( E , F ) Western blotting confirming Nf1 downregulation p - Fak1 upregulation in ( E ) DOXO - treated LKR10 ( F ) DOXO - treated LKR13 clones transduced with sgNf1 . 3 ( n = 3 biological replicates ) .

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(E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates).


( G ) FAK1 target gene expression in DOXO - treated PDKN1 transduced with PGK - control or PGK - NF1 ( n = 3 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( H , I ) Subcutaneous volumes of DOXO - treated PDKN1 PDKN2 transduced with PGK - control or PGK - NF1 ( n = 18 each ) .

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(H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each).


( J - L ) Subcutaneous volumes of ( J ) SW1573 , ( K ) LKR10 , ( L ) LKR13 transfected with sgTom or sgNf1 . 3 ( n = 18 each ) .

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(J-L) Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n=18 each).


( M ) Depictions quantitation of KrasG12D / + ; p53 + / + ( K - only ) autochthonous burden ( total / total lung ) in pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(M) Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( N ) of grades in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(N) Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( O ) of Ki - 67 - positive nuclei per mm2 in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(O) Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( A ) Glucose consumption lactate excretion in KP KPΔNF1 clones normalized by ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( B ) Glutamine consumption in KP KPΔNF1 clones normalized by ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( C ) Relative viability of KP KPΔNF1 after 72 h of GPNA treatment assayed by - titer glo ( relative luminescent units ) ( n = 4 biological replicates ) .

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(C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates).


( D ) Cumulative population doublings of KP KPΔNF1 cultured with 2 . 0 or 0 . 5 mM glutamine ( n = 4 biological replicates ) .

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(D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates).


( E ) Two - derived KRAS ; NF1 - mutant LUAD lines ( PDKN1 , PDKN2 ) , as well as control - derived KRAS - mutant / NF1 - WT LUAD ( PDK ) , were passaged for 14 population doublings prior to collection . displays most significantly upregulated genes in PDKN1 PDKN2 lines ( relative to PDK control ) , with degree of upregulation depicted by color as indicated in legend .

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(E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend.


of expression of six key altered genes in cultured PDKN1 ( passaged for 14 population doublings ) vs . their respective original samples using qPCR . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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Quantification of the expression of the six key altered genes in cultured PDKN1 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).


of expression of six key altered genes in cultured PDKN2 ( passaged for 14 population doublings ) vs . their respective original samples using qPCR . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

Quantification of the expression of the six key altered genes in cultured PDKN2 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs).


( H ) Western blotting of Psat1 expression in KP KPΔNF1 infected with sgTom or sgPsat1 following selection . GAPDH was used as a loading control .

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(H) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection. GAPDH was used as a loading control.


( I ) Cumulative population doublings of KP KPΔNF1 after transduction with sgTom - or sgPsat1 - containing vectors ( n = 4 biological replicates ) .

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(I) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).


( J - L ) Cumulative population doublings of - derived KRAS - mutant LUAD lines that are either ( J , K ) NF1 - mutant ( PDKN1 PDKN2 ) or ( L ) NF1 - WT ( PDK ) after selection with sgTom - or sgPsat1 - containing vectors ( n = 4 biological replicates ) .

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(J-L) Cumulative population doublings of patient-derived KRAS-mutant LUAD cell lines that are either (J, K) NF1-mutant (PDKN1 and PDKN2) or (L) NF1-WT (PDK) after selection with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).


Relative viability of KP KPΔNF1 clones after CB - 839 treatment ( IC50 : 18 nM ) for 72 h assayed via CellTiter - Glo ( relative luminescence units ) . data points are relative to vehicle - treated controls ( n = 4 biological replicates ) .

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Relative viability of KP and KPΔNF1 clones after CB-839 treatment (IC50: 18 nM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).


Relative viability of KP KPΔNF1 clones after AOA treatment ( IC50 : 6 µM ) for 72 h assayed via CellTiter - Glo ( relative luminescence units ) . data points are relative to vehicle - treated controls ( n = 4 biological replicates ) .

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Relative viability of KP and KPΔNF1 clones after AOA treatment (IC50: 6 µM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).


( D ) Cumulative population doublings of KP KPΔNF1 incubated with vehicle , 18 nM CB - 839 , or 6 µM AOA after 6 d in culture ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(D) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle, 18 nM CB-839, or 6 µM AOA after 6 d in culture (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( E ) Relative viability of indicated human KRAS - mutant lung cancer lines by trypan blue exclusion . were incubated with vehicle , 18 nM CB - 839 , or 6 µM AOA for 72 h ( n = 4 biological replicates ) . data points are normalized to vehicle - treated lines . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(E) Relative viability of the indicated human KRAS-mutant lung cancer cell lines by trypan blue exclusion assay. Cells were incubated with vehicle, 18 nM CB-839, or 6 µM AOA for 72 h (n = 4 biological replicates). All data points are normalized to vehicle-treated cell lines. For bar charts, data presented as means with error bars representing standard deviations (SDs).


by micro - CT of mean volumes in ( B ) CB - 839 - , or vehicle - treated KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , 20 post - infection with control sgTom or sgNf1 . 3 ( n = 21 mice 21 mice , respectively ) . Vehicle - treated KP mice are same control group for ( B )

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Quantification by micro-CT of mean tumor volumes in (B) CB-839-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (B)


by micro - CT of mean volumes in ( C ) AOA - , or vehicle - treated KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , 20 post - infection with control sgTom or sgNf1 . 3 ( n = 21 mice 21 mice , respectively ) . Vehicle - treated KP mice are same control group for ( C ) .

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Quantification by micro-CT of mean tumor volumes in (C) AOA-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (C).


by micro - CT of total volume per mouse in ( D ) CB - 839 - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , 20 post - infection . Vehicle - treated KP mice are same control group for ( D )

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Quantification by micro-CT of total tumor volume per mouse in (D) CB-839-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (D)


E ) by micro - CT of total volume per mouse in , ( E ) AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 8 , 12 , 16 , 20 post - infection . Vehicle - treated KP mice are same control group for ( E ) .

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E) Quantification by micro-CT of total tumor volume per mouse in , (E) AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (E).


Relative responses of KP mice tumors treated with ( F ) CB - 839 , or vehicle starting from week 8 post - infection measured until week 20 . Relative response = average volume with treatment / average volume with vehicle .

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Relative responses of KP mice tumors treated with (F) CB-839, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of KP mice tumors treated with ( G ) AOA , or vehicle starting from week 8 post - infection measured until week 20 . Relative response = average volume with treatment / average volume with vehicle .

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Relative responses of KP mice tumors treated with (G) AOA, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.


( H ) Depictions quantitation of total burden ( total / total lung ) in CB - 839 - , AOA - , or vehicle - treated KP mice after infection with control sgTom or sgNf1 . 3 at 20 post - infection . Vehicle - treated KP mice are same control group in both boxplots . For boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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(H) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in CB-839-, AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 20 weeks post-infection. Vehicle-treated KP mice are the same control group in both boxplots. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


Relative responses of subcutaneous KP KPΔNF1 tumors treated with vehicle , ( B ) CB - 839 , starting from day 12 measured until day 27 ( n = 6 tumors per per treatment ) Relative response = average volume with treatment / average volume with vehicle .

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Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, (B) CB-839, starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.


) Relative responses of subcutaneous KP KPΔNF1 tumors treated with vehicle , C ) AOA starting from day 12 measured until day 27 ( n = 6 tumors per per treatment ) Relative response = average volume with treatment / average volume with vehicle .

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) Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, C) AOA starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous KP KPΔNF1 tumors treated with vehicle , CB - 839 , or AOA ( D ) final masses of these tumors . or boxplots , whiskers indicate minimum maximum values , upper lower perimeters represent first third quartiles , midline represents median value , x symbol represents mean .

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Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, CB-839, or AOA (D) the final masses of these tumors. or boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


Relative responses of orthotopic KP KPΔNF1 tumors treated with vehicle , ( E ) CB - 839 starting from day 12 measured until day 27 ( n = 24 mice per per treatment ) . Relative response = average volume with treatment / average volume with vehicle . of growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 transduced with a luciferase vector .

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Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (E) CB-839 starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of orthotopic KP KPΔNF1 tumors treated with vehicle , ( F ) AOA starting from day 12 measured until day 27 ( n = 24 mice per per treatment ) . Relative response = average volume with treatment / average volume with vehicle . of growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 transduced with a luciferase vector .

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Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (F) AOA starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of subcutaneous KP - ix tumors harboring doxycycline ( DOX ) - inducible gain - of - function ( GOF ) - Fak1 cDNA treated with vehicle , ( G ) CB - 839 , with or without DOX / PIP2 ( n = 36 mice per treatment group ) . Relative response = average volume with treatment / average volume with vehicle .

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Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle, (G) CB-839, with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous KP - ix tumors harboring doxycycline ( DOX ) - inducible gain - of - function ( GOF ) - Fak1 cDNA treated with vehicle ( H ) AOA with or without DOX / PIP2 ( n = 36 mice per treatment group ) . Relative response = average volume with treatment / average volume with vehicle .

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Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle (H) AOA with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous - derived NF1 - mutant ( PDKN1 PDKN2 ) NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( I ) CB - 839 starting from day 12 measured until day 30 ( n = 6 tumors per per treatment ) . Relative response = average volume with treatment / average volume with vehicle .

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Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (I) CB-839 starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of subcutaneous - derived NF1 - mutant ( PDKN1 PDKN2 ) NF1 - WT ( PDK ) LUAD tumors treated with vehicle ( J ) AOA starting from day 12 measured until day 30 ( n = 6 tumors per per treatment ) . Relative response = average volume with treatment / average volume with vehicle .

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Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle (J) AOA starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.


Relative responses of orthotopic - derived NF1 - mutant ( PDKN1 PDKN2 ) NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( K ) CB - 839 starting from day 12 measured until day 27 ( n = 12 mice per per treatment ) . Relative response = average volume with treatment / average volume with vehicle . of growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 transduced with a luciferase vector .

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Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (K) CB-839 starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


Relative responses of orthotopic - derived NF1 - mutant ( PDKN1 PDKN2 ) NF1 - WT ( PDK ) LUAD tumors treated with vehicle , ( L ) AOA starting from day 12 measured until day 27 ( n = 12 mice per per treatment ) . Relative response = average volume with treatment / average volume with vehicle . of growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 transduced with a luciferase vector .

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Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (L) AOA starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.


A - D Proportional distribution of chymotrypsin - like activity of proteasome across nine tissues from ( n = 9 ) , ( n = 10 ) mice . Proportional distribution of trypsin - like activity of proteasome across nine tissues from ( n = 9 ) , ( n = 10 ) mice . Graphical representation of color code of tissues analyzed .

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A-D Proportional distribution of chymotrypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Proportional distribution of trypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Graphical representation of color code of tissues analyzed.


Chymotrypsin - like activity of proteasome across nine tissues in mice . Each data point ( n = 9 n = 10 ) indicates data from a single mouse performed in triplicate . Each central error bar indicate mean + / - . * indicates p < 0 . 05 by unpaired two - students t - comparing samples .

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Chymotrypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


trypsin - like activity of proteasome across nine tissues in mice . Each data point ( n = 9 n = 10 ) indicates data from a single mouse performed in triplicate . Each central error bar indicate mean + / - . * indicates p < 0 . 05 by unpaired two - students t - comparing samples .

trypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


A - D Western blot for K48Ub - linked in spinal cord ( A ) intestine ( C ) lysates from mice . ( B , D ) of panels A C , respectively . Values were normalized to actin as a loading control . Each central error bar indicate mean + / - of a biological replicates . Data information : * indicates p < 0 . 05 by unpaired two - students t - comparing samples .

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A-D Western blot analysis for K48Ub-linked proteins in spinal cord (A) and intestine (C) lysates from male and female mice. (B,D) Quantification of panels A and C, respectively. Values were normalized to actin as a loading control. Each central line and error bar indicate mean +/- SEM of a biological replicates. Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


E ) Curve of correlation between chymotrypsin - like activity Ub - K48 linked in intestine , where variability between mice is observed .

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E) Curve of correlation between chymotrypsin-like activity and Ub-K48 linked proteins in the intestine, where variability between mice is observed.


F ) Native gel followed by western of β5 subunit to distinguish unassembled catalytic ( CP ) or assembled with regulatory ( RP ) in ( n = 5 ) ( n = 5 ) mice in intestine .

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F) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and females (n=5) mice in intestine.


G ) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic ( CP ) or assembled with regulatory ( RP ) in ( n = 5 ) ( n = 5 ) spinal cord .

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G) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and female (n=5) spinal cord.


H ) of of assembled ( RP - CP ) versus unassembled ( CP ) proteasome in intestine of . Each central error bar indicate mean + / - of a biological replicates ( n = 5 per group ) . I ) of of RP - CP versus CP proteasome in spinal cord of . Each central error bar indicate mean + / - of a biological replicates ( n = 5 per group ) . Data information : * indicates p < 0 . 05 by unpaired two - students t - comparing samples .

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H) Quantification of the ratio of assembled (RP-CP) versus unassembled (CP) proteasome in the intestine of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). I) Quantification of the ratio of RP-CP versus CP proteasome in the spinal cord of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


J ) Correlation curve between chymotrypsin - like activity RP - CP / CP in ( purple ) ( blue ) intestine . K ) Correlation curve between chymotrypsin - like activity RP - CP / CP in ( purple ) ( blue ) spinal cord .

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J) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) intestine. K) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) spinal cord.


A - D Comparison of chymotrypsin - like activity from ( n = 9 ) ( n = 10 ) mice in young ( 3 - 5 months old ) or older ( 10 - 15 months old , ( n = 6 ) ( n = 7 ) ) groups . Each point indicates a single mouse . A ) Tissues that did not show significant decline , B ) Tissues that show significant decline in only , C ) Tissues that show significant decline in only or D ) in both . Each central error bar indicate mean + / - of a biological replicates ( n = 9 young , n = 6 older , n = 10 young , n = 7 older ) . P values obtained by unpaired two - students t - comparing young older samples are indicated .

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A-D Comparison of chymotrypsin-like activity from female (n=9) and male (n=10) mice in young (3-5 months old) or older (10-15 months old, female (n=6) and male (n=7)) groups. Each point indicates a single mouse. A) Tissues that did not show significant decline, B) Tissues that show significant decline in females only, C) Tissues that show significant decline in males only or D) in both males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=9 young females, n=6 older females, n=10 young males, n=7 older males). P values obtained by unpaired two-tailed students t-test comparing young and older samples are indicated.


B ) Western blot of Ub - K48 in soluble fractions of intestine lysates from mice plus or minus heat shock . C ) of panel B . Each central error bar indicate mean + / - of a biological replicates ( n = 3 biological replicates per group ) . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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B) Western blot analysis of Ub-K48 proteins in the soluble fractions of intestine lysates from mice plus or minus heat shock. C) Quantification of panel B. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group). Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


D ) Western blot of Ub - K48 in insoluble fraction of intestine lysates from mice plus or minus heat shock . E ) of D . Each central error bar indicate mean + / - of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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D) Western blot analysis of Ub-K48 proteins in the insoluble fraction of intestine lysates from mice plus or minus heat shock. E) Quantification of D. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


F ) Western blot of Ub - K48 in soluble fractions of spinal cord lysates from mice plus or minus heat shock . G ) of F . Each central error bar indicate mean + / - of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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F) Western blot analysis of Ub-K48 proteins in the soluble fractions of spinal cord lysates from mice plus or minus heat shock. G) Quantification of F. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


H ) Western blot of Ub - K48 in insoluble fraction of spinal cord lysates from mice plus or minus heat shock . I ) of H . Each central error bar indicate mean + / - of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

H) Western blot analysis of Ub-K48 proteins in the insoluble fraction of spinal cord lysates from mice plus or minus heat shock. I) Quantification of H. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


K ) Western blot of Ub - K48 in insoluble fraction of lysates of SOD1 - G93A mouse spinal cord plus or minus heat shock . L ) of panel K . Each central error bar indicate mean + / - of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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K) Western blot of Ub-K48 proteins in the insoluble fraction of protein lysates of SOD1-G93A mouse spinal cord plus or minus heat shock. L) Quantification of panel K. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


M ) Chymotrypsin - like proteasome activity in spinal cord lysates from SOD1 - G93A mice . Each central error bar indicate mean + / - of a biological replicates ( n = 5 biological replicates per group ) . Bordered points indicate biological replicates un - bordered points indicate technical replicates . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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M) Chymotrypsin-like proteasome activity in spinal cord lysates from SOD1-G93A mice. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group). Bordered points indicate biological replicates and un-bordered points indicate technical replicates. Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


N ) Filter trap of lysates from insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . O ) of panel N . Each central error bar indicate mean + / - of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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N) Filter trap assay of protein lysates from the insoluble fraction of SOD1-G93A spinal cord lysates plus or minus heat shock. O) Quantification of panel N. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


P ) Representative transmission electron of in insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . Scale bar equals 600nm . Q ) of P . ( n = 9 technical replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , * * * indicates p < 0 . 0005 by unpaired two students t - comparing samples or samples with or without heat shock .

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P) Representative transmission electron micrographs of protein aggregates in the insoluble fraction of SOD1-G93A spinal cord lysates plus or minus heat shock. Scale bar equals 600nm. Q) Quantification of P. (n=9 technical replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


( A ) Multiple alignment of Atg8 homologues . ‐ acid sequences of Atg8 its mammalian homologues are aligned using Clustal _ W program . Underlined residues represent mutated in Atg8 .

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(A) Multiple sequence alignment of Atg8 homologues. Amino‐acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal_W program. Underlined residues represent mutated amino acids in Atg8.


( B ) Alignment of Atg8 predicted three ‐ dimensional structure with its mammalian homologues . SWISS MODEL was used to predict Atg8 structure PyMol was used for presentation . Orange represents predicted Atg8 structure , red , blue green represent GATE ‐ 16 , GABARAP LC3 , respectively .

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(B) Alignment of Atg8 predicted three‐dimensional structure with its mammalian homologues. SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation. Orange represents the predicted Atg8 structure, and red, blue and green represent GATE‐16, GABARAP and LC3, respectively.


( C ) Three ‐ dimensional model of Atg8 . predicted structure contains a ubiquitin fold decorated by two further ‐ terminal helices ( Tyr 49 , Leu 50 , Phe 77 Phe 79 are marked in green ) .

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(C) Three‐dimensional model of Atg8. The predicted structure contains a ubiquitin fold decorated by two further amino‐terminal helices (amino acids Tyr 49, Leu 50, Phe 77 and Phe 79 are marked in green).


( A ) were grown to mid ‐ exponential phase then incubated in presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . Aminopeptidase I ( API ) maturation was tested in incubated in control or starvation medium by western blot using anti ‐ Atg8 ( right bottom panel ) , anti ‐ GFP ( left bottom panel ) or anti ‐ API antibodies ( upper panel ) .

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(A) Cells were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies (upper panel).


( B ) were - labelled with [ 35S ] methionine chased on non - radioactive starvation medium . Aliquots were taken at indicated times acid - soluble small peptides generated by proteolysis were determined .

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(B) Cells were pulse-labelled with [35S]methionine and chased on non-radioactive starvation medium. Aliquots were taken at the indicated times and acid-soluble small peptides generated by proteolysis were determined.


( C ) were grown in control or in starvation medium visualized by microscopy . Arrows point to pre ‐ autophagosomal structure ( PAS ) , arrowheads point to GFP within vacuoles .

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(C) Cells were grown in control or in starvation medium and visualized by light microscopy. Arrows point to the pre‐autophagosomal structure (PAS), and arrowheads point to GFP within the vacuoles.


( D ) extracts were subjected to SDS - polyacrylamide gel electrophoresis followed by immunoblot using anti ‐ GFP antibodies . GFP , green ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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(D) Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis using anti‐GFP antibodies. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) Atg8WT - HA , Atg8F77A - HA , Atg8F79A - HA or Atg8F77 / 79A - HA was transformed into Δatg8 . extracts were prepared subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) containing 6 M urea , followed by immunoblot with anti - Atg8 antibodies .

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(A) Atg8WT-HA, Atg8F77A-HA, Atg8F79A-HA or Atg8F77/79A-HA was transformed into Δatg8 cells. Cell extracts were prepared and subjected to 13.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) containing 6 M urea, followed by immunoblot analysis with anti-Atg8 antibodies.


( B ) Prediction of - interaction sites in Atg8 ( Neuvirth et al , 2004 ) . A three ‐ dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online ) with red representing predicted - binding sites , Phe 77 Phe 79 are marked in green within prediction site .

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(B) Prediction of protein-protein interaction sites in Atg8 ( Neuvirth et al, 2004). A three‐dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online) with red representing the predicted protein-protein binding sites, and Phe 77 and Phe 79 are marked in green within the prediction site.


( C ) Pull ‐ down of Atg8 mutants with Atg4 . His - Atg4 was attached to cobalt beads ( Talon metal ‐ affinity resin ) , washed incubated with total extracts of Atg8 mutants . Elution was subjected to SDS - PAGE followed by western blot using anti ‐ Atg8 anti ‐ Atg4 antibodies .

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(C) Pull‐down assay of Atg8 mutants with Atg4. His-Atg4 was attached to cobalt beads (Talon metal‐affinity resin), washed and incubated with total cell extracts of Atg8 mutants. Elution was subjected to SDS-PAGE followed by western blot analysis using anti‐Atg8 and anti‐Atg4 antibodies.


( D ) Localization of green ( GFP ) - Atg8wt GFP - Atg877 / 79 carrying Atg4 plasmid under normal ( control ) starvation conditions . Arrows point to pre ‐ autophagosomal structure , arrowheads point to GFP within vacuoles .

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(D) Localization of green fluorescent protein (GFP)-Atg8wt and GFP-Atg877/79 cells carrying Atg4 plasmid under normal (control) and starvation conditions. Arrows point to the pre‐autophagosomal structure, and arrowheads point to GFP within the vacuoles.


( E ) Aminopeptidase I ( API ) maturation was tested in incubated in control or starvation medium by western blot using anti ‐ API antibodies . mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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(E) Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐API antibodies. mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) extracts were subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis containing 6 M urea , followed by immunoblot using anti ‐ Atg8 ‐ 15N peptide antibodies .

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(A) Cell extracts were subjected to 13.5% SDS-polyacrylamide gel electrophoresis containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐15N peptide antibodies.


( B ) were grown in control or in starvation medium visualized using microscopy . Arrows point to pre ‐ autophagosomal structure , whereas arrowheads point to GFP within vacuoles .

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(B) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy. Arrows point to the pre‐autophagosomal structure, whereas arrowheads point to GFP within the vacuoles.


( C ) Aminopeptidase I ( API ) maturation GFP cleavage were tested as described above . Asterisk represents nonspecific band . GFP , green ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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(C) Aminopeptidase I (API) maturation and GFP cleavage were tested as described above. Asterisk represents nonspecific band. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) Δatg8 transformed with GFP - Atg8WT , GFP - Atg8L50A or GFP - Atg8Y49A constructs were grown to mid ‐ exponential phase then incubated in presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . API maturation was tested using anti ‐ Atg8 ( right bottom panel ) , anti ‐ GFP ( left bottom panel ) or anti ‐ API antibodies .

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(A) Δatg8 cells transformed with GFP-Atg8WT, GFP-Atg8L50A or GFP-Atg8Y49A constructs were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. API maturation was tested using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies.


( B ) Starvation ‐ induced degradation of Δatg8 harbouring different mutants was tested as described above . API , aminopeptidase I ; GFP , green ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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(B) Starvation‐induced protein degradation of Δatg8 harbouring the different mutants was tested as described above. API, aminopeptidase I; GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) GFP - Atg8 localization was determined using microscopy ( arrows depict pre ‐ autophagosomal structure , arrowheads point to GFP within vacuoles ) . were also tested for appearance of autophagic bodies by using microscopy .

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(A) GFP-Atg8 localization was determined using fluorescence microscopy (arrows depict pre‐autophagosomal structure, and arrowheads point to GFP within vacuoles). Cells were also tested for the appearance of autophagic bodies by using light microscopy.


( B ) Immunoblot of different extracts was carried out using anti ‐ GFP antibodies ( asterisk represents nonspecific band ) .

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(B) Immunoblot analysis of different cell extracts was carried out using anti‐GFP antibodies (asterisk represents nonspecific band).


( C ) Atg8WT - HA , Atg8L50A - HA or Atg8Y49A - HA was transformed into Δatg8 Δatg8Δatg4 double knockout strains extracts were subjected to SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) followed by immunoblot using anti ‐ HA antibodies ( lower panel ; asterisk represents nonspecific band ) .

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(C) Atg8WT-HA, Atg8L50A-HA or Atg8Y49A-HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblot analysis using anti‐HA antibodies (lower panel; asterisk represents nonspecific band).


( D ) Atg8WT , Atg8L50A or Atg8Y49A was transformed into Δatg8 Δatg8Δatg4 double knockout strains grown under control or starvation conditions for 4 h . extracts were subjected to 13 . 5 % SDS - PAGE containing 6 M urea , followed by immunoblot using anti ‐ Atg8 ‐ N15 antibodies . GFP , green ; WT , wild type .

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(D) Atg8WT, Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h. Cell extracts were subjected to 13.5% SDS-PAGE containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐N15 antibodies. GFP, green fluorescent protein; WT, wild type.


Overexpression of WT OGT , but not catalytic - inactive OGT mutant , increased O - GlcNAc of TDP - 43 . OGT were examined by immunoblotting . of ( G ) .

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Overexpression of WT OGT, but not catalytic-inactive OGT mutant, increased the O-GlcNAc levels of TDP-43. OGT protein levels were examined by immunoblotting.   Quantification of (G).


Yeast expressing indicated constructs were induced in galactose - containing medium , representative were shown using microscopy to visualize TDP - 43 . DIC : Differential interference contrast . TDP - 43 aggregate foci numbers from of ( B ) were quantified from over 50 each of indicated yeast strains . foci numbers in > 3 were labelled with yellow , foci numbers in < 3 are labelled with blue .

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Yeast cells expressing the indicated constructs were induced in galactose-containing medium, and representative images were shown using fluorescence microscopy to visualize TDP-43 aggregates. DIC: Differential interference contrast. TDP-43 aggregate foci numbers from individual cell of (B) were quantified from over 50 cells each of the indicated yeast strains. The foci numbers in individual cell > 3 were labelled with yellow, and the foci numbers in individual cell < 3 are labelled with blue.


SH - SY5Y treated with indicated compounds were immunostained with TDP - 43 or pTDP - 43 observed by immunofluorescence . of signal intensities of pTDP - 43 ( left ) or foci numbers per ( right ) . number counted : left panel , " Mock " n = 58 , " GlcNAc " n = 35 , " EA " n = 45 , " GlcNAc + EA " n = 52 ; right panel , " Mock " n = 1167 , " GlcNAc " n = 1051 , " EA " n = 542 , " GlcNAc + EA " n = 815 .

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SH-SY5Y cells treated with the indicated compounds were immunostained with TDP-43 or pTDP-43 and observed by immunofluorescence. Quantification of signal intensities of pTDP-43 staining (left) or foci numbers per cell (right). Cell number counted: left panel, "Mock" n = 58, "GlcNAc" n = 35, "EA" n = 45, "GlcNAc+EA" n = 52; right panel, "Mock" n = 1167, "GlcNAc" n = 1051, "EA" n = 542, "GlcNAc+EA" n = 815.


SDS - soluble insoluble fractions were isolated from SH - SY5Y expressing WT or catalytic - inactive mutant OGT treated with 20 μM EA , fractions were analyzed by immunoblotting . of ( F ) .

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SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT or catalytic-inactive mutant OGT treated with 20 μM EA, and the fractions were analyzed by immunoblotting. Quantification of (F).


expressing indicated forms of GFP - TDP - 43 were subjected to immunoprecipitation , O - GlcNAcylated of TDP - 43 were examined . of ( G ) .

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Cells expressing the indicated forms of GFP-TDP-43 were subjected to immunoprecipitation, and the O-GlcNAcylated levels of TDP-43 were examined.   Quantification of (G).


Walking speed of 10 - day old adult flies of indicated genotypes was calculated Mean ± SD . Unpaired two - t - , biological replicates : Control , n = 63 , WT , n = 45 , T199A , n = 70 , T233A , n = 87 ; 2TA , n = 43 .

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Walking speed of 10-day old adult male flies of the indicated genotypes was calculated Mean ± SD. Unpaired two-tailed t-test, biological replicates: Control, n = 63, WT, n = 45, T199A, n = 70, T233A, n = 87; 2TA, n = 43.


. Adult flies were fed with regular sucrose diet ( RS ) or high sucrose diet ( HS ) , survival of adult flies of indicated genotypes were examined . Kaplan - Meier survival , n ≥ 40 for each group .

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.Adult flies were fed with regular sucrose diet (RS) or high sucrose diet (HS), and survival percentage of adult flies of the indicated genotypes were examined. Kaplan-Meier survival analysis, n ≥ 40 for each group.


CFTR splicing was performed in with knockdown of either OGT , TDP - 43 , or combination . ACTB served as loading controls ( top panel ) . indicated were examined by immunoblotting ( bottom panel ) .

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CFTR splicing assay was performed in cells with knockdown of either OGT, TDP-43, or combination. ACTB levels served as loading controls (top panel). The indicated protein levels were examined by immunoblotting (bottom panel).


CFTR splicing was examined in siTDP - 43 overexpressing WT or mutant OGT by PCR . ACTB served as loading controls ( top panel ) . Various were examined by immunoblotting ( bottom panel ) .

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CFTR splicing assay was examined in siTDP-43 cells overexpressing the WT or mutant OGT by PCR analysis. ACTB levels served as loading controls (top panel). Various protein levels were examined by immunoblotting (bottom panel).


Representative PCR to examine expression of spliced mRNA isoform containing exon 2a of STMN2 in SH - SY5Y with indicated constructs ( top panel ) . TDP - 43 were examined by immunoblotting ( bottom panel ) .

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Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH-SY5Y cells with the indicated constructs (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).


A BreastMark microarray platform ; Kaplan - Meier curves of overall survival ( OS ) , demonstrating that high expression of ESRP1 ( red ) is associated with poor prognosis in ER + breast cancer . A log rank was used to calculate P = 2 . 8 e - 07 ( n = 934 , number of events = 216 ) .

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A BreastMark microarray platform; Kaplan-Meier curves of overall survival (OS), demonstrating that high expression of ESRP1 (red line) is associated with poor prognosis in ER+ breast cancer. A log rank test was used to calculate P=2.8 e-07 (n= 934, number of events= 216).


B BreastMark microarray platform ; Kaplan - Meier curves of overall survival ( OS ) of ER - breast cancer . A log rank was used to calculate P = 0 . 4481 ( n = 322 , number of events = 130 ) .

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B BreastMark microarray platform; Kaplan-Meier curves of overall survival (OS) of ER- breast cancer. A log rank test was used to calculate P=0.4481 (n= 322, number of events= 130).


C BreastMark microarray platform ; correlation of ESRP1 expression with overall survival in tamoxifen - treated . A log rank was used to calculate P = 1 . 218 , e - 06 ( n = 210 , number of events = 49 ) .

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C BreastMark microarray platform; correlation of ESRP1 expression with overall survival in tamoxifen-treated patients. A log rank test was used to calculate P=1.218, e-06 (n= 210, number of events= 49).


D BreastMark microarray platform ; correlation of ESRP1 expression with overall survival in chemotherapy - treated . A log rank was used to calculate P = 0 . 28 ( n = 129 , number of events = 21 )

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D BreastMark microarray platform; correlation of ESRP1 expression with overall survival in chemotherapy-treated patients. A log rank test was used to calculate the P=0.28 (n= 129, number of events= 21)


E Cancer Genome Atlas Breast Invasive Carcinoma ( TCGA - BRCA ) - seq dataset ; Kaplan - Meier curves of overall survival ( OS ) in ER + breast cancer , demonstrating that high expression of ESRP1 ( red ) is associated with poor prognosis in ER + . A log rank was used to calculate P = 0 . 00011 ( n = 656 , number of events = 62 ) .

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E The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) RNA-seq dataset; Kaplan-Meier curves of overall survival (OS) in ER+ breast cancer, demonstrating that high expression of ESRP1 (red line) is associated with poor prognosis in ER+. A log rank test was used to calculate P=0.00011 (n= 656, number of events= 62).


F Cancer Genome Atlas Breast Invasive Carcinoma ( TCGA - BRCA ) - seq dataset ; Kaplan - Meier curves of overall survival ( OS ) in ER - breast cancer . Red - high ESRP1 expression ; Black - low ESRP1 expression . A log rank was used to calculate P = 0 . 19 ( n = 100 , number of events = 17 ) .

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F The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) RNA-seq dataset; Kaplan-Meier curves of overall survival (OS) in ER- breast cancer. Red-high ESRP1 expression; Black-low ESRP1 expression. A log rank test was used to calculate P=0.19 (n=100, number of events=17).


A RT - qPCR confirmed stable knockdown of ESRP1 expression in endocrine - resistant using a shRNA lentiviral system ( Mission TRC human shRNA constructs - Sigma ) ; 2 - control ( LCC2 transduced with pLKO . 1 control vector ) 2C1 2C3 ( LCC2 transduced with pLKO . 1 shRNA ESRP1 ) . Clones were selected for expression verification ( clone 1 clone 3 ) ; 9 - control ( LCC9 transduced with pLKO . 1 control vector ) , 9C2 9C3 ( LCC9 transduced with pLKO . 1 shRNA ESRP1 . Clones were selected for expression verification ( clone 2 clone 3 ) . * represents P < 0 . 05 ; statistically significant . Data ( mean ± SD ) were calculated using two - way ANOVA on three independent biological replicates .

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A RT-qPCR confirmed the stable knockdown of ESRP1 expression in endocrine-resistant cells using a shRNA lentiviral system (Mission TRC human shRNA constructs-Sigma); 2-control (LCC2 cells transduced with pLKO.1 control vector) and 2C1 and 2C3 (LCC2 cells transduced with pLKO.1 shRNA ESRP1). Clones were selected for expression verification (clone 1 and clone 3); 9-control (LCC9 cells transduced with pLKO.1 control vector), 9C2 and 9C3 (LCC9 cells transduced with pLKO.1 shRNA ESRP1. Clones were selected for expression verification (clone 2 and clone 3). * represents P <0.05; statistically significant. Data (mean ± SD) were calculated using two-way ANOVA based on three independent biological replicates.


B Western blot using ESRP1 antibody GTX 131373 ( GeneTex ) . GAPDH is used as reference control .

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B Western blot analysis using ESRP1 antibody GTX 131373 (GeneTex). GAPDH is used as the reference control.


C Relative density was determined by crystal violet . were treated with TAM ( 10 - 6 M ) or ICI ( 10 - 9 M ) for 6 days in presence absence of E2 ( β - estradiol ; 10 - 10 M ) . Data ( mean + SD ) were calculated using two - way ANOVA on three independent biological replicates .

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C Relative cell density was determined by crystal violet assay. Cells were treated with TAM (10-6 M) or ICI (10-9 M) for 6 days in the presence and absence of E2 (β-estradiol; 10-10 M). Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates.


D viability was determined using Biolabs CytoSelect ™ Transformation using 2 - control , 2C3 , 9 - control 9C2 in CSM - charcoal stripped media , E2 ( β - estradiol ; 10 - 10 M ) 10 % bovine serum . Data ( mean + SD ) were calculated using two - way ANOVA on three independent biological replicates .

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D Cell viability was determined using Cell Biolabs CytoSelect™ Cell Transformation Assay using 2-control, 2C3, 9-control and 9C2 cells in CSM-charcoal stripped media, E2 (β-estradiol; 10-10 M) and 10% fetal bovine serum. Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates.


E Impact of ESRP1 knockdown on in vivo growth . Five million ( 2 - control , 2C3 , 9 - control 9C2 ) were implanted into mammary fat pads of athymic mice ( five mice per group ) in presence of supplemental estrogen . Tumors were measured weekly using calipers for external measurements . volume was calculated as L x W2 / 2 , where L is W is width ( note different scale for volumes ) . Data ( mean ± SD ) were calculated using two - way ANOVA ( n = 5 mice per group ) .

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E Impact of ESRP1 knockdown on in vivo tumor growth. Five million cells (2-control, 2C3, 9-control and 9C2) were implanted into mammary fat pads of athymic mice (five mice per group) in the presence of supplemental estrogen. Tumors were measured weekly using calipers for external measurements. Tumor volume was calculated as L x W2/2, where L is length and W is width (note the different scale for tumor volumes). Data (mean ± SD) were calculated using two-way ANOVA (n= 5 mice per group).


A using high - power magnification ( 40x ) of cultured lines under a microscope in LCC2 model - 2 - control 2C3 . Scale bar : 50 µm .

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A Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC2 model-2-control and 2C3 cells. Scale bar: 50 µm.


B using high - power magnification ( 40x ) of cultured lines under a microscope in LCC9 model - 9 - control 9C2 . Scale bar : 50 µm .

B Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC9 model-9-control and 9C2 cells. Scale bar: 50 µm.


C - D expression of key EMT - TFs in ESRP1 knockdown ( 2C3 9C2 ) compared to endocrine resistant control lines ( 2 - control 9 - control ) using Western blotting .

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C-D Protein expression of key EMT-TFs in ESRP1 knockdown cells (2C3 and 9C2) compared to endocrine resistant control cell lines (2-control and 9-control) using Western blotting analysis.


E expression of key EMT - TFs in ESRP1 knockdown ( T - 47D - kESRP1 ) ESRP1 - overexpressing MCF - 7 compared to their control counterparts ( T - 47D - control MCF - 7 - control , respectively ) .

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E Protein expression of key EMT-TFs in ESRP1 knockdown cells (T-47D-kESRP1) and ESRP1-overexpressing MCF-7 cells compared to their control counterparts (T-47D-control and MCF-7-control, respectively).


A Doughnut chart for - seq showing differentially regulated alternative splicing events ( ASEs ) their corresponding genes in ESRP1 knockdown compared to control ; SE : skipped exon , RI : retained intron , MXE : mutually exclusive exons , A5SS : alternative 5 ' splice site , A3SS : alternative 3 ' splice site ) . " spliced in " ( PSI or ψ ) value was estimated . Differences in inclusion ( ∆ ψ ) between samples their significance were calculated ( P < 0 . 05 IΔψI > 0 . 1 ) . Circle 1 : ASEs ; Circle 2 : genes corresponding to ASEs ; Circle 3 : significant ASEs ; Circle 4 : significant genes corresponding to significantly altered ASEs ; Circle 5 : significant ASEs with ESRP1 motifs ; Circle 6 : significant genes corresponding to significantly altered ASEs with ESRP1 motifs . ESRP - binding motifs were obtained from Dittmar et al . 31 [ ] analyzed on following assumption : presence of motif in downstream region of an exon results in inclusion of exon , while upstream or intraexonic location leads to exclusion of exon .

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A Doughnut chart for RNA-seq analysis showing the differentially regulated alternative splicing events (ASEs) and their corresponding genes in ESRP1 knockdown cells compared to control cells; SE: skipped exon, RI: retained intron, MXE: mutually exclusive exons, A5SS: alternative 5' splice site, and A3SS: alternative 3' splice site). The "percent spliced in" (PSI or ψ) value was estimated. Differences in inclusion levels (∆ψ) between samples and their significance were calculated (P<0.05 and IΔψI>0.1). Circle 1: all ASEs; Circle 2: all genes corresponding to all ASEs; Circle 3: significant ASEs; Circle 4: significant genes corresponding to significantly altered ASEs; Circle 5: significant ASEs with ESRP1 motifs; Circle 6: significant genes corresponding to significantly altered ASEs with ESRP1 motifs. ESRP-binding motifs were obtained from Dittmar et al.31[] and analyzed based on the following assumption: the presence of the motif in the downstream region of an exon results in inclusion of the exon, while upstream or intraexonic location leads to exclusion of the exon.


B Radar plot showing that EMT gene signature was not altered at gene level in ESRP1 knockdown model ( LCC2 set ) . C Radar plot showing that EMT gene signature was altered at ASEs level ( SE ) in ESRP1 knockdown model ( LCC2 set ) .

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B Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC2 set). C Radar plot showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC2 set).


D Radar plot showing that EMT gene signature was not altered at gene level in ESRP1 knockdown model ( LCC9 set ) . E Radar plots showing that EMT gene signature was altered at ASEs level ( SE ) in ESRP1 knockdown model ( LCC9 set ) .

D Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC9 set). E Radar plots showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC9 set).


B TCGA Splicegraphs for EMT signature genes in ESRP1high versus ESRP1low tumors . A splice graph of gene ' s exons is shaded on expression level shows selected splice event outlined in red .

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B TCGA Splicegraphs for the EMT signature genes in ESRP1high versus ESRP1low tumors. A splice graph of the gene's exons is shaded based on expression level and shows the selected splice event outlined in red.


A Volcano plots showing distribution of significant gene expression changes using HTA 2 . 0 platform .

A Volcano plots showing the distribution of significant gene expression changes using the HTA 2.0 platform.


C Validation of of FASN , SCD1 PHGDH in ESRP1 knockdown versus control resistant lines using Western blot . GAPDH was used as loading control . data are representative of three biological replicates .

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C Validation of the protein levels of FASN, SCD1 and PHGDH protein levels in ESRP1 knockdown versus control resistant cell lines using Western blot analysis. GAPDH was used as the loading control. The data are representative of three individual biological replicates.


B . Diluting concentrations of 4D7 to bound full - 30 acid peptide compared to scrambled , C - terminal N - terminal 11 acid peptides as shown in A ( n = 3 biological replicates ) .

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B. Diluting concentrations of 4D7 to bound full-length 30 amino acid peptide compared to scrambled, C-terminal and N-terminal 11 amino acid peptides as shown in A (n=3 biological replicates).


F . Proliferation curves of factor - independent TF - 1 TpoR CALRdel61 cultured with 2 , 10 or 20 µg / mL 4D7 or 20 µg / mL of control IgG antibody ( n = 3 biological replicates ) . G Proliferation curves of factor - independent TF - 1 TpoR CALRdel52 cultured with 10 or 20 µg / mL 4D7 or 20 µg / mL of control IgG antibody ( n = 3 biological replicates ) . Data information : bars represent standard error of mean summarizing 3 biological replicates performed in triplicate , with a one - way ANOVA with Bonferroni correction used to determine statistical significance .

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F. Proliferation curves of factor-independent TF-1 TpoR CALRdel61 cells cultured with 2, 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). G Proliferation curves of factor-independent TF-1 TpoR CALRdel52 cells cultured with 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). Data information: bars represent standard error of the mean summarizing 3 biological replicates performed in triplicate, with a one-way ANOVA with Bonferroni correction used to determine statistical significance.


B . Similar experiment using TPO - independent TF - 1 TpoR CALRdel61 . C . Similar experiment using TPO - independent TF - 1 TpoR CALRdel52 at 8 hours . D . Similar experiment using PBMNCs from CALRdel52 PMF at 8 hours . Additionally , were treated with 280 nM of ruxolitinib as a positive control .

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B. Similar experiment using TPO-independent TF-1 TpoR CALRdel61 cells. C. Similar experiment using TPO-independent TF-1 TpoR CALRdel52 cells at 8 hours. D. Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 hours. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control.


F . Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF - 1 TpoR . An increase in caspase 3 is observed after 48 hours of treatment with 20 µg / mL 4D7 in TF - 1 TpoR CALRdel61 TF - 1 TpoR CALRdel52 .

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F. Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF-1 TpoR cells. An increase in cleaved caspase 3 is observed after 48 hours of treatment with 20 µg/mL 4D7 in TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel52 cells.


G . Western blot of TpoR immunoprecipitation under non - reducing conditions showing associated CALR 50 kDa monomers 100 kDa dimers ( red arrowheads ) present only in TF - 1 TpoR CALRdel61 disrupted by 8 - hour treatment with 20 µg / mL 4D7 but not PBS or 20 µg / mL IgG . CALR monomers & dimers are detectable by polyclonal anti - wild type CALR or anti - mutant CALR monoclonal antibodies . Red arrowheads , detected mutant CALR ; brown arrowheads , detected wildtype CALR , asterisk , non - specific bands .

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G. Western blot of TpoR immunoprecipitation under non-reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF-1 TpoR CALRdel61 disrupted by 8-hour treatment with 20 µg/mL 4D7 but not PBS or 20 µg/mL IgG. CALR monomers & dimers are detectable by polyclonal anti-wild type CALR or anti-mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wildtype CALR protein, asterisk, non-specific bands.


B . Graph showing decreased fold change of CD41 + CD61 + megakaryocytes cultured in 4D7 compared to IgG . FACS - sorted CD34 + from myelofibrosis with CALR or JAK2 mutation were cultured over 12 days without TPO in presence of SCF , IL - 6 IL - 9 . Black columns show JAK2V617F mutation positive samples ( n = 4 technical replicates of samples from different ) . Data information : Unpaired students t - used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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B. Graph showing the decreased fold change of CD41+CD61+ megakaryocytes cultured in 4D7 compared to IgG. FACS-sorted CD34+ from myelofibrosis patients with CALR or JAK2 mutation were cultured over 12 days without TPO in the presence of SCF, IL-6 and IL-9. Black columns show JAK2V617F mutation positive samples (n= 4 technical replicates of samples from different patients). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


C . Number of CD41 + megakaryocytes derived from isolated CD34 + progenitors from myelofibrosis . Number of CD41 / CD61 + counted on day 12 using trypan blue exclusion ( n = 1 technical replicate ) . D . Summary of fold change reduction of CD41 / CD61 + megakaryocytes by 4D7 in tested CALR mutated samples compared to CALR wild type , normalised to IgG ( n = 11 samples from different , with 4 technical replicates per sample ) . Data information : Bars represent standard error of means Unpaired students t - used to determine statistical significance * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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C. Number of CD41+ megakaryocytes derived from isolated CD34+ progenitors from myelofibrosis patients. Number of CD41/CD61+ cells counted on day 12 using trypan blue exclusion (n=1 technical replicate). D. Summary of fold change reduction of CD41/CD61+ megakaryocytes by 4D7 in all tested CALR mutated patient samples compared to CALR wild type, normalised to IgG (n=11 samples from different patients, with 4 technical replicates per sample). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


E Number of CD41 + megakaryocyte colonies from samples after 4D7 treatment . CD34 + from with myelofibrosis with CALRdel52 or CALRins5 were plated on collagen - matrix in presence of 20 µg / mL 4D7 or IgG control ( n = 3 biological replicates ) . Data information : Unpaired students t - used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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E Number of CD41+ megakaryocyte colonies from patient samples after 4D7 treatment. CD34+ from patients with myelofibrosis with CALRdel52 or CALRins5 were plated on collagen-based matrix in presence of 20 µg/mL 4D7 or IgG control (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


F . Representative showing CD41 + colonies in pink CD41 - colonies in blue after treatment with 4D7 or IgG at 100 X or 40X magnification . Scale bar indicates 100µm . G . Summary of fold change reduction of CD41 + megakaryocytes in CALR mutated samples cultured MegaCult treated with 4D7 compared to IgG ( n = 4 biological replicates ) . Data information : Bars represent standard error of means Unpaired students t - used to determine statistical significance * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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F. Representative micrographs showing CD41+ colonies in pink and CD41- colonies in blue after treatment with 4D7 or IgG at 100 X or 40X magnification. Scale bar indicates 100µm. G. Summary of fold change reduction of CD41+ megakaryocytes in CALR mutated samples cultured MegaCult treated with 4D7 compared to IgG (n= 4 biological replicates). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


H . Number of megakaryocyte colony forming units grouped according to colony number after 4D7 treatment in a mutant CALRins5 sample ( n = 3 biological replicates ) . Data information : Unpaired students t - used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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H. Number of megakaryocyte colony forming units grouped according to colony cell number after 4D7 treatment in a mutant CALRins5 sample (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


B . Effect of 4D7 on TPO - dependent megakaryocyte differentiation of healthy CD34 + cultured with 20 µg / mL 4D7 or IgG control ( n = 2 biological replicates , with 2 technical replicates ) . Data information : Error bars indicate standard error of mean .

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B. Effect of 4D7 on TPO-dependent megakaryocyte differentiation of healthy CD34+ cells cultured with 20 µg/mL 4D7 or IgG control (n= 2 biological replicates, with 2 technical replicates). Data information: Error bars indicate standard error of mean.


C . Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG . CFU - GM , colony forming unit - granulocyte macrophage ; BFU - E , blast forming unit - erythroid ; CFU - GEMM , colony forming unit granulocyte , erythroid , monocyte , megakaryocyte ( n = 3 biological replicates ) . Data information : Error bars indicate standard error of mean .

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C. Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG. CFU-GM, colony forming unit- granulocyte macrophage; BFU-E, blast forming unit-erythroid; CFU-GEMM, colony forming unit granulocyte, erythroid, monocyte, megakaryocyte (n=3 biological replicates). Data information: Error bars indicate standard error of mean.


F . Western blot showing signalling in ruxolitinib - resistant TF - 1 TpoR CALRdel61 compared to ruxolitinib sensitive TF - 1 TpoR CALRdel61 after treatment with 100nM ruxolitinib for 16 hours or 30 minutes blotted for phospho - STAT5 , total STAT5 , phospho - ERK , total ERK actin as indicated . Ruxolitinib - sensitive were non - viable after 16 hours of treatment .

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F. Western blot showing signalling in ruxolitinib-resistant TF-1 TpoR CALRdel61 compared to ruxolitinib sensitive TF-1 TpoR CALRdel61 cells after treatment with 100nM ruxolitinib for 16 hours or 30 minutes and blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin as indicated. Ruxolitinib-sensitive cells were non-viable after 16 hours of treatment.


G . Comparison of growth after DMSO , 20 µg / mL 4D7 , IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib - resistant TF - 1 TpoR CALRdel61 . counted using Trypan Blue exclusion ( n = 3 biological replicates with 3 technical replicates ) . H . Number of colonies observed from plated in MethoCult following 72 hours of treatment with DMSO , 20 µg / mL 4D7 , IgG or 100nM ruxolitinib performed in ruxolitinib - resistant TF - 1 TpoR CALRdel61 ( n = 3 biological replicates with 3 technical replicates ) . Data information : Error bars on represent standard deviation . P values are unpaired Student ' s unpaired t - .

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G. Comparison of cell growth after DMSO, 20 µg/mL 4D7, IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib-resistant TF-1 TpoR CALRdel61. Cells counted using Trypan Blue exclusion (n=3 biological replicates with 3 technical replicates). H. Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO, 20 µg/mL 4D7, IgG or 100nM ruxolitinib performed in ruxolitinib-resistant TF-1 TpoR CALRdel61 cells (n=3 biological replicates with 3 technical replicates). Data information: Error bars on represent standard deviation. All P values are unpaired Student's unpaired t-test.


B . Pharmacokinetic measurements of serum of 4D7 in mice after intraperitoneal injection at 0 , 1 , 24 , 48 , 72 110 hours since administration .

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B. Pharmacokinetic measurements of serum levels of 4D7 in mice after intraperitoneal injection at 0, 1, 24, 48, 72 and 110 hours since administration.


D . Kaplan - Meier survival curve of bone marrow engraftment model of TF - 1 TpoR CALRdel61 showing improved survival of mice treated with 4D7 compared with IgG control commencing 1 week after tail vein injection ( n = 5 mice per treatment ) . Data information : For survival curves log - rank Mantel - Cox P value is shown .

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D. Kaplan-Meier survival curve of bone marrow engraftment model of TF-1 TpoR CALRdel61 cells showing improved survival of mice treated with 4D7 compared with IgG control commencing 1 week after tail vein injection (n=5 mice per treatment). Data information: For all survival curves the log-rank Mantel-Cox test P value is shown.


F . Kaplan - Meier survival curve of TF - 1 TpoR CALRdel61 chloroma mice treated with 4D7 or IgG until humane killing due to tumour diameter > 30 mm or ulceration . ( n = 3 mice per treatment ) G . Kaplan - Meier survival curve of NSG mice engrafted with ruxolitinib - resistant TF - 1 TpoR CALRdel61 treated with 12 . 5 mg / kg 4D7 or IgG twice weekly ( n = 5 6 mice for IgG 4D7 respectively ) . Data information For survival curves log - rank Mantel - Cox P value is shown .

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F. Kaplan-Meier survival curve of TF-1 TpoR CALRdel61 chloroma mice treated with 4D7 or IgG until humane killing due to tumour diameter > 30 mm or ulceration. (n=3 mice per treatment) G. Kaplan-Meier survival curve of NSG mice engrafted with ruxolitinib-resistant TF-1 TpoR CALRdel61 treated with 12.5 mg/kg 4D7 or IgG twice weekly (n=5 and 6 mice for IgG and 4D7 respectively). Data information For all survival curves the log-rank Mantel-Cox test P value is shown.


A . RT - PCR of CNPase ( left ) MBP ( right ) mRNAs expression in MO3 . 13 precursors under control conditions following 10 - 200 μM D - Asp exposure for 3 days . Graphs show of of CNPase , MBP to L19 Data information : values represent means ± S . E . M . Level of significance was determined by using : in A , left panel , one - way ANOVA P < 0 . 0001 followed by Tukey ' s post hoc , * P < 0 . 05 versus control ( n = 3 ) ; A , right panel , one - way ANOVA P < 0 . 0001 followed by Tukey ' s post hoc , * P < 0 . 05 versus control ( n = 3

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A. RT-PCR of CNPase (left) and MBP (right) mRNAs expression in MO3.13 precursors under control conditions and following 10-200 μM D-Asp exposure for 3 days. Graphs show quantification of ratio of CNPase, and MBP to L19 Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, left panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control (n=3); A, right panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P<0.05 versus control (n=3


B . Western blotting ( left ) densitometric ( right ) of MBP expression in absence or in presence of 10 - 200 μΜ D - Asp exposure for 5 days Data information : values represent means ± S . E . M . Level of significance was determined by using ) ; B , one - way ANOVA P = 0 . 0001 followed by Bonferroni post hoc , * P < 0 . 05 versus control ( n =

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B. Western blotting (left) and densitometric analysis (right) of MBP expression in absence or in presence of 10-200 μΜ D-Asp exposure for 5 days Data information: The values represent the means ± S.E.M. Level of significance was determined by using ); B, one-way ANOVA P=0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control (n=3