Figure 1A DNA content of mCFU - E and BaF3 - EpoR cells in response to different Epo concentrations . [ 3H ] - Thymidine incorporation was measured after 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR ) . Data represented as mean ± standard deviation , N = 3 . Lines represent sigmoidal regression . EC50 values are given . B Cell doubling with time in response to 50 U / ml Epo . Cell numbers were determined by manual counting using trypan blue exclusion assay . Data represented as mean ± standard deviation , N = 3 . Lines represent exponential regression . Doubling time is indicated . C Size determinations of mCFU - E and BaF3 - EpoR cells . Exemplary fluorescence microscopy pictures upon Hoechst staining for nucleus visualization with 60x objective . The bar respresents 10 µm distance ( upper panel ) . Cell diameter was measured by Imaging flow cytometry . Cytoplasm was stained with Calcein , nuclei were stained with DRAQ5 . Probability density function of size distribution with indicated mean diameter of mCFU - E and BaF3EpoR cells . All cells were growth - factor deprived and unstimulated . D Epo - induced phosphorylation of EpoR , AKT and ERK of mCFU - E and BaF3 - EpoR cells . Above each panel the number of growth - factor deprived cells examined per time point is indicated as well as the concentration of Epo applied for stimulation . To the left the position of the molecular weight marker is indicated in kDa and arrowheads indicate the position of the protein of interest . For the detection of the EpoR , 5x106 mCFU - E cells were stimulated with 2 . 5 U / ml Epo and 5x106 BaF3 - EpoR cells were stimulated with 5 U / ml Epo . Cells were lysed , subjected to immunoprecipitation with anti - EpoR and were analyzed by immunoblotting using either anti - pTyr ( pEpoR ) or anti - EpoR ( EpoR ) antibodies . For the detection of AKT , mCFU - E cells were stimulated with 2 . 5 U / ml Epo and BaF3 - EpoR cells were stimulated with 5 U / ml Epo . Per time point cellular lysates equivalent to 5x105 mCFU - E cells and 1x106 BaF3 - EpoR cells were analyzed by immunoblotting using anti - pAKT and anti - AKT antibodies . For the detection of ERK , cells were stimulated with 50 U / ml Epo . Per time point cellular lysates equivalent to 8x105 cells were analyzed by immunoblotting . Immunoblot detection was performed with chemiluminescence utilizing a CCD camera device ( ImageQuant ) . E Absolute concentrations of pEpoR , pAKT and ppERK in mCFU - E and BaF3 - EpoR cells with time in response to 50 U / ml Epo . Experimental data of a representative experiment is depicted with filled circles , dashed lines represent splines . Error bars indicate standard deviation estimated by an error model . F Expression of cell - cycle indicator genes of mCFU - E and BaF3 - EpoR cells in response to 5 U / ml Epo . Genes were selected based on microarray analysis . Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation , N = 3 . Welch Modified Two - Sample t - Test , * p < 0 . 05 . G Fold change of fractions of cells in S / G2 / M phase of the cell cycle with respect to 0 U / ml Epo . Growth - factor deprived cells were stimulated with indicated Epo doses for given time . Fractions in sub - G1 , G1 , S , G2 / M were determined by propidium iodide ( PI ) staining for DNA content . N = 3 . Welch Modified Two - Sample t - Test , n . s . = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . The percentage of cells in S / G2 / M phase in unstimulated cells is additionally indicated .

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

Figure 1A DNA content of mCFU-E and BaF3-EpoR cells in response to different Epo concentrations. [3H]-Thymidine incorporation was measured after 14 h (mCFU-E) or 38 h (BaF3-EpoR). Data represented as mean ± standard deviation, N=3. Lines represent sigmoidal regression. EC50 values are given.B Cell doubling with time in response to 50 U/ml Epo. Cell numbers were determined by manual counting using trypan blue exclusion assay. Data represented as mean ± standard deviation, N=3. Lines represent exponential regression. Doubling time is indicated.C Size determinations of mCFU-E and BaF3-EpoR cells. Exemplary fluorescence microscopy pictures upon Hoechst staining for nucleus visualization with 60x objective. The bar respresents 10 µm distance (upper panel). Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of mCFU-E and BaF3EpoR cells. All cells were growth-factor deprived and unstimulated.D Epo-induced phosphorylation of EpoR, AKT and ERK of mCFU-E and BaF3-EpoR cells. Above each panel the number of growth-factor deprived cells examined per time point is indicated as well as the concentration of Epo applied for stimulation. To the left the position of the molecular weight marker is indicated in kDa and arrowheads indicate the position of the protein of interest. For the detection of the EpoR, 5x106 mCFU-E cells were stimulated with 2.5 U/ml Epo and 5x106 BaF3-EpoR cells were stimulated with 5 U/ml Epo. Cells were lysed, subjected to immunoprecipitation with anti-EpoR and were analyzed by immunoblotting using either anti-pTyr (pEpoR) or anti-EpoR (EpoR) antibodies. For the detection of AKT, mCFU-E cells were stimulated with 2.5 U/ml Epo and BaF3-EpoR cells were stimulated with 5 U/ml Epo. Per time point cellular lysates equivalent to 5x105 mCFU-E cells and 1x106 BaF3-EpoR cells were analyzed by immunoblotting using anti-pAKT and anti-AKT antibodies. For the detection of ERK, cells were stimulated with 50 U/ml Epo. Per time point cellular lysates equivalent to 8x105 cells were analyzed by immunoblotting. Immunoblot detection was performed with chemiluminescence utilizing a CCD camera device (ImageQuant).E Absolute concentrations of pEpoR, pAKT and ppERK in mCFU-E and BaF3-EpoR cells with time in response to 50 U/ml Epo. Experimental data of a representative experiment is depicted with filled circles, dashed lines represent splines. Error bars indicate standard deviation estimated by an error model.F Expression of cell-cycle indicator genes of mCFU-E and BaF3-EpoR cells in response to 5 U/ml Epo. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, * p<0.05.G Fold change of fractions of cells in S/G2/M phase of the cell cycle with respect to 0 U/ml Epo. Growth-factor deprived cells were stimulated with indicated Epo doses for given time. Fractions in sub-G1, G1, S, G2/M were determined by propidium iodide (PI) staining for DNA content. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05 , ** p < 0.01 , *** p < 0.005. The percentage of cells in S/G2/M phase in unstimulated cells is additionally indicated.


Figure 2B - G Model calibration with time - resolved quantitative immunoblot data of mCFU - E cells in blue and BaF3 - EpoR cells in black . Growth - factor deprived mCFU - E cells ( 5x106 cells per condition ) and BaF3 - EpoR cells ( 1x107 cells per condition ) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR ( B ) , pAKT ( C ) , ppERK ( D ) . The scale for pS6 ( E ) was estimated in arbitrary units . GTP - Ras ( F ) and ppERK were determined upon stimulation with indicated , color - coded Epo doses . pEpoR was analyzed by immunoprecipitation followed by immunoblotting , GTP - Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting . For pAkt and ppERK , cellular lysates were subjected to quantitative immunoblotting . Calibrator proteins were used for EpoR , AKT , GTP - Ras and ERK to facilitate the conversion to nM concentrations . Experimental data represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories .

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Figure 2B-G Model calibration with time-resolved quantitative immunoblot data of mCFU-E cells in blue and BaF3-EpoR cells in black. Growth-factor deprived mCFU-E cells (5x106 cells per condition) and BaF3-EpoR cells (1x107 cells per condition) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR (B), pAKT (C), ppERK (D). The scale for pS6 (E) was estimated in arbitrary units.GTP-Ras (F) and ppERK were determined upon stimulation with indicated, color-coded Epo doses. pEpoR was analyzed by immunoprecipitation followed by immunoblotting, GTP-Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting. For pAkt and ppERK, cellular lysates were subjected to quantitative immunoblotting. Calibrator proteins were used for EpoR, AKT, GTP-Ras and ERK to facilitate the conversion to nM concentrations. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.


Figure 3A Model calibration with mCFU - E wild - type cells , and mCFU - E cells overexpressing SHIP1 or PTEN , and BaF3 - EpoR wild - type cells . Experimental data represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories . B Model prediction of pAKT dynamics in BaF3 - EpoR cells overexpressing PTEN or SHIP1 . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model . D Experimental validation of basal DUSP expression in mCFU - E and BaF3 - EpoR . Quantitative RT - PCR was performed with all samples on the same plate , allowing a direct comparison of mRNA levels between mCFU - E and BaF3 - EpoR cells . Data is normalized to the Rpl32 gene . Ratios of expression in BaF3 - EpoR cells compared to mCFU - E cells are shown as box plots . N = 10 . For details , see Appendix K .

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Figure 3A Model calibration with mCFU-E wild-type cells, and mCFU-E cells overexpressing SHIP1 or PTEN, and BaF3-EpoR wild-type cells. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.B Model prediction of pAKT dynamics in BaF3-EpoR cells overexpressing PTEN or SHIP1. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model.D Experimental validation of basal DUSP expression in mCFU-E and BaF3-EpoR. Quantitative RT-PCR was performed with all samples on the same plate, allowing a direct comparison of mRNA levels between mCFU-E and BaF3-EpoR cells. Data is normalized to the Rpl32 gene. Ratios of expression in BaF3-EpoR cells compared to mCFU-E cells are shown as box plots. N=10. For details, see Appendix K.


Figure 4A Model prediction and experimental validation for pAKT , ppERK and pS6 dynamics of 32D - EpoR cells in response to 50 U / ml Epo . Simulations are based on measured , cell - type - specific protein abundance of 32D - EpoR cells and global kinetic rates estimated from mCFUE and BaF3 - EpoR cells . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model . C Model prediction and experimental validation for RSK , AKT , ERK and S6 activation upon RSK overexpression ( oe ) and 5 U / ml Epo stimulation in 32DEpoR cells . Simulations are based on measured , cell - type - specific protein abundance of 32D - EpoR cells and global kinetic rates estimated from mCFUE and BaF3 - EpoR cells . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model . Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D - EpoR wild - type ( wt ) cells , N = 4 ( lower right panel ) . Two - Sample t - Test , * * * p < 0 . 005 . D Impact of AKT , Ras or PTEN overexpression on BaF3 - induced pS6 dynamics in EpoR and BaF3 - PTEN cells . Quantitative AKT upon overexpression of PTEN , Ras , a constitutive active BaF3 protein , or the empty vector control in EpoR cells or BaF3 - mCFU - E cells . BaF3 - factor deprived mCFU - E cells ( 5x106 cells per condition ) and Epo - immunoblotting cells ( 1x107 cells per condition ) were stimulated with 5 U / ml Epo for indicated time points . Cellular lysates were analyzed by S6 employing sequential reprobing anti - pAKT , anti - ppERK , anti - pS6 , anti - Epo and to ensure equal loading with anti - beta - actin antibodies . Detection was performed with chemiluminescence using a CCD camera device ( ImageQuant ) . Quantification of pS6 on the right is depicted as fold change to wild - type samples at 30 minutes after Epo stimulation . Error bars represent standard deviation . oe : overexpression . N = 3 . Welch Modified Two - Sample t - Test , n . s . = not significant , * p < 0 .

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 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 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

Figure 4A Model prediction and experimental validation for pAKT, ppERK and pS6 dynamics of 32D-EpoR cells in response to 50 U/ml Epo. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model.C Model prediction and experimental validation for RSK, AKT, ERK and S6 activation upon RSK overexpression (oe) and 5 U/ml Epo stimulation in 32DEpoR cells. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model. Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D-EpoR wild-type (wt) cells, N=4 (lower right panel). Two-Sample t-Test, *** p<0.005.D Impact of AKT, Ras or PTEN overexpression on BaF3-induced pS6 dynamics in EpoR and BaF3-PTEN cells. Quantitative AKT upon overexpression of PTEN, Ras, a constitutive active BaF3 protein, or the empty vector control in EpoR cells or BaF3-mCFU-E cells. BaF3-factor deprived mCFU-E cells (5x106 cells per condition) and Epo-immunoblotting cells (1x107 cells per condition) were stimulated with 5 U/ml Epo for indicated time points. Cellular lysates were analyzed by S6 employing sequential reprobing anti-pAKT, anti-ppERK, anti-pS6, anti-Epo and to ensure equal loading with anti-beta-actin antibodies. Detection was performed with chemiluminescence using a CCD camera device (ImageQuant). Quantification of pS6 on the right is depicted as fold change to wild-type samples at 30 minutes after Epo stimulation. Error bars represent standard deviation. oe: overexpression. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05


Figure 5A Epo - induced signaling upon AKT or MEK inhibitor treatment . Growth - factor deprived cells were pretreated for half an hour with AKT VIII or U0126 , respectively , and subsequently stimulated with 5 U / ml Epo . PDI served as loading control . B Model calibration with integrated pAKT , ppERK and pS6 data upon inhibitor treatment and 5 U / ml Epo stimulation of mCFU - E , 32D - EpoR and BaF3 - EpoR cells . Area under curve from time - resolved , quantitative immunoblotting data was calculated and is represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories . C Analysis of cell - cycle indicator genes upon AKT VIII and U0126 treatment . Growth - factor deprived cells were pretreated for half an hour indicated doses of a single inhibitor , followed by stimulation with 5 U / ml Epo for 0 h and 3 h . The expression of cyclinD2 , cyclinG2 and p27 was measured by quantitative RT - PCR and normalized to the Rpl32 gene . Genes were selected based on microarray analysis . Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation , N = 3 . Welch Modified Two - Sample t - Test , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A Epo-induced signaling upon AKT or MEK inhibitor treatment. Growth-factor deprived cells were pretreated for half an hour with AKT VIII or U0126, respectively, and subsequently stimulated with 5 U/ml Epo. PDI served as loading control.B Model calibration with integrated pAKT, ppERK and pS6 data upon inhibitor treatment and 5 U/ml Epo stimulation of mCFU-E, 32D-EpoR and BaF3-EpoR cells. Area under curve from time-resolved, quantitative immunoblotting data was calculated and is represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.C Analysis of cell-cycle indicator genes upon AKT VIII and U0126 treatment. Growth-factor deprived cells were pretreated for half an hour indicated doses of a single inhibitor, followed by stimulation with 5 U/ml Epo for 0 h and 3 h. The expression of cyclinD2, cyclinG2 and p27 was measured by quantitative RT-PCR and normalized to the Rpl32 gene. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, n.s. not significant, * p<0.05, ** p<0.01, *** p<0.005.


Figure 6B Linear regression fit for the respective cell - cycle indicator of experimental data . Data points represent mean ± standard deviation , N = 3 . Solid lines represent the linear regression fit .

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Figure 6B Linear regression fit for the respective cell-cycle indicator of experimental data. Data points represent mean ± standard deviation, N=3. Solid lines represent the linear regression fit.


Figure 7Lower panel : Experimental validation of PTEN and SHIP1 overexpression effects on Epo - dependent proliferation . Proliferation was assessed using [ 3H ] - Thymidine incorporation 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR ) after retroviral transduction with PTEN or SHIP1 construct for overexpression . Data represented as mean ± standard deviation , N = 3 . EC50 values are given . Lower panel : Experimental validation of proliferation upon combined inhibitor treatment and 5 U / ml Epo stimulation . Proliferation was measured as cell numbers after 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR , 32D - EpoR ) with Coulter Counter . Maximum was scaled to

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Figure 7Lower panel: Experimental validation of PTEN and SHIP1 overexpression effects on Epo-dependent proliferation. Proliferation was assessed using [3H]-Thymidine incorporation 14 h (mCFU-E) or 38 h (BaF3-EpoR) after retroviral transduction with PTEN or SHIP1 construct for overexpression. Data represented as mean ± standard deviation, N=3. EC50 values are given.Lower panel: Experimental validation of proliferation upon combined inhibitor treatment and 5 U/ml Epo stimulation. Proliferation was measured as cell numbers after 14 h (mCFU-E) or 38 h (BaF3-EpoR, 32D-EpoR) with Coulter Counter. Maximum was scaled to 1


Figure 8A Venn diagramm representing overlap of hCFU - E proteome from three independent healthy donors . In total 6 , 925 proteins were quantifiedB Size determinations of hCFU - E compared to mCFU - E cells . Cell diameter was measured by Imaging flow cytometry . Cytoplasm was stained with Calcein , nuclei were stained with DRAQ5 . Probability density function of size distribution with indicated mean diameter of hCFU - E and mCFU - E cells . All cells were growth - factor deprived and unstimulated . C Cytoplasmic volumes of BaF3 - EpoR , 32D - EpoR , mCFU - E and the hCFU - E cells . All volumes were determined using imaging flow cytometry as described in ( B ) . D Correlation between protein molecules per cell for key signaling components determined by quantiative immunoblotting or quantitative mass spectrometry and the " proteomic ruler " method ( Wiśniewski et al , 2014 ) in BaF3 - EpoR and mCFU - E cells . Diagonal line as guide to the eye . Spearman ' s rank - based coefficient of correlation ρ = 0 . 88 . E Protein abundance of key signaling components were determined by mass spectrometry of whole - cell lysates of hCFU - E cells . Copy numbers of proteins per cell were obtained by the " proteomic ruler " method and converted to cytoplasmic concentrations with the volumes calculated from ( B ) and shown in ( C ) . F Model prediction and experimental validation of proliferation upon single or combined inhibitor treatment with AKT VIII and U0126 upon 5 U / ml Epo stimulation . Maximum proliferation was scaled to 1 . Proliferation was measured as cell numbers , counted with a hemocytometer after 96 hours by trypan blue exclusion assay . Solid lines represent model trajectories . Experimental data is represented as mean ± standard deviation . N =

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

Figure 8A Venn diagramm representing overlap of hCFU-E proteome from three independent healthy donors. In total 6,925 proteins were quantifiedB Size determinations of hCFU-E compared to mCFU-E cells. Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of hCFU-E and mCFU-E cells. All cells were growth-factor deprived and unstimulated.C Cytoplasmic volumes of BaF3-EpoR, 32D-EpoR, mCFU-E and the hCFU-E cells. All volumes were determined using imaging flow cytometry as described in (B).D Correlation between protein molecules per cell for key signaling components determined by quantiative immunoblotting or quantitative mass spectrometry and the "proteomic ruler" method (Wiśniewski et al, 2014) in BaF3-EpoR and mCFU-E cells. Diagonal line as guide to the eye. Spearman's rank-based coefficient of correlation ρ=0.88.E Protein abundance of key signaling components were determined by mass spectrometry of whole-cell lysates of hCFU-E cells. Copy numbers of proteins per cell were obtained by the "proteomic ruler" method and converted to cytoplasmic concentrations with the volumes calculated from (B) and shown in (C).F Model prediction and experimental validation of proliferation upon single or combined inhibitor treatment with AKT VIII and U0126 upon 5 U/ml Epo stimulation. Maximum proliferation was scaled to 1. Proliferation was measured as cell numbers, counted with a hemocytometer after 96 hours by trypan blue exclusion assay. Solid lines represent model trajectories. Experimental data is represented as mean ± standard deviation. N=3


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 .

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

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 .

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

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

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 ) .

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

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 .

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 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

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 .

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

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 1Representative grey - scale images of primary rat hippocampal neurons ( DIV 10 ) transfected with GFP and the indicated miRNA mimics . Quantification of the mean number of intersections by Sholl analysis . GFP - only transfected conditions were set to one in each experiment . Representative grey - scale images of GFP - transfected Cacna1c + / + ( WT ) or Cacna1c + / - primary rat hippocampal neurons ( DIV 10 ) . Scale bars = 50 μm . Quantification of the mean number of intersections by Sholl analysis . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Unpaired two - sample t - Test , * * p = 0 . 0096 ) . Fold changes represent changes in dendritic complexity relative to the control condition .

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Figure 1Representative grey-scale images of primary rat hippocampal neurons (DIV 10) transfected with GFP and the indicated miRNA mimics. Quantification of the mean number of intersections by Sholl analysis. GFP-only transfected conditions were set to one in each experiment.Representative grey-scale images of GFP-transfected Cacna1c+/+ (WT) or Cacna1c+/- primary rat hippocampal neurons (DIV 10). Scale bars = 50 μm.   Quantification of the mean number of intersections by Sholl analysis. Data are represented as scattered dot plots with bar, mean±S.D. (n=3 independent experiments; Unpaired two-sample t-Test, **p=0.0096). Fold changes represent changes in dendritic complexity relative to the control condition.


Figure 2Representative images of DIV 10 hippocampal neurons co - transfected with control or miR - 499 - 5p mimics and a pMT2 - CACNB2 expression plasmid ( CACNB2 ) . Scale bars = 50 μm . Quantification of the mean number of intersections by Sholl analysis . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Two - way ANOVA : no main effect of the CACNB2 transfection p = 0 . 5027 or the miRNA mimics p = 0 . 0805 , main effect of the CACBN2 transfection by miRNA mimics interaction p = 0 . 0213 , Tukey ' s HSD : * p = 0 . 0303 ) . Fold changes represent the changes in dendritic complexity of transfected neurons compared to non - treated control neurons . ns = not significant .

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Figure 2Representative images of DIV 10 hippocampal neurons co-transfected with control or miR-499-5p mimics and a pMT2-CACNB2 expression plasmid (CACNB2). Scale bars = 50 μm.   Quantification of the mean number of intersections by Sholl analysis. Data are represented as scattered dot plots with bar, mean±S.D. (n=3 independent experiments; Two-way ANOVA: no main effect of the CACNB2 transfection p=0.5027 or the miRNA mimics p=0.0805, main effect of the CACBN2 transfection by miRNA mimics interaction p=0.0213, Tukey's HSD: *p=0.0303). Fold changes represent the changes in dendritic complexity of transfected neurons compared to non-treated control neurons. ns = not significant.


Figure 3A ) Representative images of DIV 19 rat hippocampal neurons co - transfected with GFP ( green channel ) and Cav1 . 2 - HA ( red channel ) , together with either control or miR - 499 - 5p mimics . After 12 - 13 days of expression , labeling with Anti - HA antibodies was performed in live conditions to identify cell surface Cav1 . 2 channels . Whole cells : scale bars = 20 μm ; cell body and dendrite insets : scale bars = 5 μm . B ) Quantification of the area occupied by surface Cav1 . 2 fluorescence normalized to the total cell area ( GFP fluorescence ) from non - permeabilized neurons transfected as in A ) . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 4 independent experiments ; Paired two - sample t - Test * p = 0 . 0318 ) .

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Figure 3A) Representative images of DIV 19 rat hippocampal neurons co-transfected with GFP (green channel) and Cav1.2-HA (red channel), together with either control or miR-499-5p mimics. After 12-13 days of expression, labeling with Anti-HA antibodies was performed in live conditions to identify cell surface Cav1.2 channels. Whole cells: scale bars = 20 μm; cell body and dendrite insets: scale bars= 5 μm. B) Quantification of the area occupied by surface Cav1.2 fluorescence normalized to the total cell area (GFP fluorescence) from non-permeabilized neurons transfected as in A). Data are represented as scattered dot plots with bar, mean±S.D. (n=4 independent experiments; Paired two-sample t-Test *p=0.0318).


Figure 4D ) Representative Western blot images of CACNB2 ( upper panel ) and GAPDH ( lower panel ) protein expression levels in the hippocampus of WT rats and Cacna1c + / - rats injected with the AAV - Control or AAV - miR - 499 hairpin viruses . GAPDH was used as a loading control E ) Quantification of CACNB2 Western blots using hippocampal protein lysate from wild type rats injected with either AAV - control or AAV - miR - 499 . Data are represented as box plot with whiskers and data points ( + : mean , line : median ; whiskers : minimum and maximum values ) . ( * p = 0 . 0103 , Unpaired two - sample t - Test ; n = 7 rats in the AAV - Control group , n = 6 rats in the AAV - miR - 499 group ) . F ) Quantification of CACNB2 Western blots using hippocampal protein lysate from Cacna1c + / - rats injected with either AAV - control or AAV - miR - 499 . Data are represented as box plot with whiskers and data points ( + : mean , line : median ; whiskers : minimum and maximum values ) . ( * * p = 0 . 0044 , Unpaired two - sample t - Test ; n = 8 rats in the AAV - Control group , n = 7 rats in the AAV - miR - 499 group ) .

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Figure 4D) Representative Western blot images of CACNB2 (upper panel) and GAPDH (lower panel) protein expression levels in the hippocampus of WT rats and Cacna1c +/- rats injected with the AAV-Control or AAV-miR-499 hairpin viruses. GAPDH was used as a loading control E) Quantification of CACNB2 Western blots using hippocampal protein lysate from wild type rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (*p=0.0103, Unpaired two-sample t-Test; n=7 rats in the AAV-Control group, n=6 rats in the AAV-miR-499 group). F) Quantification of CACNB2 Western blots using hippocampal protein lysate from Cacna1c +/- rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (**p=0.0044, Unpaired two-sample t-Test; n=8 rats in the AAV-Control group, n=7 rats in the AAV-miR-499 group).


Figure 5miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 26 , male = 31 ) , BD ( female = 26 , male = 37 ) , or MDD ( female = 18 , male = 24 ) subjects . Wilcoxon rank sum test after correction for age and antidepressant treatment ( linear model of the form Fold Change ~ Group * Sex + Age + Antidepressant treatment ) . 2 - way ANOVA with correction for sex , age and antidepressant treatment ( linear model of the form Fold Change ~ Group * Sex + Age + Antidepressant treatment ) . Control vs . BD : * * * * p = 8 . 89e - 07 ; control vs . MDD : n . s . p = 0 . 12674 . Data are presented as violin plots with median , quartiles and data points . miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 8 , male = 10 ) and SZ ( female = 8 , male = 15 ) subjects . 2 - way ANOVA , Main effect Sex : p = 0 . 9785 ; main effect Group : p = 0 . 0924 ; main effect Interaction : p = 0 . 9802 . Tukey ' s HSD : p = 0 . 0924 , ns . Data are presented as violin plots with median , quartiles and data points . miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 26 , male = 31 ) , BD I ( female = 9 , male = 9 ) , or BDI II ( female = 12 , male = 16 ) subjects . 2 - way ANOVA , Main effect Sex : p = 0 . 174 ; main effect Group : * * * * p = 4 . 07e - 09 ; Interaction Group x Sex : p = 0 . 311 . Tukey ' s HSD : control vs . BDI : * * * * p = 0 . 0000037 . control vs . BDII : p = 0 . 0000002 . Data are presented as violin plots with median , quartiles and data points . miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control subjects ( female = 26 , male = 31 ) , and BD subjects in different mood states ( depressive ( female = 7 , male = 11 ) , euthymic ( female = 7 , male = 4 ) , hypomanic ( female = 2 , male = 6 ) , manic ( female = 2 , male = 1 ) , mixed ( female = 1 , male = 1 ) . 2 - way ANOVA , main effect Sex : p = 0 . 260 ; main effect Group : p = 1 . 26e - 07 ; Interaction Group x Sex : p = 0 . 059 ; Tukey ' s HSD : control vs . depressive * * * p = 0 . 0002621 , control vs . euthymic * * * p = 0 . 0008533 , control vs . hypomanic * * * * p = 0 . 0000470 . Data are presented as violin plots with median , quartiles and data points . miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects ( - CMT , n = 17 ) and healthy subjects with a history of childhood maltreatment ( + CMT , n = 17 ) . ( * * * * p < 0 . 0001 , Mann - Whitney U test ) . CMT : Childhood maltreatment . Data are presented as violin plots with median , quartiles and data points .

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Figure 5miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD (female= 26, male= 37), or MDD (female= 18, male=24) subjects. Wilcoxon rank sum test after correction for age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). 2-way ANOVA with correction for sex, age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). Control vs. BD: ****p=8.89e-07; control vs. MDD: n.s. p=0.12674. Data are presented as violin plots with median, quartiles and data points.miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 8, male= 10) and SZ (female= 8, male= 15) subjects. 2-way ANOVA, Main effect Sex: p=0.9785; main effect Group: p= 0.0924; main effect Interaction: p= 0.9802. Tukey's HSD: p=0.0924, ns. Data are presented as violin plots with median, quartiles and data points.miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD I (female= 9, male= 9), or BDI II (female= 12, male=16) subjects. 2-way ANOVA, Main effect Sex: p=0.174; main effect Group: ****p=4.07e-09; Interaction Group x Sex: p=0.311. Tukey's HSD: control vs. BDI: ****p=0.0000037. control vs. BDII:p=0.0000002. Data are presented as violin plots with median, quartiles and data points.miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control subjects (female= 26, male= 31), and BD subjects in different mood states (depressive (female= 7, male=11), euthymic (female= 7, male=4), hypomanic (female=2, male=6), manic (female=2, male=1), mixed (female=1, male=1). 2-way ANOVA, main effect Sex: p=0.260; main effect Group: p=1.26e-07; Interaction Group x Sex: p=0.059; Tukey's HSD: control vs. depressive ***p=0.0002621, control vs. euthymic ***p=0.0008533, control vs. hypomanic ****p=0.0000470. Data are presented as violin plots with median, quartiles and data points.miR-499-5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects (-CMT, n=17) and healthy subjects with a history of childhood maltreatment (+CMT, n=17). (****p<0.0001, Mann-Whitney U test). CMT: Childhood maltreatment. Data are presented as violin plots with median, quartiles and data points.


Figure 6Association of miR - 499 - 5p expression and GMV in HC ( n = 23 ) : miR - 499 - 5p levels were negatively correlated with a GMV cluster comprising parts of the left Wernicke language area ( i . e . transverse temporal gyrus , the left parietal operculum and the left superior temporal gyrus ) ( in blue ) at p < . 05 cluster - level family wise error - corrected ( FWE ) for multiple comparisons after an initial threshold of p < . 001 uncorrected ( k = 1090 , x / y / z = - 42 / - 30 / 15 , t = 4 . 7 , p = . 045 ) .

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Figure 6Association of miR-499-5p expression and GMV in HC (n=23): miR-499-5p levels were negatively correlated with a GMV cluster comprising parts of the left Wernicke language area (i.e. transverse temporal gyrus, the left parietal operculum and the left superior temporal gyrus) (in blue) at p<.05 cluster-level family wise error-corrected (FWE) for multiple comparisons after an initial threshold of p<.001 uncorrected (k=1090, x/y/z=-42/-30/15, t= 4.7, p=.045).


Figure 1 ( B ) Plaque formation was monitored by automated scanning Levin - Reisman et al . , 2010 ( ) on a lawn of infected PY79 ( WT ) ( 10 - 6 PFU / ml ) cells spread over an MB agar plate . Shown are images captured at the indicated time points ( hrs ) . Scale bar 2 mm . Corresponds to Movie EV2 . ( C - D ) The dynamic of SPP1 ( C ) and Phi29 ( D ) plaque formation following PY79 ( WT ) infection was monitored as described in ( B ) . Shown is the diameter of individual plaques for each phage ( n ≥ 12 ) , with the average highlighted in red .

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Figure 1(B) Plaque formation was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected PY79 (WT) (10-6 PFU/ml) cells spread over an MB agar plate. Shown are images captured at the indicated time points (hrs). Scale bar 2 mm. Corresponds to Movie EV2. (C-D) The dynamic of SPP1 (C) and Phi29 (D) plaque formation following PY79 (WT) infection was monitored as described in (B). Shown is the diameter of individual plaques for each phage (n ≥ 12), with the average highlighted in red.


Figure 2 ( A ) The indicated strains were infected with SPP1 ( 10 - 6 PFU / ml ) , spread over MB plates , and plaque diameter was monitored after 20 hrs of incubation . Shown is average plaque diameter and SD for each strain ( n ≥ 54 ) . ( D ) PY79 ( WT ) and ET19 ( ∆ sigX ) cells were infected with SPP1 at either high ( phages : bacteria 1 : 1 ) or low ( 1 : 20 ) MOI , and OD600nm was followed at 2 min intervals . Shown is a representative experiment out of 6 biological repeats , and the average values and SD of 8 technical repeats . ( E ) BDR2637 ( Pveg - mCherry ) ( WT , purple ) and ET191 ( PrrnE - gfp , ∆ sigX ) ( ∆ sigX , cyan ) cells were mixed , infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy . Shown are overlay images from mCherry ( purple ) and GFP ( cyan ) signals of the bacterial lawn captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 µm . Corresponds to Movie EV4 . ( F ) ET27 ( PsigX - gfp ) cells were infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are fluorescence from GFP ( upper panels ) and corresponding phase contrast images ( lower panels ) , captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 µm .

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Figure 2(A) The indicated strains were infected with SPP1 (10-6 PFU/ml), spread over MB plates, and plaque diameter was monitored after 20 hrs of incubation. Shown is average plaque diameter and SD for each strain (n ≥ 54).(D) PY79 (WT) and ET19 (∆sigX) cells were infected with SPP1 at either high (phages:bacteria 1:1) or low (1:20) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, and the average values and SD of 8 technical repeats.(E) BDR2637 (Pveg-mCherry) (WT, purple) and ET191 (PrrnE-gfp, ∆sigX) (∆sigX, cyan) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from mCherry (purple) and GFP (cyan) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm. Corresponds to Movie EV4.(F) ET27 (PsigX-gfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluorescence from GFP (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.


Figure 3 ( C ) ET26 ( PsigX - sigX - gfp ) cells were visualized by fluorescence microscopy . Shown are signal from SigX - GFP ( cyan ) ( left panel ) , and an overlay image of phase contrast ( grey ) and signal from SigX - GFP ( cyan ) ( right panel ) . Scale bar 1 μm . ( D ) BDR2637 ( Pveg - mCherry ) ( WT , purple ) and ET261 ( ∆ yueB , PsigX - sigX - gfp ) ( cyan ) cells were mixed , infected with SPP1 at 5 : 1 ( phages : bacteria ) MOI , placed on an agarose pad and followed by time lapse fluorescence microscopy . Shown are overlay images from mCherry ( purple ) , SigX - GFP ( cyan ) , and phase contrast ( grey ) , captured at the indicated time points post infection . Scale bar 1 μm .

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

Figure 3(C) ET26 (PsigX-sigX-gfp) cells were visualized by fluorescence microscopy. Shown are signal from SigX-GFP (cyan) (left panel), and an overlay image of phase contrast (grey) and signal from SigX-GFP (cyan) (right panel). Scale bar 1 μm.(D) BDR2637 (Pveg-mCherry) (WT, purple) and ET261 (∆yueB, PsigX-sigX-gfp) (cyan) cells were mixed, infected with SPP1 at 5:1 (phages:bacteria) MOI, placed on an agarose pad and followed by time lapse fluorescence microscopy. Shown are overlay images from mCherry (purple), SigX-GFP (cyan), and phase contrast (grey), captured at the indicated time points post infection. Scale bar 1 μm.


Figure 4 ( B ) ET29 ( Pveg - mCherry , PIPTG - sigX ) ( purple ) cells were grown in the presence of IPTG and mixed with PY79 ( WT ) cells . The mixture was infected with SPP1 - lysin - yfp 5 : 1 ( phages : bacteria ) MOI , placed on an IPTG - containing agarose pad , and followed by time lapse fluorescence microscopy . Shown are overlay images of phase contrast ( grey ) , signal from mCherry labeled cells ( purple ) , and signal from Lysin - SPP1 - YFP ( cyan ) , captured at the indicated time points post infection ( upper panels ) . Corresponding signal from Lysin - SPP1 - YFP ( cyan ) is shown separately ( lower panels ) . Arrows highlight the delayed infection of ET29 cells . Scale bar 1 μm . ( E ) OF83 ( Ppen - lacIΔ11 - cfp ) ( WT ) and ET40 ( Ppen - lacIΔ11 - cfp , PIPTG - sigX ) cells , grown in the presence or absence of IPTG , were infected with SPP1 - delX110lacO64 at 5 : 1 ( phages : bacteria ) MOI . The formation of LacI - CFP foci on phage DNA was monitored 10 min post infection . Non - infected OF83 cells were used for comparison . Shown are overlay images of phase contrast ( grey ) and signal from LacI - CFP ( cyan ) . Scale bar 1 µm . ( F ) Quantification of the experiment described in ( E ) . Shown is the percentage of LacI - CFP foci 10 min post infection of OF83 and ET40 cells by SPP1 , with average values and SD ( n ≥ 850 cells for each population ) .

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 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

Figure 4(B) ET29 (Pveg-mCherry, PIPTG-sigX) (purple) cells were grown in the presence of IPTG and mixed with PY79 (WT) cells. The mixture was infected with SPP1-lysin-yfp 5:1 (phages:bacteria) MOI, placed on an IPTG-containing agarose pad, and followed by time lapse fluorescence microscopy. Shown are overlay images of phase contrast (grey), signal from mCherry labeled cells (purple), and signal from Lysin-SPP1-YFP (cyan), captured at the indicated time points post infection (upper panels). Corresponding signal from Lysin-SPP1-YFP (cyan) is shown separately (lower panels). Arrows highlight the delayed infection of ET29 cells. Scale bar 1 μm.(E) OF83 (Ppen-lacIΔ11-cfp) (WT) and ET40 (Ppen-lacIΔ11-cfp, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1-delX110lacO64 at 5:1 (phages:bacteria) MOI. The formation of LacI-CFP foci on phage DNA was monitored 10 min post infection. Non-infected OF83 cells were used for comparison. Shown are overlay images of phase contrast (grey) and signal from LacI-CFP (cyan). Scale bar 1 µm. (F) Quantification of the experiment described in (E). Shown is the percentage of LacI-CFP foci 10 min post infection of OF83 and ET40 cells by SPP1, with average values and SD (n ≥ 850 cells for each population).


Figure 5 ( A ) ET28 ( PIPTG - sigX ) and ET42 ( ∆ dltA , PIPTG - sigX ) cells , grown in the presence or absence of IPTG , were infected with SPP1 at 1 : 20 ( phages : bacteria ) MOI , and OD600nm was followed at 2 min intervals . Shown is a representative experiment out of 6 biological repeats , with the average values and SD of 8 technical repeats . ( D ) PY79 ( WT ) and ET41 ( ΔdltA ) cells were infected with SPP1 ( 10 - 6 PFU / ml ) , spred over an MB agar plate , and plaque diameter was monitored after 20 hrs of incubation . Shown is plaque diameter distributions for each strain ( n ≥ 40 ) . ( F ) AR16 ( PrrnE - gfp ) ( WT , cyan ) and ET411 ( Pveg - mCherry , ∆ dltA ) ( ∆ dltA , purple ) cells were mixed , infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are overlay images from GFP ( cyan ) and mCherry ( purple ) signals of the bacterial lawn captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 150 µm . ( G ) ET43 ( dltA - yfp ) cells were infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are fluoresce from DltA - YFP signal ( upper panels ) and corresponding phase contrast images ( lower panels ) , captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 µm .

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 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 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 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

Figure 5(A) ET28 (PIPTG-sigX) and ET42 (∆dltA, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1 at 1:20 (phages:bacteria) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, with the average values and SD of 8 technical repeats.(D) PY79 (WT) and ET41 (ΔdltA) cells were infected with SPP1 (10-6 PFU/ml), spred over an MB agar plate, and plaque diameter was monitored after 20 hrs of incubation. Shown is plaque diameter distributions for each strain (n ≥ 40).(F) AR16 (PrrnE-gfp) (WT, cyan) and ET411 (Pveg-mCherry, ∆dltA) (∆dltA, purple) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from GFP (cyan) and mCherry (purple) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 150 µm.(G) ET43 (dltA-yfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluoresce from DltA-YFP signal (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.


Figure 1A , Representative images of controls from the siRNA screen for HU - induced RPA accumulation . GFP - RPA1 and RFP - PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU ( 3 mM ) for 2 hrs prior to pre - extraction , fixation and imaging . B , Gene ranking based on siRNA scores from two independent screens . 219 genes were targeted by three individual siRNAs and ranked based on P - values derived from RSA and Fisher ' s analysis . Selected hits are highlighted . C , Validation of selected genes by single cell analysis of GFP - RPA1 intensity in pre - extracted cells . Only S - phase cells positive for RFP - PCNA were analysed . a , b , c denote independent siRNAs ; Ctrl , Control . Median with interquartile range of relative intensity per cell is shown , n > 4000 . Mann - Whitney : * * * p < 10 - 4 , n . s . non - significant . One representative experiment out of two biological replicas is shown . D , Analysis of DNA synthesis rate . Cells were pulsed 15 min with EdU and EdU intensity was analysed in S - phase cells positive for RFP - PCNA . EdU incorporation is shown relative to siCtrl .

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 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 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

Figure 1A, Representative images of controls from the siRNA screen for HU-induced RPA accumulation. GFP-RPA1 and RFP-PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU (3 mM) for 2 hrs prior to pre-extraction, fixation and imaging.B, Gene ranking based on siRNA scores from two independent screens. 219 genes were targeted by three individual siRNAs and ranked based on P-values derived from RSA and Fisher's analysis. Selected hits are highlighted.C, Validation of selected genes by single cell analysis of GFP-RPA1 intensity in pre-extracted cells. Only S-phase cells positive for RFP-PCNA were analysed. a, b, c denote independent siRNAs; Ctrl, Control. Median with interquartile range of relative intensity per cell is shown, n > 4000. Mann-Whitney: *** p < 10-4, n.s. non-significant. One representative experiment out of two biological replicas is shown.D, Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl.


Figure 2A , DNA replication measured by EdU incorporation . RPF - PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min . S - phase cells positive for RFP - PCNA were analysed . n > 150 . Error bars , SD ; n = 3 biological replicas . B , Complementation analysis of DNA synthesis . Stable cell lines expressing BRPF3 resistant to the BRPF3 / a siRNA and control ( lacZ - V5 ) were siRNA transfected and analysed for EdU incorporation as in A . Error - bars , SD ; n = 6 biological replicas . Two - tailed t test , * * p < 10 - 2 . C , Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells . ( left ) mass - spec analysis ( spectral counts / total peptides identified ) and ( right ) verification of associated protein by western blot . D , Immunoprecipitation analysis of BRPF3 deletion mutants . One representative experiment out of two is shown . E , Histone acetyltransferase assay ( HAT ) with purified BRPF1 / 3 complexes on free histones and mononucleosomes . F , HAT assay with purified BRPF1 / 3 complexes on H3 peptides unmodified or acetylated ( ac ) / methylated ( me ) at K14 and K27 , respectively . G , Analysis of histone acetylation in BRPF3 depleted cells . ( left ) Western blot of siRNA treated U - 2 - OS cells . TSA treatment ( 1 hr ) was included as a positive control . 2x , double amount of extract loaded in 1x . ( right ) H3K14 acetylation levels quantified relative to total H3 . Error bars , SD ; n = 3 biological replicates . H , Complementation analysis of H3K14ac . Total cell extracts of siRNA treated lacZ - V5 and BRPF3 - V5 ( siBRPF3 / a resistant ) expressing cells were analysed by western blotting . One representative experiment out of four biological replicas is shown .

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

Figure 2A, DNA replication measured by EdU incorporation. RPF-PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min. S-phase cells positive for RFP-PCNA were analysed. n > 150. Error bars, SD; n= 3 biological replicas.B, Complementation analysis of DNA synthesis. Stable cell lines expressing BRPF3 resistant to the BRPF3/a siRNA and control (lacZ-V5) were siRNA transfected and analysed for EdU incorporation as in A. Error-bars, SD; n = 6 biological replicas. Two-tailed t test, ** p<10-2.C, Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells. (left) mass-spec analysis (spectral counts/total peptides identified) and (right) verification of associated protein by western blot.D, Immunoprecipitation analysis of BRPF3 deletion mutants. One representative experiment out of two is shown.E, Histone acetyltransferase assay (HAT) with purified BRPF1/3 complexes on free histones and mononucleosomes.F, HAT assay with purified BRPF1/3 complexes on H3 peptides unmodified or acetylated (ac)/methylated (me) at K14 and K27, respectively.G, Analysis of histone acetylation in BRPF3 depleted cells. (left) Western blot of siRNA treated U-2-OS cells. TSA treatment (1 hr) was included as a positive control. 2x, double amount of extract loaded in 1x. (right) H3K14 acetylation levels quantified relative to total H3. Error bars, SD; n = 3 biological replicates.H, Complementation analysis of H3K14ac. Total cell extracts of siRNA treated lacZ-V5 and BRPF3-V5 (siBRPF3/a resistant) expressing cells were analysed by western blotting. One representative experiment out of four biological replicas is shown.


Figure 3A - B , Single - molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse - labelled with IdU ( 10 min ) and CIdU ( 20 min ) . ( A ) Distribution of inter - track distances . Bars represent the median . n > 100 . Mann - Whitney : * * * P < 10 - 3 . One representative experiment out of two biological replicas is shown . ( B ) Size distribution of CldU track length . Bars represent the median . n > 300 . Mann - Whitney : * * * P < 10 - 3 . One representative experiment out of two biological replicas is shown . C , Distribution of intertrack distances measured by IdU / CIdU pulse labelling and DNA fiber analysis . Bars represent the median . n > 110 . Mann - Whitney : * * * * P < 10 - 4 . One representative experiment out of two biological replicas is shown . D , Scatter plot of chromatin - bound GFP - RPA1 intensity per cell . GFP - RPA1 and RFP - PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN - 01 ( 300 nM ) prior to pre - extraction and fixation . GFP - RPA1 levels were measured in PCNA positive cells . Lines represent medians . n > 150 . t - tests : * * * * P < 10 - 4 , * P < 0 . 05 ; a . u . , arbitrary unit . E , Analysis of replication factor loading . Soluble proteins from siRNA transfected cells treated as in D where removed by CSK - T extraction and chromatin pellets analysed by western blotting . One representative experiment out of three independent biological replicas is shown . F , Quantification of chromatin - bound MCM2 ( left ) and CDC45 ( right ) from the three independent experiments described in ( E ) . MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control . t - tests : * * * P < 10 - 3 , * * P < 0 . 01 , * P < 0 . 05 , n . s . non - significant . G , Analysis of chromatin - bound proteins in HBO1 depleted cells as in E . One representative experiment out of two biological replicas is shown .

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 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 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

Figure 3A-B, Single-molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse-labelled with IdU (10 min) and CIdU (20 min). (A) Distribution of inter-track distances. Bars represent the median. n > 100. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. (B) Size distribution of CldU track length. Bars represent the median. n > 300. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. C, Distribution of intertrack distances measured by IdU/CIdU pulse labelling and DNA fiber analysis. Bars represent the median. n > 110. Mann-Whitney: **** P < 10-4. One representative experiment out of two biological replicas is shown.D, Scatter plot of chromatin-bound GFP-RPA1 intensity per cell. GFP-RPA1 and RFP-PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN-01 (300 nM) prior to pre-extraction and fixation. GFP-RPA1 levels were measured in PCNA positive cells. Lines represent medians. n > 150. t-tests: **** P < 10-4, * P < 0.05; a.u., arbitrary unit.E, Analysis of replication factor loading. Soluble proteins from siRNA transfected cells treated as in D where removed by CSK-T extraction and chromatin pellets analysed by western blotting. One representative experiment out of three independent biological replicas is shown. F, Quantification of chromatin-bound MCM2 (left) and CDC45 (right) from the three independent experiments described in (E). MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control. t-tests: *** P < 10-3, ** P < 0.01, * P < 0.05, n.s. non-significant.G, Analysis of chromatin-bound proteins in HBO1 depleted cells as in E. One representative experiment out of two biological replicas is shown.