mice treated with anti-IGF2R using anti-IGF1R, anti-pospho-IGF1R and GAPDH. Densitometry analysis of data expressed as the pIGF1R/IGF1R ratio normalized to GAPDH content in arbitrary units. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group) (b). All values are expressed as the mean±SEM.


Figure 7Corrosion casting results showing the linearization of capillaries in IGF2R - treated VM mdx muscles . Pericytes around capillaries are shown in the insert box ( a ) . Scale bars = 100μm . Representative immunofluorescence results ( b ) of longitudinal sections of VM mdx muscles for the CD146 - and NG2 - positive capillaries are shown . Representative immunofluorescence quantification ( c ) of longitudinal sections of VM mdx muscles for the CD146 - and NG2 - positive capillaries are shown . Scale bars = 75μm . One - way ANOVA . * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) . All values are expressed as the mean ± SEM . ( d ) Representative WB showing CD146 and NG2 expression in total protein lysates of TA and VM muscles of mdx mice treated with anti - IGF2R . Densitometry analysis of data expressed as the ratio of the indicated antibody / β - actin in arbitrary units . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) . All values are expressed as the mean ± SEM . ( e ) Representative Ve - cadherin ( red ) and CD31 ( green ) expression in small vessels of VM muscles from untreated mdx and mdx treated with low dose of anti - IGF2R for 4 weeks . Scale bars = 25 μm .

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Figure 7Corrosion casting results showing the linearization of capillaries in IGF2R-treated VM mdx muscles. Pericytes around capillaries are shown in the insert box (a). Scale bars=100μm.Representative immunofluorescence results (b) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown.Representative immunofluorescence quantification (c) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown. Scale bars=75μm. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean± SEM.(d) Representative WB showing CD146 and NG2 expression in total protein lysates of TA and VM muscles of mdx mice treated with anti-IGF2R. Densitometry analysis of data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean ± SEM.(e) Representative Ve-cadherin (red) and CD31 (green) expression in small vessels of VM muscles from untreated mdx and mdx treated with low dose of anti-IGF2R for 4 weeks. Scale bars=25 μm.


Figure 1C , Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A . Total protein extracts ( ZT13 ) from 10 - day - old seedlings grown under 12h / 12h light / dark cycles were subject to heat ( 30ºC 20min ) , pyrophosphatase ( PP ) and NaF / Na3VO4 ( phosphatase inhibitors ) treatments . TOC1 - GFP at different phosphorylation states were separated by 8 % SDS - PAGE ( acrylamide : bisacrylamide 150 : 1 ) and subsequently detected by α - GFP .

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Figure 1C, Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A. Total protein extracts (ZT13) from 10-day-old seedlings grown under 12h/12h light/dark cycles were subject to heat (30ºC 20min), pyrophosphatase (PP) and NaF/Na3VO4 (phosphatase inhibitors) treatments. TOC1-GFP at different phosphorylation states were separated by 8% SDS-PAGE (acrylamide:bisacrylamide 150:1) and subsequently detected by α-GFP.


Figure 2A - F Shown are hypocotyl lengths of cRL - grown 4 - day - old seedlings of toc1 complemented with wild - type TOC1 ( A ) , 5X ( B ) , T135A ( C ) , S175A ( D ) driven by TOC1 native promoter and wild - type TOC1 ( E ) , 5X ( F ) driven by 35S promoter at sequential fluent rates . Different letters indicate statistically significant differences between genotypes ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) , error bars indicate SEM . Data are representative of three biological trials with similar results .

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Figure 2A-F Shown are hypocotyl lengths of cRL-grown 4-day-old seedlings of toc1 complemented with wild-type TOC1 (A), 5X (B), T135A (C), S175A (D) driven by TOC1 native promoter and wild-type TOC1 (E), 5X (F) driven by 35S promoter at sequential fluent rates. Different letters indicate statistically significant differences between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test), error bars indicate SEM. Data are representative of three biological trials with similar results.


Figure 3Hypocotyl lengths in SD condition of Col - 0 , toc1 - 101 and two independent native or 35S promoter lines of wild - type TOC1 and each phosphosite mutants . The middle line of the box represents the median , the x in the box represents the mean . The bottom line and the top line of the box represent 1st and 3rd quartile , respectively . The whiskers extend from the ends of the box to the minimum value and maximum value . Data information : 18 to 27 seedlings per line were entrained in 12h / 12h light / dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2 - h intervals for 1 week . C - E , Average CCA1 : LUC bioluminescence traces of indicated plant lines . Data information : 18 to 27 seedlings per line were entrained in 12h / 12h light / dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2 - h intervals for 1 week . White and gray regions indicate subjective light and dark period . Data are representative of at least two independent trials with similar results . Different letters indicate statistically significant differences in period between genotypes ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) .

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Figure 3Hypocotyl lengths in SD condition of Col-0, toc1-101 and two independent native or 35S promoter lines of wild-type TOC1 and each phosphosite mutants. The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week.C-E, Average CCA1:LUC bioluminescence traces of indicated plant lines. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week. White and gray regions indicate subjective light and dark period. Data are representative of at least two independent trials with similar results. Different letters indicate statistically significant differences in period between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test).


Figure 4A - C , PIL1 , AT5G02580 and CDF5 expression pattern in 3 - d - old SD - grown seedlings from indicated plant lines at night . Data is representative of 3 biological trials .

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Figure 4A-C, PIL1, AT5G02580 and CDF5 expression pattern in 3-d-old SD-grown seedlings from indicated plant lines at night. Data is representative of 3 biological trials.


Figure 5A , Co - IP assay of PIF3 - TAP with TOC1 - GFP vs . 5X - GFP in N . benthamiana . Protein extracts were immunoprecipitated by IgG resins followed by HRV - 3C protease digestion . PIF3 and TOC1 were detected by anti - MYC and anti - GFP , respectively . B , Quantification of protein interaction in A . Results from 3 independent trials were averaged . Error bars indicate SEM , asterisks indicate significant difference ( * p < 0 . 05 , student ' s t test ) . C , ChIP - qPCR assay of TOC1 binding to promoter of pre - dawn - phased PIF3 target genes at ZT14 in Col - 0 , native promoter TOC1 and 5X lines . D , ChIP - qPCR assay of the chromatin residence of native promoter TOC1 - GFP at target promoters in toc1 vs . pif3 / 4 / 5 toc1 ( pif345t1 ) mutant backgrounds . Data information : 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 - GFP was immunoprecipitated by α - GFP and magnetic protein G beads . Data from 3 independent trials were averaged , error bars indicate SEM . Asterisks indicates significant differences ( * * p < 0 . 001 , * * * p < 0 . 0001 , Student ' s t test ) , n . s = not significant .

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Figure 5A, Co-IP assay of PIF3-TAP with TOC1-GFP vs. 5X-GFP in N. benthamiana. Protein extracts were immunoprecipitated by IgG resins followed by HRV-3C protease digestion. PIF3 and TOC1 were detected by anti-MYC and anti-GFP, respectively. B, Quantification of protein interaction in A. Results from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant difference (*p<0.05, student's t test). C, ChIP-qPCR assay of TOC1 binding to promoter of pre-dawn-phased PIF3 target genes at ZT14 in Col-0, native promoter TOC1 and 5X lines. D, ChIP-qPCR assay of the chromatin residence of native promoter TOC1-GFP at target promoters in toc1 vs. pif3/4/5 toc1 (pif345t1) mutant backgrounds. Data information: 3-d-old SD-grown seedlings were harvested at ZT14, TOC1-GFP was immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicates significant differences (** p<0.001, *** p<0.0001, Student's t test), n.s=not significant.


Figure 6B , Measurement of TOC1 - GFP protein turn - over in TOC1 , 5X , T135A , S175A native promoter lines by cycloheximide ( CHX ) treatment . 10 - d - old seedlings grown in 12h / 12h light / dark cycles were subject to cycloheximide ( CHX ) or ethanol ( Mock ) treatment for indicated time length . Data from 3 independent trials were fitted to non - linear Weibull regression curves . Data information : TOC1 protein was detected by α - GFP , ADK was used as loading control . TOC1 / ADK ratio was calculated by the intensities of TOC1 - GFP bands normalized by the intensities of ADK bands using ImageJ . C , Estimates of TOC1 - GFP protein half - life in B . Error bars indicate SEM , statistical analyses were performed with student ' s t test , different letters indicate significant differences ( p < 0 . 05 ) . Data from 3 independent trials were averaged .

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 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

Figure 6B, Measurement of TOC1-GFP protein turn-over in TOC1, 5X, T135A, S175A native promoter lines by cycloheximide (CHX) treatment. 10-d-old seedlings grown in 12h/12h light/dark cycles were subject to cycloheximide (CHX) or ethanol (Mock) treatment for indicated time length. Data from 3 independent trials were fitted to non-linear Weibull regression curves. Data information: TOC1 protein was detected by α-GFP, ADK was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK bands using ImageJ.C, Estimates of TOC1-GFP protein half-life in B. Error bars indicate SEM, statistical analyses were performed with student's t test, different letters indicate significant differences (p<0.05). Data from 3 independent trials were averaged.


Figure 7B , Quantification of interactions between TOC1 / 5X - TAP and NF - YC proteins in presence or absence of NF - YB subunits . Proteins were transiently expressed in N . benthamiana . TOC1 / 5X were immunoprecipitated with IgG resins followed by HRV - 3C protease digestion . TOC1 / 5X and NF - YB / C were detected by α - MYC and α - HA , respectively . Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co - immunoprecipitated NF - YC to immunoprecipitated TOC1 / 5X to NF - YC input . Results from 3 independent trials were averaged . Error bars indicate SEM , different letters denote significant differences ( p < 0 . 05 , Student ' s t test ) . C , ChIP - qPCR assay of native promoter driven TOC1 - GFP and 5X - GFP ' s residence at target promoters in toc1 vs . nf - yc3 / 4 / 9 toc1 ( c349t1 ) mutants . 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 and 5X were immunoprecipitated by α - GFP and magnetic protein G beads . Data from 3 independent trials were averaged . Error bars indicate SEM , asterisks indicate significant differences ( * p < 0 . 05 , * * * p < 0 . 0001 , Student ' s t test ) , n . s = not significant .

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 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

Figure 7B, Quantification of interactions between TOC1/5X-TAP and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TOC1/5X were immunoprecipitated with IgG resins followed by HRV-3C protease digestion. TOC1/5X and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated TOC1/5X to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, Student's t test).C, ChIP-qPCR assay of native promoter driven TOC1-GFP and 5X-GFP's residence at target promoters in toc1 vs. nf-yc3/4/9 toc1 (c349t1) mutants. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant differences (* p<0.05, *** p<0.0001, Student's t test), n.s= not significant.


Figure 8A , Co - IP assay of HDA15 - TAP with TOC1 - GFP and 5X - GFP in N . benthamiana . HDA15 - TAP was immunoprecipitated with IgG resins and digested by HRV - 3C protease . HDA15 and TOC1 were detected by α - MYC and α - GFP , respectively . B , Quantification of the interaction strength in A . Results from 5 independent trials were averaged . Error bar indicates SEM , asterisk indicates significant difference ( * p < 0 . 05 , student ' s t test ) . C and D , Hypocotyl lengths and free - running periods of indicated plants lines . Data of ~ 75 plants from 3 independent experiments were averaged , different letters indicate statistically significant differences ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) . The middle line of the box represents the median , the x in the box represents the mean . The bottom line and the top line of the box represent 1st and 3rd quartile , respectively . The whiskers extend from the ends of the box to the minimum value and maximum value . E , Expression of PIL1 , AT5G02580 and CDF5 in 3 - d - old SD - grown seedlings at ZT18 . Data from 3 independent experiments were averaged . Error bars indicate SEM . Different letters denote statistically significant differences based on Wilcoxon test ( p < 0 . 05 ) .

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

Figure 8A, Co-IP assay of HDA15-TAP with TOC1-GFP and 5X-GFP in N. benthamiana. HDA15-TAP was immunoprecipitated with IgG resins and digested by HRV-3C protease. HDA15 and TOC1 were detected by α-MYC and α-GFP, respectively. B, Quantification of the interaction strength in A. Results from 5 independent trials were averaged. Error bar indicates SEM, asterisk indicates significant difference (*p<0.05, student's t test). C and D, Hypocotyl lengths and free-running periods of indicated plants lines. Data of ~75 plants from 3 independent experiments were averaged, different letters indicate statistically significant differences (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test). The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value.E, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 independent experiments were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).


Figure 9A and B , ChIP - qPCR assay of HDA15 ' s ( A ) and NF - YC9 ' s ( B ) residence at target promoters in presence or absence of TOC1 and PRR5 . GFP - HDA15 and GFP - NF - YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col - 0 , toc1 single and prr5 toc1 double mutant grown in SD condition . Results were normalized by % input of PIL1 coding region ( PIL1 CDS ) . Data from at least 3 independent experiments were averaged , error bars indicate SEM . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 001 , * * * p < 0 . 0001 , Student ' s t test ) . C and D , Dual luciferase assay of the repression activity of GFP - TOC1 and GFP - 5X on promoter of AT5G02580 ( C ) and CDF5 ( D ) in protoplasts of toc1 and hda15toc1 ( h15t1 ) mutants . AT5G02580 : LUC , CDF5 : LUC reporters and the effectors ( empty GFP , GFP - TOC1 , and GFP - 5X ) were co - transfected to toc1 and hda15 toc1 protoplasts with 35S : Renilla - LUC as internal control . Data are means ± SEM ( n = 5 biological trials ) relative to empty GFP which is set as 1 . Different letters indicate statistically significant differences ( student ' s t test , p < 0 . 05 ) .

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 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

Figure 9A and B, ChIP-qPCR assay of HDA15's (A) and NF-YC9's (B) residence at target promoters in presence or absence of TOC1 and PRR5. GFP-HDA15 and GFP-NF-YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col-0, toc1 single and prr5 toc1 double mutant grown in SD condition. Results were normalized by % input of PIL1 coding region (PIL1 CDS). Data from at least 3 independent experiments were averaged, error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.001, *** p<0.0001, Student's t test).C and D, Dual luciferase assay of the repression activity of GFP-TOC1 and GFP-5X on promoter of AT5G02580 (C) and CDF5 (D) in protoplasts of toc1 and hda15toc1 (h15t1) mutants. AT5G02580:LUC, CDF5:LUC reporters and the effectors (empty GFP, GFP-TOC1, and GFP-5X) were co-transfected to toc1 and hda15 toc1 protoplasts with 35S:Renilla-LUC as internal control. Data are means ± SEM (n = 5 biological trials) relative to empty GFP which is set as 1. Different letters indicate statistically significant differences (student's t test, p<0.05).


Figure 10A , Quantification of interactions between TAP - PRR5 and NF - YC proteins in presence or absence of NF - YB subunits . Proteins were transiently expressed in N . benthamiana . TAP - PRR5 was immunoprecipitated with IgG resins followed by HRV - 3C protease digestion . PRR5 and NF - YB / C were detected by α - MYC and α - HA , respectively . Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co - immunoprecipitated NF - YC to immunoprecipitated PRR5 to NF - YC input . Results from 3 independent trials were averaged . Error bars indicate SEM , different letters denote significant differences ( p < 0 . 05 , student ' s t test ) . C , ChIP - qPCR assay of native promoter TOC1 - GFP and 5X - GFP binding to the target promoters at ZT14 in toc1 vs . prr5 toc1 ( p5t1 ) mutant . 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 and 5X were immunoprecipitated by α - GFP . Data from 3 independent trials were averaged , error bars indicate SEM . Asterisks indicate significant differences ( Student ' s t test ; * p < 0 . 05 ) , n . s = not significant . D , Expression of PIL1 , AT5G02580 and CDF5 in 3 - d - old SD - grown seedlings at ZT18 . Data from 3 biological trials were averaged . Error bars indicate SEM . Different letters denote statistically significant differences based on Wilcoxon test ( p < 0 . 05 ) .

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 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

Figure 10A, Quantification of interactions between TAP-PRR5 and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TAP-PRR5 was immunoprecipitated with IgG resins followed by HRV-3C protease digestion. PRR5 and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated PRR5 to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, student's t test).C, ChIP-qPCR assay of native promoter TOC1-GFP and 5X-GFP binding to the target promoters at ZT14 in toc1 vs. prr5 toc1 (p5t1) mutant. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicate significant differences (Student's t test; * p<0.05), n.s= not significant.D, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 biological trials were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).


Figure 1A . Rabies virus infection kinetics in the central nervous system of infected mice . Tha - RABV load ( copies / µg of RNA ) detected in the thoracolumbar spinal cord , in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time - points post - infection ( n = 4 per time - point ) . nd : not detected . Horizontal lines indicate the median . B . Follow - up of the clinical signs of mice under different treatments . Heatmaps were established based on a progressive 0 - 7 clinical score scale ( 0 : no apparent changes | 1 : ruffled fur | 2 : slow movement , hind limb ataxia | 3 : apathy | 4 : monoplegia | 5 : hind limb paralysis , tremors | 6 : paralysis , conjunctivitis / keratitis , urine staining of the haircoat of the perineum | 7 : death ) . Each line represents one animal throughout time . C . Body weight progression of mice under different treatments . Mice were weighted on a daily basis during the treatment administration , and then twice a week up to 100 days post - infection . All the mice were equipped with iPrecio pumps weighting ca . 3 . 3 g . Each line represents one animal throughout time . D - E . Mouse behavioral testing . The tested animals were non - infected ( n = 3 mice with iPrecio pump + n = 4 age - related mice without iPrecio pump ) , infected non - treated ( n = 5 ) , infected and treated at 7 dpi ( n = 5 , mice # 9 to # 13 ) , and infected and treated at 8 dpi ( n = 5 , mice # 24 to # 28 ) . ( D ) Open field test performed at 7 dpi ( prodromal phase ) and after the remission of clinical signs for the survivors ( 80 dpi ) . ( E ) Rotarod motor performance at 7 dpi ( prodromal phase ) , 8 dpi ( symptomatic phase ) and after the remission of clinical signs for the survivors ( 20 , 30 and 60 dpi ) . Horizontal lines indicate the median . Kruskal - Wallis followed by the Dunn ' multiple comparisons test ( 7 and 8 days post - infection ) and Mann - Whitney test ( 20 , 30 and 60 days post - infection ) . The rotarod performance presented a negative correlation ( Spearman r = - 0 . 768 ) with the clinical score at 8 dpi . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Treatments description = NI : non - infected ( n = 5 ) ; NT : infected , non - treated ( n = 5 ) ; infected , treated at 6 dpi ( 2 + 2 mg / kg ) ( n = 4 ) ; infected , treated at 7 dpi ( 2 + 2 mg / kg ) ( n = 9 ) ; infected , treated at 8 dpi ( 2 + 2 mg / kg ) ( n = 15 ) ; infected , treated at 8 dpi ( 10 + 10 mg / kg ) ( n = 4 ) .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rabies virus infection kinetics in the central nervous system of infected mice. Tha-RABV load (copies/µg of RNA) detected in the thoracolumbar spinal cord, in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time-points post-infection (n=4 per time-point). nd: not detected. Horizontal lines indicate the median.B. Follow-up of the clinical signs of mice under different treatments. Heatmaps were established based on a progressive 0-7 clinical score scale (0: no apparent changes | 1: ruffled fur | 2: slow movement, hind limb ataxia | 3: apathy | 4: monoplegia | 5: hind limb paralysis, tremors | 6: paralysis, conjunctivitis/ keratitis, urine staining of the haircoat of the perineum | 7: death). Each line represents one animal throughout time.C. Body weight progression of mice under different treatments. Mice were weighted on a daily basis during the treatment administration, and then twice a week up to 100 days post-infection. All the mice were equipped with iPrecio pumps weighting ca. 3.3 g. Each line represents one animal throughout time.D-E. Mouse behavioral testing. The tested animals were non-infected (n=3 mice with iPrecio pump + n=4 age-related mice without iPrecio pump), infected non-treated (n=5), infected and treated at 7 dpi (n=5, mice #9 to #13), and infected and treated at 8 dpi (n=5, mice #24 to #28). (D) Open field test performed at 7 dpi (prodromal phase) and after the remission of clinical signs for the survivors (80 dpi). (E) Rotarod motor performance at 7 dpi (prodromal phase), 8 dpi (symptomatic phase) and after the remission of clinical signs for the survivors (20, 30 and 60 dpi). Horizontal lines indicate the median. Kruskal-Wallis followed by the Dunn' multiple comparisons test (7 and 8 days post-infection) and Mann-Whitney test (20, 30 and 60 days post-infection). The rotarod performance presented a negative correlation (Spearman r = -0.768) with the clinical score at 8 dpi. *P<0.05, **P<0.01, ***P<0.001. Treatments description= NI: non-infected (n=5); NT: infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).


Figure 2A . Cumulative Kaplan - Meier survival curves of mice treated at different time - points post - infection . Log - rank ( Mantel - Cox ) test to compare treated groups with the infected , non - treated group . * * P < 0 . 01 , * * * P < 0 . 001 . Treatments description = infected , non - treated ( n = 5 ) ; infected , treated at 6 dpi ( 2 + 2 mg / kg ) ( n = 4 ) ; infected , treated at 7 dpi ( 2 + 2 mg / kg ) ( n = 9 ) ; infected , treated at 8 dpi ( 2 + 2 mg / kg ) ( n = 15 ) ; infected , treated at 8 dpi ( 10 + 10 mg / kg ) ( n = 4 ) . B . Cumulative Kaplan - Meier survival curves of all the treated animals that presented with clinical signs ( n = 31 , all treatments combined ) . Log - rank ( Mantel - Cox ) test to compare treated groups with the infected , non - treated group . * * * * P < 0 . 0001 . C . Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups . D . Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi ( LOD : limit of detection = 0 . 02 µg / mL ) . E . Viral load in the brain of mice from different experimental groups . The samples were either collected at the time of death , or at 100 dpi for the survivors . Cytokines and innate immune mediators ' profile in the brain of mice from different experimental groups : ( F ) IFN - β1 , ( G ) ISG15 , ( H ) Mx1 , detected in one brain hemisphere of mice from different experimental groups . The expression of the genes of interest was normalized to the GAPDH housekeeping gene . Cytokines and innate immune mediators ' profile in the brain of mice from different experimental groups : , ( I ) IFN - γ , ( J ) CXCL10 , ( K ) CCL5 , ( L ) IL - 6 , ( M ) TNF - α and ( N ) IL - 1β , detected in one brain hemisphere of mice from different experimental groups . The expression of the genes of interest was normalized to the GAPDH housekeeping gene .

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

Figure 2A. Cumulative Kaplan-Meier survival curves of mice treated at different time-points post-infection. Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. **P<0.01, ***P<0.001. Treatments description= infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).B. Cumulative Kaplan-Meier survival curves of all the treated animals that presented with clinical signs (n=31, all treatments combined). Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. ****P<0.0001.C. Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups.D. Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi (LOD: limit of detection = 0.02 µg/mL).E. Viral load in the brain of mice from different experimental groups. The samples were either collected at the time of death, or at 100 dpi for the survivors.Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: (F) IFN-β1, (G) ISG15, (H) Mx1, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: , (I) IFN-γ, (J) CXCL10, (K) CCL5, (L) IL-6, (M) TNF-α and (N) IL-1β, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.