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 .

O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O O O O O O O O O O O O

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 ) .

O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O B-GENEPROD O 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-GENEPROD O O O O O O O O O O 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-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_LINE O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E 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 .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F 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 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-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G 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.


B - 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 .

O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-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 .

O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A 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 .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 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 .

O O O O O O O B-CELL_LINE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-CELL_LINE O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O

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.


A 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 .

O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

A 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 .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O B-CELL_LINE O O B-CELL_TYPE I-CELL_TYPE O O B-CELL_LINE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O 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-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D 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


A 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 .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O

A 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 .

O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O 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 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 O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


B 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 .

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


Lower 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 .

O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

Lower 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

O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O O O

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


A Venn diagramm representing overlap of hCFU - E proteome from three independent healthy donors . In total 6 , 925 proteins were

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O

A Venn diagramm representing overlap of hCFU-E proteome from three independent healthy donors. In total 6,925 proteins were quantified


B 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 .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O O

B 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 ) .

O O O O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 .

O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O

D 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 ) .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E 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 O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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


( D ) Immunofluorescence staining of the spinal cord at GW7 , GW8 , GW10 , GW11 , and GW23 for RBFOX3 ( red ) , OLIG2 ( green ) , and GFAP ( white ) . The appearance of astrocytes and oligodendrocytes was observed from GW8 onward . Scale bars : 100 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O

(D) Immunofluorescence staining of the spinal cord at GW7, GW8, GW10, GW11, and GW23 for RBFOX3 (red), OLIG2 (green), and GFAP (white). The appearance of astrocytes and oligodendrocytes was observed from GW8 onward. Scale bars: 100 μm.


( C ) Immunofluorescence staining of the spinal cord from GW7 , GW8 , and GW10 for a dorsal marker ( PAX7 , green ) , neuronal precursor marker ( ASCL1 , red ) , and nuclei marker ( DAPI , blue ) . Images show the neuronal differentiation process on the dorsal side of the spinal cord . Scale bar : 100 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O O O O O O

(C) Immunofluorescence staining of the spinal cord from GW7, GW8, and GW10 for a dorsal marker (PAX7, green), neuronal precursor marker (ASCL1, red), and nuclei marker (DAPI, blue). Images show the neuronal differentiation process on the dorsal side of the spinal cord. Scale bar: 100 μm.


( C ) Violin plot showing the expression of selected genes . EGFR + Cluster 14 is circled with a red dotted line .

O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O

(C) Violin plot showing the expression of selected genes. EGFR+ Cluster 14 is circled with a red dotted line.


( D ) Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR , DLL1 , MIAT , and FAM189A2 in cluster 14 with development based on snRNA counts data .

O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O

(D) Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR, DLL1, MIAT, and FAM189A2 in cluster 14 with development based on snRNA counts data.


( B ) Immunofluorescence staining of spinal cord sections at GW7 , GW11 , GW17 , and GW23 for EGFR ( green ) and nuclei ( DAPI , blue ) . EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord . EGFR expression on the dorsal side was observed in GW11 , GW17 , and GW23 spinal cord . Dorsal EGFR expression was mainly concentrated within the dorsal horn area . Scale bar : 100 μm . ( Top ) Higher - magnification view of boxes at bottom . White dashed lines indicate dorsal horn borders .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE B-GENEPROD B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE 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-TISSUE I-TISSUE O O

(B) Immunofluorescence staining of spinal cord sections at GW7, GW11, GW17, and GW23 for EGFR (green) and nuclei (DAPI, blue). EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord. EGFR expression on the dorsal side was observed in GW11, GW17, and GW23 spinal cord. Dorsal EGFR expression was mainly concentrated within the dorsal horn area. Scale bar: 100 μm. (Top) Higher-magnification view of boxes at bottom. White dashed lines indicate dorsal horn borders.


( D ) Representative immunofluorescence staining of a GW11 spinal cord section . Most dorsal EGFR - expressing cells at GW11 co - expressed GFAP and SOX9 , but not OLIG2 . Scale bars : 100 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O B-TISSUE B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O

(D) Representative immunofluorescence staining of a GW11 spinal cord section. Most dorsal EGFR-expressing cells at GW11 co-expressed GFAP and SOX9, but not OLIG2. Scale bars: 100 μm.


GW11 EGFR - expressing cells exhibited radial glia morphology and most co - expressed SOX9 ( A ) but not OLIG2 ( B ) . However , EGFR co - expression with SOX9 or OLIG2 was found in GW17 EGFR - expressing cells ( C , D ) . White dashed lines marked dorsal horn borders . Higher - magnification view of boxes on left . Scale bars : 100 μm .

O B-GENEPROD O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O

GW11 EGFR-expressing cells exhibited radial glia morphology and most co-expressed SOX9 (A) but not OLIG2 (B). However, EGFR co-expression with SOX9 or OLIG2 was found in GW17 EGFR-expressing cells (C, D). White dashed lines marked dorsal horn borders. Higher-magnification view of boxes on left. Scale bars: 100 μm.


( A , B ) ( A ) Representative IHC images and ( B ) semi quantification of CRMP4 protein in human spinal cords ( SC ) cross sections from 2 control patients and 3 ALS patients . We analyzed total of 7 SC sections of controls and 14 SC sections of ALS patients , Data presented as mean ± SE . DAB : labeled CRMP4 . Scale bar : left images 20 µm , right insets 10 µm . Mann - Whitney test * * * p = 0 . 0003 .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-ORGANISM B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-ORGANISM O O B-DISEASE B-ORGANISM O O O O O O B-TISSUE O O O O O B-TISSUE O O B-DISEASE B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) (A) Representative IHC images and (B) semi quantification of CRMP4 protein in human spinal cords (SC) cross sections from 2 control patients and 3 ALS patients. We analyzed total of 7 SC sections of controls and 14 SC sections of ALS patients, Data presented as mean ±SE. DAB: labeled CRMP4. Scale bar: left images 20 µm, right insets 10 µm. Mann-Whitney test ***p = 0.0003.


( C ) Representative images of P90 SC cross sections of SOD1G93A and WT mice . Red : denotes NeuN , Green : denotes CRMP4 . Scale bar : 10 μm . ( D ) Quantification of the percentage of CRMP4 positive SC neurons in 3 WT VS . 3 SOD1G93A mice . We monitored CRMP4 expression in total of 108 cells in WT condition and 123 cells in SOD1G93A , an average of 36 or 41 cells in each repeat respectively . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0161 .

O O O O O O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-TISSUE B-CELL_TYPE O O O O O O B-GENEPROD B-ORGANISM O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images of P90 SC cross sections of SOD1G93A and WT mice. Red: denotes NeuN, Green: denotes CRMP4. Scale bar: 10 μm. (D) Quantification of the percentage of CRMP4 positive SC neurons in 3 WT VS. 3 SOD1G93A mice. We monitored CRMP4 expression in total of 108 cells in WT condition and 123 cells in SOD1G93A, an average of 36 or 41 cells in each repeat respectively. Student's t-test, n = 3, Data presented as mean ±SE, *p = 0.0161.


( A ) Representative images of ALS patient or non - ALS human control intra - muscular nerves . Red : denotes NFH , Green : denotes CRMP4 , White : denotes co - localization area using Imaris software . Scale bar : 20 μm . ( B ) Quantification of CRMP4 intensity levels in NFH positive intra - muscular distal nerves from 5 non - ALS controls and 4 sALS patients . We analyzed 40 terminal axons from the healthy samples ( ~ 8 axons per sample ) and 36 terminal axons from sALS samples ( ~ 8 axons per sample ) . Data presented as mean ± SE . Student ' s t - test , * p = 0 . 0475 .

O O O O O O B-DISEASE B-ORGANISM O O O B-DISEASE B-ORGANISM O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O B-DISEASE O O O O B-ORGANISM O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of ALS patient or non-ALS human control intra-muscular nerves. Red: denotes NFH, Green: denotes CRMP4, White: denotes co-localization area using Imaris software. Scale bar: 20 μm. (B) Quantification of CRMP4 intensity levels in NFH positive intra-muscular distal nerves from 5 non-ALS controls and 4 sALS patients. We analyzed 40 terminal axons from the healthy samples (~8 axons per sample) and 36 terminal axons from sALS samples (~8 axons per sample). Data presented as mean ±SE. Student's t-test, *p = 0.0475.


( C ) Representative images of SOD1G93A / ChAT : : tdTomato or WTChAT : : tdTomato neuromuscular junctions at P90 . White : denotes BTX , Red : denotes direct ChAT , Green : denotes CRMP4 , Yellow : denotes Z projection of 3D Imaris co - localization of CRMP4 and ChAT . Scale bar : 10 µm . ( D ) Quantification of CRMP4 positive NMJs in gastrocnemius muscles from 3 WT or 3 SOD1G93A P90 mice . Total of 44 NMJ ' s in WT condition and 60 NMJ ' s in SOD1G93A condition . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0157 . ( E ) Quantification of the percent of partially denervated NMJ ' s in the presence or absence of CRMP4 immunostaining in 3 different SOD1G93A mice . We counted 24 NMJ ' s in SOD1G93A CRMP4 negatives and 67 NMJ ' s in SOD1G93A CRMP4 positives . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0352 .

O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-TISSUE O B-TISSUE I-TISSUE O O O O O B-GENEPROD O B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O O O O O O O O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images of SOD1G93A/ChAT::tdTomato or WTChAT::tdTomato neuromuscular junctions at P90. White: denotes BTX, Red: denotes direct ChAT, Green: denotes CRMP4, Yellow: denotes Z projection of 3D Imaris co-localization of CRMP4 and ChAT. Scale bar: 10 µm. (D) Quantification of CRMP4 positive NMJs in gastrocnemius muscles from 3 WT or 3 SOD1G93A P90 mice. Total of 44 NMJ's in WT condition and 60 NMJ's in SOD1G93A condition. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0157. (E) Quantification of the percent of partially denervated NMJ's in the presence or absence of CRMP4 immunostaining in 3 different SOD1G93A mice. We counted 24 NMJ's in SOD1G93A CRMP4 negatives and 67 NMJ's in SOD1G93A CRMP4 positives. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0352.


( F ) Representative images of P90 SOD1G93A and WT sciatic nerves . Red : denotes NFH , Blue : denotes GFAP and green denotes CRMP4 , Yellow : denotes the Z projection of 3D Imaris co - localization of CRMP4 and NFH . Scale bar : 5 μm . ( G ) Quantification of the co - localization area of CRMP4 with NFH in the sciatic nerve in 3 SOD1G93A mice compared to 3 WT mice using Imaris analysis . 14 WT sciatic nerve sections and 11 SOD1G93A sections were monitored . Data presented as mean ± SE . Student ' s t - test , n = 3 , * * * * p < 0 . 0001 .

O O O O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O B-GENEPROD B-ORGANISM O O O O B-ORGANISM O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Representative images of P90 SOD1G93A and WT sciatic nerves. Red: denotes NFH, Blue: denotes GFAP and green denotes CRMP4, Yellow: denotes the Z projection of 3D Imaris co-localization of CRMP4 and NFH. Scale bar: 5 μm. (G) Quantification of the co-localization area of CRMP4 with NFH in the sciatic nerve in 3 SOD1G93A mice compared to 3 WT mice using Imaris analysis. 14 WT sciatic nerve sections and 11 SOD1G93A sections were monitored. Data presented as mean ±SE. Student's t-test, n=3, ****p<0.0001.


( A ) Representative images of healthy or C9orf72 iPSC - derived MNs treated with Sema3A or untreated in the distal compartment , 6 hours post treatment . Red : denotes Tubulin , Green : denotes CRMP4 . Scale bar : 5 μm . ( B ) Quantification of CRMP4 intensity levels in healthy or C9orf72 iPSC - derived MNs with Sema3A treatment or untreated . 14 untreated healthy axons , 12 healthy axons with Sema3A treatment , 49 untreated C9orf72 axons and 31 C9orf72 axons with Sema3A treatment were monitored from 3 different chambers . One - way ANOVA , Tukey ' s multiple comparisons test , n = 3 , Data presented as mean ± SE , * p = 0 . 0338 ; * * * * p < 0 . 0001 .

O O O O O O O O B-GENEPROD B-CELL_TYPE O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD B-CELL_TYPE O O B-CELL_TYPE O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of healthy or C9orf72 iPSC-derived MNs treated with Sema3A or untreated in the distal compartment, 6 hours post treatment. Red: denotes Tubulin, Green: denotes CRMP4. Scale bar: 5 μm. (B) Quantification of CRMP4 intensity levels in healthy or C9orf72 iPSC-derived MNs with Sema3A treatment or untreated. 14 untreated healthy axons, 12 healthy axons with Sema3A treatment, 49 untreated C9orf72 axons and 31 C9orf72 axons with Sema3A treatment were monitored from 3 different chambers. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p = 0.0338; ****p<0.0001.


( D ) Representative images of healthy or C9orf72 human - derived MN cell somata with Sema3A treatment , Sema3A + dynein inhibitor treatment , or untreated . Gray : denotes CTX , Green : denotes CRMP4 . Scale bar : 5μm . ( E ) Quantification of CRMP4 intensity ( normalized to GAPDH + mCherry / area ) levels at the somata of healthy or C9orf72 human - derived MN after Sema3A treatment , Sema3A + dynein inhibitor treatment , or untreated . Analysis performed in 3 independent chambers per condition . 19 healthy untreated cell somata , 26 healthy cell somata with Sema3A treatment , 20 healthy cell somata with Sema3A + dynein inhibitor treatment and 14 C9orf72 cell somata from each condition were monitored . One - way ANOVA , Newman - Keuls multiple comparisons test , n = 3 , Data presented as mean ± SE , * p = 0 . 0207 ; * * * p = 0 . 0004 .

O O O O O O O O B-GENEPROD B-ORGANISM O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O O O B-GENEPROD B-ORGANISM O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Representative images of healthy or C9orf72 human-derived MN cell somata with Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Gray: denotes CTX, Green: denotes CRMP4. Scale bar: 5μm. (E) Quantification of CRMP4 intensity (normalized to GAPDH+mCherry/area) levels at the somata of healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Analysis performed in 3 independent chambers per condition. 19 healthy untreated cell somata, 26 healthy cell somata with Sema3A treatment, 20 healthy cell somata with Sema3A + dynein inhibitor treatment and 14 C9orf72 cell somata from each condition were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n=3, Data presented as mean ±SE, *p=0.0207; ***p=0.0004.


( F ) Representative images of healthy or C9orf72 human - derived MN proximal axons with Sema3A treatment , Sema3A + dynein inhibitor treatment or untreated control . Green : denotes CRMP4 , Red : denotes GAPDH . Scale bar : 5μm . ( G ) Quantification of CRMP4 intensity levels ( normalized to GAPDH + mCherry / area ) at the proximal axons in healthy or C9orf72 human - derived MN after Sema3A treatment , Sema3A + dynein inhibitor treatment , or untreated control . Analysis performed from 3 independent chambers in each condition . 21 healthy untreated proximal axons , 24 healthy proximal axons with Sema3A treatment , 16 healthy proximal axons with Sema3A + dynein inhibitor treatment , 12 C9orf72 untreated proximal axons , 8 C9orf72 proximal axons with Sema3A treatment and 13 C9orf72 proximal axons with Sema3A + dynein inhibitor treatment were monitored . One - way ANOVA , Newman - Keuls multiple comparisons test , n = 3 , Data presented as mean ± SE , * p = 0 . 0334 , * * * * p < 0 . 0001 .

O O O O O O O O B-GENEPROD B-ORGANISM O O B-CELL_TYPE B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD B-ORGANISM O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Representative images of healthy or C9orf72 human-derived MN proximal axons with Sema3A treatment, Sema3A + dynein inhibitor treatment or untreated control. Green: denotes CRMP4, Red: denotes GAPDH. Scale bar: 5μm. (G) Quantification of CRMP4 intensity levels (normalized to GAPDH+mCherry/area) at the proximal axons in healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated control. Analysis performed from 3 independent chambers in each condition. 21 healthy untreated proximal axons, 24 healthy proximal axons with Sema3A treatment, 16 healthy proximal axons with Sema3A + dynein inhibitor treatment, 12 C9orf72 untreated proximal axons, 8 C9orf72 proximal axons with Sema3A treatment and 13 C9orf72 proximal axons with Sema3A + dynein inhibitor treatment were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n = 3, Data presented as mean ±SE, *p =0.0334, ****p<0.0001.


( F ) Upper panel - Immunoprecipitation assay with anti ‐ Flag antibody followed by Western blot analysis of dynactin ( p150 ) in COS7 cells overexpressing Flag - CRMP4 ( size of ~ 65 KDa ) . Lower panel - Total input ( size of ~ 150 KDa ) . ( G ) Quantification of the blot in F . The dynactin intensity band was normalized to the Flag - CRMP4 intensity band in each repeat . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0299 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O 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-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Upper panel - Immunoprecipitation assay with anti‐Flag antibody followed by Western blot analysis of dynactin (p150) in COS7 cells overexpressing Flag-CRMP4 (size of ~65 KDa). Lower panel - Total input (size of ~150 KDa). (G) Quantification of the blot in F. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0299.


( H ) Immunoprecipitation of DIC ( size of ~ 75 KDa ) followed by Western blot analysis of CRMP4 ( size of ~ 64 KDa ) in COS7 cells that were transfected with CRMP4 and AAV9 - 50aa or its control . IgG antibody was used as a control . ( I ) Quantification of the blot in H from 3 independent repeats . The CRMP4 intensity band was normalized to the DIC intensity band in each repeat . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0479 .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Immunoprecipitation of DIC (size of ~75 KDa) followed by Western blot analysis of CRMP4 (size of ~64 KDa) in COS7 cells that were transfected with CRMP4 and AAV9-50aa or its control. IgG antibody was used as a control. (I) Quantification of the blot in H from 3 independent repeats. The CRMP4 intensity band was normalized to the DIC intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0479.


( E ) Representative images from the proximity ligation assay ( For explanation of PLA technique ; please refer to method section ) for CRMP4 and dynein in SODG93A and WT primary MNs axons that were exposed to either control or Sema3A 8h post treatment . Scale bar : 5µm . ( F ) Quantification of the CRMP4 - DIC puncta number per primary motor neuron axon in each condition . We analyzed ~ 20 axons per condition from 3 independent chambers per group ( One - way ANOVA , Tukey ' s multiple comparisons test , n = 3 , Data presented as mean ± SE , * * p = 0 . 01 * p = 0 . 04 )

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(E) Representative images from the proximity ligation assay (For explanation of PLA technique; please refer to method section) for CRMP4 and dynein in SODG93A and WT primary MNs axons that were exposed to either control or Sema3A 8h post treatment. Scale bar: 5µm. (F) Quantification of the CRMP4-DIC puncta number per primary motor neuron axon in each condition. We analyzed ~20 axons per condition from 3 independent chambers per group (One-way ANOVA, Tukey's multiple comparisons test, n=3, Data presented as mean ±SE, **p=0.01 *p=0.04)


( G ) Representative images of proximity ligation assay for CRMP4 and dynein in healthy and C9orf72 human - derived proximal axons post peptides treatment , Sema3A treatment , Sema3A + peptides treatment or untreated controls . Scale bar : 5µm . ( H ) Quantification of the CRMP4 - DIC puncta number per axon in healthy or C9orf72 human - derived MN proximal axons after Sema3A treatment , Sema3A + peptides treatment or untreated controls . Data collected from 3 independent chambers in each condition . Total of 37 healthy untreated proximal axons , 61 healthy proximal axons with peptides treatment , 59 healthy proximal axons with Sema3A treatment and 52 healthy proximal axons with Sema3A + peptides treatment . 67 C9orf72 untreated proximal axons , 63 C9orf72 proximal axons with peptides treatment , 41 C9orf72 proximal axons with Sema3A treatment and 50 C9orf72 proximal axons with Sema3A + peptides treatment monitored . Data presented as mean ± SE . One - way ANOVA , Tukey ' s multiple comparisons test , n = 3 , * * * * p < 0 . 0001 .

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(G) Representative images of proximity ligation assay for CRMP4 and dynein in healthy and C9orf72 human-derived proximal axons post peptides treatment, Sema3A treatment, Sema3A + peptides treatment or untreated controls. Scale bar: 5µm. (H) Quantification of the CRMP4-DIC puncta number per axon in healthy or C9orf72 human-derived MN proximal axons after Sema3A treatment, Sema3A + peptides treatment or untreated controls. Data collected from 3 independent chambers in each condition. Total of 37 healthy untreated proximal axons, 61 healthy proximal axons with peptides treatment, 59 healthy proximal axons with Sema3A treatment and 52 healthy proximal axons with Sema3A + peptides treatment. 67 C9orf72 untreated proximal axons, 63 C9orf72 proximal axons with peptides treatment, 41 C9orf72 proximal axons with Sema3A treatment and 50 C9orf72 proximal axons with Sema3A + peptides treatment monitored. Data presented as mean ±SE. One-way ANOVA, Tukey's multiple comparisons test, n=3, ****p<0.0001.


( A ) Representative images of CTX signal in healthy and C9orf72 human IPSC - derived MNs before and after Sema3A application . Green : denotes CTX - positive cells . Yellow circles are numbered CTX positive cells . Purple circles are cells that are missing post Sema3A treatment . Scale bar : 40 μm . ( B - C ) Quantification of CTX signal in healthy and C9orf72 IPSC - derived MNs before and 3 days after applying Sema3A to distal compartment , compared with untreated control . 3 independent chambers in each condition were analyzed . Average of ~ 150 neurons per condition monitored . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p < 0 . 05 .

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(A) Representative images of CTX signal in healthy and C9orf72 human IPSC-derived MNs before and after Sema3A application. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 40 μm. (B-C) Quantification of CTX signal in healthy and C9orf72 IPSC-derived MNs before and 3 days after applying Sema3A to distal compartment, compared with untreated control. 3 independent chambers in each condition were analyzed. Average of ~150 neurons per condition monitored. Student's t-test, n=3, Data presented as mean ±SE, *p<0.05.


( D ) Representative images of CTX signal in WT or SOD1G93A primary MNs before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor + Sema3A , Dynasore + Sema3A or untreated . Green : denotes CTX - positive cells . Yellow circles are numbered CTX positive cells . Purple circles are cells that are missing post Sema3A treatment . Scale bar : 30 μm . ( E ) Quantification of CTX signal in a SOD1G93A explant before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor + Sema3A , Dynasore + Sema3A . 3 independent chambers in each condition were analyzed . ~ 200 neurons were monitored per each condition . One - way ANOVA , Tukey ' s multiple comparisons test , n = 3 , Data presented as mean ± SE , * p < 0 . 05 , * * p < 0 . 01 . Dynein inhibitor and Dynasore treatments were used as a negative control .

O O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O

(D) Representative images of CTX signal in WT or SOD1G93A primary MNs before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A or untreated. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 30 μm. (E) Quantification of CTX signal in a SOD1G93A explant before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A. 3 independent chambers in each condition were analyzed. ~200 neurons were monitored per each condition. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p<0.05, **p<0.01. Dynein inhibitor and Dynasore treatments were used as a negative control.


( A ) Representative images and insets of P90 WT and SOD1G93A SC cross sections at P90 . Blue : denotes DAPI , Red : denoted NeuN , and White : denotes activated caspase 3 . Scale bar : 20 µm .

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(A) Representative images and insets of P90 WT and SOD1G93A SC cross sections at P90. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 20 µm.


( C ) Representative images of P90 SOD1G93A mice SC cross sections that were injected with AAV9 - GFP / AAV9 - 50aa - GFP . Blue : denotes DAPI , Red : denoted NeuN , and White : denotes activated caspase 3 . Scale bar : 10 µm .

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(C) Representative images of P90 SOD1G93A mice SC cross sections that were injected with AAV9-GFP/AAV9-50aa-GFP. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 10 µm.


Kaplan Meier plots representing the survival difference between SKCM patients stratified according to the 40 % highest or lowest BNIP3 expression .

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Kaplan Meier plots representing the survival difference between SKCM patients stratified according to the 40% highest or lowest BNIP3 expression.


Representative images of BNIP3 immunostaining in a benign nevus ( n = 5 ) , primary melanoma and their paired metastasis ( n = 7 ) . In the representative pictures , nevi or tumor tissue is circled in yellow and highlighted with a yellow asterisk ( * ) . Histopathological scoring of BNIP3 protein levels in nevi and melanoma patients with paired primary and metastases harboring weak and in gradient ( + / - ) , weak and homogeneous ( + ) or strong and homogeneous ( + + ) BNIP3 protein levels based on histopathological staining intensity and distribution scoring . Scale bars represent 150µm .

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Representative images of BNIP3 immunostaining in a benign nevus (n=5), primary melanoma and their paired metastasis (n=7). In the representative pictures, nevi or tumor tissue is circled in yellow and highlighted with a yellow asterisk (*). Histopathological scoring of BNIP3 protein levels in nevi and melanoma patients with paired primary and metastases harboring weak and in gradient (+/-), weak and homogeneous (+) or strong and homogeneous (++) BNIP3 protein levels based on histopathological staining intensity and distribution scoring. Scale bars represent 150µm.


Representative images of BNIP3 immunohistochemistry in TMAs containing ( n = 158 ) melanoma metastases and related Kaplan Meier plot of survival difference comparing melanoma patients with different BNIP3 histopathological scores . Scale bars represent 50µm .

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Representative images of BNIP3 immunohistochemistry in TMAs containing (n=158) melanoma metastases and related Kaplan Meier plot of survival difference comparing melanoma patients with different BNIP3 histopathological scores. Scale bars represent 50µm.


Growth curves of B16 - F10 tumors generated with cells transduced with either empty vector ( CntlKD ) , shRNA against BNIP3 ( BNIP3KD ) or ATG5 ( ATG5KD ) ( n = 7 per condition ) and analyzed with a two - way ANOVA with Tukey ' s multiple comparisons test .

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Growth curves of B16-F10 tumors generated with cells transduced with either empty vector (CntlKD), shRNA against BNIP3 (BNIP3KD) or ATG5 (ATG5KD) (n=7 per condition) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test.


Haematoxylin and eosin staining of sections representative of CntlKD , BNIP3KD or ATG5KD B16 - F10 tumors . Necrotic areas are highlighted with an asterisk ( * ) . Quantification of TUNEL + tumor area ( representing the fraction of dying / dead cell population ) of CntlKD , BNIP3KD or ATG5KD B16 - F10 tumors ( n = 5 per condition ) . Quantification of the Ki67 + cells within the tumor area ( representing the fraction of proliferative cell populations in non - necrotic areas ) of CntlKD ( n = 7 ) , BNIP3KD ( n = 6 ) and ATG5KD ( n = 8 ) B16 - F10 tumors .

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Haematoxylin and eosin staining of sections representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors. Necrotic areas are highlighted with an asterisk (*). Quantification of TUNEL+ tumor area (representing the fraction of dying/dead cell population) of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors (n= 5 per condition). Quantification of the Ki67+ cells within the tumor area (representing the fraction of proliferative cell populations in non-necrotic areas) of CntlKD (n=7), BNIP3KD (n=6) and ATG5KD (n=8) B16-F10 tumors.


Immunofluorescent staining for Ki67 ( red ) representative of CntlKD , BNIP3KD or ATG5KD B16 - F10 tumor sections . Immunofluorescent staining for LC3B ( orange granularity ) representative of CntlKD , BNIP3KD or ATG5KD B16 - F10 tumor sections . A zoomed - in area is shown in the lower - left corner .

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Immunofluorescent staining for Ki67 (red) representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumor sections. Immunofluorescent staining for LC3B (orange granularity) representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumor sections. A zoomed-in area is shown in the lower-left corner.


Confocal microscopy images of co - localization analysis of the BNIP3 - proficient ( shCntl , n = 27 cells ) or deficient ( shBNIP3 , n = 31 cells ) B16 - F10 cells transiently transfected with GFP - LC3 and immunostained for TOMM20 ( mitochondrial marker ) . Colocalization events of GFP - LC3 puncta ( green ) with TOMM20 ( red ) are highlighted with white arrows . The percentage of GFP - LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann - Whitney ' s non - parametric test . Scale bars represent 10µm .

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Confocal microscopy images of co-localization analysis of the BNIP3-proficient (shCntl, n=27 cells) or deficient (shBNIP3, n=31 cells) B16-F10 cells transiently transfected with GFP-LC3 and immunostained for TOMM20 (mitochondrial marker). Colocalization events of GFP-LC3 puncta (green) with TOMM20 (red) are highlighted with white arrows. The percentage of GFP-LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann-Whitney's non-parametric test. Scale bars represent 10µm.


Glut1 , Hk2 , Pkm1 , Pkm2 , Pdk1 , Mct1 , Ldha , Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16 - F10 cell lysates . Transcript expression is represented as the fold - change relative to their corresponding shCntl value . Heatmap shows the average values in each condition ( n = 4 ) and they were analyzed with a one - sample T - test only against shCntl , except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test .

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Glut1, Hk2, Pkm1, Pkm2, Pdk1, Mct1, Ldha, Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16-F10 cell lysates. Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average values in each condition (n=4) and they were analyzed with a one-sample T-test only against shCntl, except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA and ACTIN protein levels from lysates of B16 - F10 cells ( n = 4 ) or B16 - F10 tumors [ CntlKD ( n = 5 ) , BNIP3KD ( n = 5 ) and ATG5KD ( n = 7 ) ] ) . The HIF - 1α - OH / HIF - 1α ratio is shown in a bar graph below . Densitometric quantifications relative to ACTIN levels are shown below each corresponding band . Analysis was performed as indicated in the Data Information section , unless for HIF - 1α / Actin ratio ( in vivo ) , which was analyzed with Kruskal - Wallis non - parametric test with Dunn ' s multiple comparisons test , and LDHA / Actin ratio for shATG5 condition ( in vitro ) , which was analyzed with Wilcoxon ' s rank test .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from lysates of B16-F10 cells (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=5) and ATG5KD (n=7)]). The HIF-1α-OH /HIF-1α ratio is shown in a bar graph below. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. Analysis was performed as indicated in the Data Information section, unless for HIF-1α/Actin ratio (in vivo), which was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test, and LDHA/Actin ratio for shATG5 condition (in vitro), which was analyzed with Wilcoxon's rank test.


Immunohistochemical staining for HIF - 1α ( brown ) representative from CntlKD , BNIP3KD and ATG5KD , B16 - F10 tumor tissues . Higher magnification of the highlighted ( with a white square ) stained sections is shown below .

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Immunohistochemical staining for HIF-1α (brown) representative from CntlKD, BNIP3KD and ATG5KD, B16-F10 tumor tissues. Higher magnification of the highlighted (with a white square) stained sections is shown below.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA , PHD2 and ACTIN protein levels from normoxic lysates of B16 - F10 cells transfected in presence of non - targeting siRNA sequences ( Scr ) or siRNA against PHD2 .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, PHD2 and ACTIN protein levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against PHD2.


Relative intracellular Fe2 + levels in normoxic B16 - F10 cells ( n = 4 ) . Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one - way ANOVA ( Geisser - Greenhouse correction ) with Holm - Sidak ' s multiple comparisons test . Scale bars represent 50µm .

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Relative intracellular Fe2+ levels in normoxic B16-F10 cells (n=4). Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Scale bars represent 50µm.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA , NCOA4 and ACTIN levels from normoxic lysates of B16 - F10 cells transfected in presence of non - targeting siRNA sequences ( Scr ) or siRNA against NCOA4 ( n = 4 ) .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, NCOA4 and ACTIN levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4).


Growth curves of B16 - F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase ( Luc ) or an undegradable HIF - 1α ( HIF - 1α - AA ) construct ( n = 7 for CntlKD Luc / BNIP3KD HIF - 1α - AA , n = 8 CntlKD HIF - 1α - AA and n = 5 for BNIP3KD Luc ) and analyzed with a two - way ANOVA with Tukey ' s multiple comparisons test . Representative double immunostaining for CD31 and αSMA in B16 - F10 tumors ( n = 5 per condition ) . Scale bars represent 50µm .

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Growth curves of B16-F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase (Luc) or an undegradable HIF-1α (HIF-1α-AA) construct (n=7 for CntlKD Luc/BNIP3KD HIF-1α-AA, n=8 CntlKD HIF-1α-AA and n=5 for BNIP3KD Luc) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test. Representative double immunostaining for CD31 and αSMA in B16-F10 tumors (n=5 per condition). Scale bars represent 50µm.


B Observed dsDNA processing patterns of Sgs1 , including an overview over consecutive bursts and pauses ( upper panel ) as well as detailed views into individual bursts containing multiple unwinding events ( lower panels ) . A typical unwinding event of Sgs1 starts with slow , gradual unwinding of the dsDNA followed by DNA rehybridization , that can be almost instant ( 76 % of events , see blue sections in lower right panel ) or contain slow rewinding sections ( 24 % of events , see orange sections in lower right panel ) .

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B Observed dsDNA processing patterns of Sgs1, including an overview over consecutive bursts and pauses (upper panel) as well as detailed views into individual bursts containing multiple unwinding events (lower panels). A typical unwinding event of Sgs1 starts with slow, gradual unwinding of the dsDNA followed by DNA rehybridization, that can be almost instant (76% of events, see blue sections in lower right panel) or contain slow rewinding sections (24% of events, see orange sections in lower right panel).


C Histograms of the observed unwinding and rewinding velocities for Sgs1 . The mean unwinding rate was 65 ± 2 bp / s ( N = 899 ) . The mean rewinding rate was 115 ± 6 bp / s ( N = 287 ) . We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections .

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C Histograms of the observed unwinding and rewinding velocities for Sgs1. The mean unwinding rate was 65 ± 2 bp/s (N = 899). The mean rewinding rate was 115 ± 6 bp/s (N = 287). We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections.


B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses ( upper panel ) and detailed views of single bursts ( lower panels ) . Unwinding events are always followed by slow rewinding events ( orange sections ) .

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B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses (upper panel) and detailed views of single bursts (lower panels). Unwinding events are always followed by slow rewinding events (orange sections).


C Histograms of the observed unwinding and rewinding velocities for Sgs1 in the presence of RPA ( brown bars ) . The mean unwinding rate was 51 ± 3 bp / s ( N = 2139 ) . The mean rewinding rate was 66 ± 3 bp / s ( N = 2091 ) . White bars show for comparison the distributions of unwinding and rewinding velocities for Sgs1

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C Histograms of the observed unwinding and rewinding velocities for Sgs1 in the presence of RPA (brown bars). The mean unwinding rate was 51 ± 3 bp/s (N = 2139). The mean rewinding rate was 66 ± 3 bp/s (N = 2091). White bars show for comparison the distributions of unwinding and rewinding velocities for Sgs1 alone


D Force - dependence of the Sgs1 unwinding ( black squares ) ( N = 2139 ) and rewinding rates ( black diamonds ) ( N = 2091 ) in the presence of RPA ( errors given as standard deviations ) . Red circles represent the force - dependent DNA opening and closure rates measured for RPA

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D Force-dependence of the Sgs1 unwinding (black squares) (N = 2139) and rewinding rates (black diamonds) (N = 2091) in the presence of RPA (errors given as standard deviations). Red circles represent the force-dependent DNA opening and closure rates measured for RPA alone


B Typical dsDNA processing event . The dashed red line connects the minima at the end of the rewinding sections The inset shows a DNA unwinding event in the presence of nuclease - dead Dna2 E675A and RPA .

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B Typical dsDNA processing event. The dashed red line connects the minima at the end of the rewinding sections The inset shows a DNA unwinding event in the presence of nuclease-dead Dna2 E675A and RPA.


C Histograms of the observed unwinding and rewinding rates ( green bars ) with mean values of 69 ± 3 bp / s ( N = 365 ) and 74 ± 5 bp / s ( N = 282 ) , respectively . White bars show for comparison the unwinding and rewinding velocities of Sgs1 in the presence of

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C Histograms of the observed unwinding and rewinding rates (green bars) with mean values of 69 ± 3 bp/s (N = 365) and 74 ± 5 bp/s (N = 282), respectively. White bars show for comparison the unwinding and rewinding velocities of Sgs1 in the presence of RPA


B dsDNA processing events observed for Sgs1 in the presence of Top3 - Rmi1 . Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone .

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B dsDNA processing events observed for Sgs1 in the presence of Top3-Rmi1. Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone.


C Histogram of the unwinding rate of Sgs1 in the presence of Top3 - Rmi1 ( violet bars ) with a mean rate of 86 ± 3 bp / s ( N = 1101 ) . For comparison , the distribution of unwinding rates for Sgs1 alone is depicted with white

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C Histogram of the unwinding rate of Sgs1 in the presence of Top3-Rmi1 (violet bars) with a mean rate of 86 ± 3 bp/s (N = 1101). For comparison, the distribution of unwinding rates for Sgs1 alone is depicted with white bars


E dsDNA processing by Sgs1 in the presence of Top3 - Rmi1 and RPA . In contrast to the absence of RPA , DNA closure is seen as a slow rewinding .

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E dsDNA processing by Sgs1 in the presence of Top3-Rmi1 and RPA. In contrast to the absence of RPA, DNA closure is seen as a slow rewinding.


F Histograms of unwinding and rewinding rates of Sgs1 in the presence of Top3 - Rmi1 and RPA ( purple bars ) . The mean rates for unwinding and rewinding are 66 ± 2 bp / s ( N = 502 ) and 70 ± 4 bp / s ( N = 369 ) , respectively . For comparison , the velocity distributions for Sgs1 in the presence of RPA are shown as white

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F Histograms of unwinding and rewinding rates of Sgs1 in the presence of Top3-Rmi1 and RPA (purple bars). The mean rates for unwinding and rewinding are 66 ± 2 bp/s (N = 502) and 70 ± 4 bp/s (N = 369), respectively. For comparison, the velocity distributions for Sgs1 in the presence of RPA are shown as white bars


A Mean duration of bursts ( violet bars ) ( N > 40 ) and pauses ( green bars ) ( N > 40 ) for the different enzyme combinations . Error bars represent standard error of the mean .

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A Mean duration of bursts (violet bars) (N > 40) and pauses (green bars) (N > 40) for the different enzyme combinations. Error bars represent standard error of the mean.


B Mean duration of a unidirectional unwinding event ( N > 350 ) . Error bars represent standard error of the mean .

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B Mean duration of a unidirectional unwinding event (N > 350). Error bars represent standard error of the mean.


C Mean processivity of unidirectional DNA unwinding ( N > 350 ) . Error bars represent standard error of the mean .

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C Mean processivity of unidirectional DNA unwinding (N > 350). Error bars represent standard error of the mean.


C : Time course of PIKfyve downregulation in brains of prion - infected mice ( 3 mice / time point ) . PIKfyve ( but not FIG4 , calnexin , NeuN and synaptophysin ) was suppressed between 60 and 120 dpi . T : terminal disease stage .

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C: Time course of PIKfyve downregulation in brains of prion-infected mice (3 mice/time point). PIKfyve (but not FIG4, calnexin, NeuN and synaptophysin) was suppressed between 60 and 120 dpi. T: terminal disease stage.


E : Prion - infected Gt1 cells ( 75 dpi ) stained with Alexa - Fluor647 - succinimidyl ester which stains vacuoles negatively ( arrows ) .

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E: Prion-infected Gt1 cells (75 dpi) stained with Alexa-Fluor647-succinimidyl ester which stains vacuoles negatively (arrows).


G : tga20 COCS were treated with pooled IgG or POM1 ( 72 hours ) . POM1 suppressed PIKfyve but not FIG4 and VAC14 . Right : Quantification of immunoblots . Each dot represents an independent experiment . * * : p < 0 . 01 ; * * * : p < 0 . 001 ; unpaired t - test Error bars represent s . e . m .

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G: tga20 COCS were treated with pooled IgG or POM1 (72 hours). POM1 suppressed PIKfyve but not FIG4 and VAC14. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; ***: p<0.001; unpaired t-test Error bars represent s.e.m.


H : Immunoblots of human CJD brains ( red : sampled region ) . Gt1Sc : prion - infected Gt1 cells . Loading controls : actin and calnexin ( Cnx )

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H: Immunoblots of human CJD brains (red: sampled region). Gt1Sc: prion-infected Gt1 cells. Loading controls: actin and calnexin (Cnx)


C : Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment . Each dot represents an individual experiment . Statistics : Unpaired t - test . * * : p < 0 . 01 .

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C: Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment. Each dot represents an individual experiment. Statistics: Unpaired t-test. **: p<0.01.


G : Left : partially restored PIKfyve levels in prion - infected tga20 mice treated with LIN5044 ( LCP ) or vehicle . Right : quantification of the western blot . Each dot represents an individual biological replicate . * : p0 . 05 ; unpaired t - test Error bars represent s . e . m .

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G: Left: partially restored PIKfyve levels in prion-infected tga20 mice treated with LIN5044 (LCP) or vehicle. Right: quantification of the western blot. Each dot represents an individual biological replicate. *: p0.05; unpaired t-test Error bars represent s.e.m.


H : Left , tga20 COCS were inoculated with RML or NBH , optionally treated with GSK2606414 starting at 21 dpi , and lysed at 35 dpi . Depletion of PIKfyve in prion - infected samples was largely rescued by GSK2606414 ( GSK ) . Right , quantification of western blot . Each dot represents an individual biological replicate . Statistics : Unpaired t - test . * * : p < 0 . 01 ; unpaired t - test ; Error bars represent s . e . m .

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H: Left, tga20 COCS were inoculated with RML or NBH, optionally treated with GSK2606414 starting at 21 dpi, and lysed at 35 dpi. Depletion of PIKfyve in prion-infected samples was largely rescued by GSK2606414 (GSK). Right, quantification of western blot. Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01; unpaired t-test; Error bars represent s.e.m.


E , F : Acyl - rac of Gt1 cells subjected to siRNA against zDHHC9 , zDHHC21 or both ( 72 hours post transfection ) , showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 ( quantified in F ) . Each dot represents a separate experiment ( unpaired t - test ) . * * : p < 0 . 01 . Error bars represent s . e . m .

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E, F: Acyl-rac of Gt1 cells subjected to siRNA against zDHHC9, zDHHC21 or both (72 hours post transfection), showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 (quantified in F). Each dot represents a separate experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m.


H - I : Flag - tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding . After 72 hours , zDHHC9 / 21 ( magenta ) were localized to the Golgi apparatus ( GM130 + , cyan ) in NBH - exposed cells but not in prion - infected cells . n = 3 individual biological replicates .

O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

H-I: Flag-tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding. After 72 hours, zDHHC9/21 (magenta) were localized to the Golgi apparatus (GM130+, cyan) in NBH-exposed cells but not in prion-infected cells. n=3 individual biological replicates.


A : Upper row : prion - infected Gt1 cells ( 75 dpi ) immunostained for LAMP1 showing prominent vacuoles . Control : NBH - inoculated cells . Middle row : Gt1 cells transfected with shRNA targeting PIKfyve or luciferase ( control ) for 5 days and immunostained for LAMP1 . Depletion of PIKfyve resulted in LAMP1 + vacuoles ( yellow ) . DAPI : nuclear stain . Lower row : prion - infected and NBH treated cells ( 75 dpi ) contained LAMP2 - vacuoles . n = 3 individual biological replicates .

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A: Upper row: prion-infected Gt1 cells (75 dpi) immunostained for LAMP1 showing prominent vacuoles. Control: NBH-inoculated cells. Middle row: Gt1 cells transfected with shRNA targeting PIKfyve or luciferase (control) for 5 days and immunostained for LAMP1. Depletion of PIKfyve resulted in LAMP1+ vacuoles (yellow). DAPI: nuclear stain. Lower row: prion-infected and NBH treated cells (75 dpi) contained LAMP2- vacuoles. n=3 individual biological replicates.


D : tga20 COCS treated with POM1 or control IgG ( 10 days ) and immunostained with NeuN and LAMP1 . Nuclei : DAPI . POM1 - treated COCS showed increased LAMP1 expression ( quantification : Appendix Fig . S4B ) . NeuN + cells exhibited vacuoles ( arrowheads ) .

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D: tga20 COCS treated with POM1 or control IgG (10 days) and immunostained with NeuN and LAMP1. Nuclei: DAPI. POM1-treated COCS showed increased LAMP1 expression (quantification: Appendix Fig. S4B). NeuN+ cells exhibited vacuoles (arrowheads).


G : Lysosomal enzymes , but not β - actin , were elevated in brains of terminally scrapie - sick mice . Panels show independent triplicates . Statistics : ANOVA . . * * : p < 0 . 01 . Error bars represent s . e . m .

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G: Lysosomal enzymes, but not β-actin, were elevated in brains of terminally scrapie-sick mice. Panels show independent triplicates. Statistics: ANOVA.. **: p<0.01. Error bars represent s.e.m.


A : Time course of TFEB phosphorylation in prion - infected C57BL / 6 mice . Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥ 90 days . Quantification : Each dot represents an individual biological replicate . Statistics : Unpaired t - test . * : p0 . 05 ; * * : p < 0 . 01 . Error bars represent s . e . m .

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A: Time course of TFEB phosphorylation in prion-infected C57BL/6 mice. Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥90 days. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. *: p0.05; **: p<0.01. Error bars represent s.e.m.


B : TFEB - controlled transcripts in brains of terminally scrapie - sick C57BL / 6 mice were measured by qPCR . Prion infection led to upregulation of these genes , but not of STAT3 . Control : NBH - inoculated mice . ANOVA on independent triplicates . * * * : p < 0 . 001 . Error bars represent s . e . m .

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B: TFEB-controlled transcripts in brains of terminally scrapie-sick C57BL/6 mice were measured by qPCR. Prion infection led to upregulation of these genes, but not of STAT3. Control: NBH-inoculated mice. ANOVA on independent triplicates. ***: p<0.001. Error bars represent s.e.m.


E : Gt1 cells ( as in D ) were fixed and stained with anti - TFEB antibody . Prion - infected cells showed nuclear translocation of TFEB . DAPI : Blue . Quantification : Each dot represents an individual biological replicate ( 30 images were quantified per replicate ) . Statistics : Unpaired t - test . * * : p < 0 . 01 . Error bars represent s . e . m .

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E: Gt1 cells (as in D) were fixed and stained with anti-TFEB antibody. Prion-infected cells showed nuclear translocation of TFEB. DAPI: Blue. Quantification: Each dot represents an individual biological replicate (30 images were quantified per replicate). Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m.


F : TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions ( 75 dpi ) . Panels depict independent triplicates . Statistics : ANOVA . * : p0 . 05 . Error bars represent s . e . m . G : TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl ( shRNA against luciferase ) . Panels depict independent triplicates . Statistics : ANOVA . * : p0 . 05 . Error bars represent s . e . m .

O O B-GENEPROD O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

F: TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions (75 dpi). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m. G: TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl (shRNA against luciferase). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m.


C : Prion - infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi . Control : empty lentiviral vector . At 79 dpi , GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels . Right : Quantification of western blots ( n = 3 ; unpaired t - test ) . Each dot represents an individual experiment . * * : p < 0 . 01 . Error bars represent s . e . m .

O O B-GENEPROD O O B-CELL_LINE O O B-ORGANISM O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C: Prion-infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi. Control: empty lentiviral vector. At 79 dpi, GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels. Right: Quantification of western blots (n=3; unpaired t-test). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.