L , M MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 2 µM BYL719 or DMSO as a vehicle control for the indicated times . Cells were lysed and immunblotted with LC3B , pAKTS473 , AKT ( pan ) , pS6KT389 or S6K antibodies and GAPDH antibodies as a loading control ( L ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to DMSO - treated GFP - vector cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( M ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc test in M ,

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L, M MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 2 µM BYL719 or DMSO as a vehicle control for the indicated times. Cells were lysed and immunblotted with LC3B, pAKTS473, AKT(pan), pS6KT389 or S6K antibodies and GAPDH antibodies as a loading control (L). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (M). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in M,


N , O MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 1 µM or 10 µM SAR405 or DMSO as a vehicle control for 4 hours . Cells were lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control ( N ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to DMSO - treated GFP - vector cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( O ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc test in 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N, O MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 1 µM or 10 µM SAR405 or DMSO as a vehicle control for 4 hours. Cells were lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (N). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (O). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in O,


A , B MCF - 7 cells expressing NT , INPP4B # 1 or INPP4B # 2 shRNA were fixed and immunostained with recombinant GST - 2xFYVE and LAMP2 antibodies , and co - stained with DAPI ( A ) . Data represent the proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 40 cells / experiment ) ( B ) . Arrows indicate co - localization between 2xFYVE and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in A , p values determined by one - way ANOVA with Tukey post hoc test in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (A). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >40 cells/experiment) (B). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by one-way ANOVA with Tukey post hoc test in B


C MCF - 7 cells were treated with 0 . 02 % ( w / v ) saponin for 30 seconds , then fixed and immunostained with INPP4B and either LAMP1 or CD63 antibodies , and co - stained with DAPI . Arrows indicate co - localization between INPP4B and CD63 . Data information : The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in C ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 MCF-7 cells were treated with 0.02% (w/v) saponin for 30 seconds, then fixed and immunostained with INPP4B and either LAMP1 or CD63 antibodies, and co-stained with DAPI. Arrows indicate co-localization between INPP4B and CD63. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C,


D , E MCF - 7 cells co - expressing GFP - INPP4B or GFP - vector , and Hrs or NT shRNA , were fixed and immunostained with recombinant GST - 2xFYVE and LAMP2 antibodies , and co - stained with DAPI ( D ) . Data represent the proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 30 cells / experiment ) ( E ) . Arrows indicate co - localization between 2xFYVE and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in D , p values determined by one - way ANOVA with Tukey post hoc test

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (D). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >30 cells/experiment) (E). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D, p values determined by one-way ANOVA with Tukey post hoc test in


F , G MCF - 7 cells co - expressing GFP - INPP4B or GFP - vector , and Hrs or NT shRNA , were fixed and immunostained with recombinant GST - 2xFYVE and CD63 antibodies , and co - stained with DAPI ( F ) . Data represent the proportion of 2xFYVE + late endosomes ( n = 3 experiments , > 30 cells / experiment ) ( G ) . Arrows indicate co - localization between 2xFYVE and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in F . p values determined by one - way ANOVA with Tukey post hoc test in G ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were fixed and immunostained with recombinant GST-2xFYVE and CD63 antibodies, and co-stained with DAPI (F). Data represent the proportion of 2xFYVE+ late endosomes (n=3 experiments, >30 cells/experiment) (G). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F. p values determined by one-way ANOVA with Tukey post hoc test in G,


H , I MCF - 7 cells co - expressing GFP - INPP4B or GFP - vector , and Hrs or NT shRNA , were lysed and immunoblotted with LC3B antibodies , and GAPDH antibodies as a loading control ( H ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to GFP - vector ; NT shRNA cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( I ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in I .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, I MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were lysed and immunoblotted with LC3B antibodies, and GAPDH antibodies as a loading control (H). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to GFP-vector;NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (I). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in I.


A , B MCF - 7 cells expressing GFP - INPP4B or GFP - vector were fixed and immunostained with LAMP1 or LAMP2 antibodies , and co - stained with DAPI and phalloidin ( A ) . Data represent the number of LAMP1 + or LAMP2 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 50 cells / experiment ) ( B ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in A , p values determined by two - tailed unpaired t test in B ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 MCF-7 cells expressing GFP-INPP4B or GFP-vector were fixed and immunostained with LAMP1 or LAMP2 antibodies, and co-stained with DAPI and phalloidin (A). Data represent the number of LAMP1+ or LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (B). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in A, p values determined by two-tailed unpaired t test in B,


C , D Snapshots of Magic Red cathepsin B substrate and Hoechst 33342 captured in live MCF - 7 cells expressing GFP - INPP4B or GFP - vector ( C ) . Data represent the number of Magic Red cathepsin B + puncta per cell ( n = 3 experiments , > 50 cells / experiment ) ( D ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in C , p values determined by two - tailed unpaired t test in D ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D Snapshots of Magic Red cathepsin B substrate and Hoechst 33342 captured in live MCF-7 cells expressing GFP-INPP4B or GFP-vector (C). Data represent the number of Magic Red cathepsin B+ puncta per cell (n=3 experiments, >50 cells/experiment) (D). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in C, p values determined by two-tailed unpaired t test in D,


E , F MCF - 7 cells expressing NT or INPP4B # 1 shRNA were cultured in growth media or EBSS for the indicated times , then fixed and immunostained with LAMP1 antibodies and co - stained with DAPI and phalloidin ( E ) . Data represent the number of LAMP1 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 30 cells per experiment ) ( F ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in p values determined by one - way ANOVA with Tukey post hoc test in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F MCF-7 cells expressing NT or INPP4B #1 shRNA were cultured in growth media or EBSS for the indicated times, then fixed and immunostained with LAMP1 antibodies and co-stained with DAPI and phalloidin (E). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >30 cells per experiment) (F). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in p values determined by one-way ANOVA with Tukey post hoc test in F


G , H MCF - 7 cells expressing GFP - INPP4B or GFP - vector were transfected with NT , PIK3CA # 1 or PIK3CA # 2 siRNA . After 24 hours , cells were fixed and immunostained with LAMP1 antibodies , and co - stained with DAPI and phalloidin ( G ) . Data represent the number of LAMP1 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 40 cells per experiment ) ( H ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in G . p values determined by one - way ANOVA in H .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H MCF-7 cells expressing GFP-INPP4B or GFP-vector were transfected with NT, PIK3CA #1 or PIK3CA #2 siRNA. After 24 hours, cells were fixed and immunostained with LAMP1 antibodies, and co-stained with DAPI and phalloidin (G). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (H). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in G. p values determined by one-way ANOVA in H.


A , B Snapshots of LAMP1 - GFP , Magic Red cathepsin B substrate and Hoechst 33342 captured in live HeLa cells transfected with INPP4B or NT siRNA ( A ) . Data represent the number of LAMP1 + / Magic Red Cathepsin B + and LAMP1 + / Magic Red Cathepsin B − lysosomes ( terminal storage lysosomes ) per cell ( n = 3 experiments , > 50 cells / experiment ) ( B ) . Yellow arrows indicate LAMP1 + / MR Cathepsin B + lysosomes , and white arrows indicate terminal storage lysosomes . Data information : Data is presented as mean ± SD . The insets at the lower right of each image are higher power regions of the boxed areas . Scale bar is 10 µm in A , p values determined by two - way ANOVA with Holm - Sidak post - hoc test in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Snapshots of LAMP1-GFP, Magic Red cathepsin B substrate and Hoechst 33342 captured in live HeLa cells transfected with INPP4B or NT siRNA (A). Data represent the number of LAMP1+/Magic Red Cathepsin B+ and LAMP1+/Magic Red Cathepsin B− lysosomes (terminal storage lysosomes) per cell (n=3 experiments, >50 cells/experiment) (B).Yellow arrows indicate LAMP1+/MR Cathepsin B+ lysosomes, and white arrows indicate terminal storage lysosomes. Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by two-way ANOVA with Holm-Sidak post-hoc test in B


C Timelapse snapshots of LAMP1 - mCherry signals from MCF - 7 cells expressing GFP - INPP4B or GFP - vector captured by spinning disk microscopy . Maximum intensity projections of 3 z - planes taken 0 . 27 µm apart are shown . Arrows indicate lysosome reformation . Data information : The insets at the lower right of each image are higher power regions of the boxed areas . Scale bar is 10 µm in C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Timelapse snapshots of LAMP1-mCherry signals from MCF-7 cells expressing GFP-INPP4B or GFP-vector captured by spinning disk microscopy. Maximum intensity projections of 3 z-planes taken 0.27 µm apart are shown. Arrows indicate lysosome reformation. Data information: The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C.


E Data represent the number of LAMP1 + reformation events per cell per minute ( n = 12 GFP - vector cells , n = 15 GFP - INPP4B cells ) . Data information : Data is presented as mean ± SD . p values determined by two - tailed unpaired t test in E .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Data represent the number of LAMP1+ reformation events per cell per minute (n=12 GFP-vector cells, n=15 GFP-INPP4B cells). Data information: Data is presented as mean ± SD. p values determined by two-tailed unpaired t test in E.


A , B MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 5 µM YM201636 for 2 hours . Cells were fixed and immunostained with recombinant GST - 2xFYVE and LAMP2 antibodies , and co - stained with DAPI ( A ) . Data represent the proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 30 cells / experiment ) ( B ) . Arrows show co - localization between 2xFYVE and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in A p values determined by one - way ANOVA in

A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 for 2 hours. Cells were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (A). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >30 cells/experiment) (B). Arrows show co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A p values determined by one-way ANOVA in B


D - F MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours , then YM201636 was washed out for 1 , 2 or 4 hours . Cells were fixed and immunostained with LAMP1 antibodies , and co - stained with DAPI and phalloidin ( D ) . Data represent the number of LAMP1 + puncta relative to cell area ( µm2 ) ( E ) , and the lysosome fold change relative to the YM201636 - treated condition for each cell line ( F ) ( n = 3 experiments , > 30 cells per experiment ) . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in D . p values determined by one - way ANOVA in E , or by two - tailed unpaired t test of the area under the curve in F .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours, then YM201636 was washed out for 1, 2 or 4 hours. Cells were fixed and immunostained with LAMP1 antibodies, and co-stained with DAPI and phalloidin (D). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (E), and the lysosome fold change relative to the YM201636-treated condition for each cell line (F) (n=3 experiments, >30 cells per experiment). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D. p values determined by one-way ANOVA in E, or by two-tailed unpaired t test of the area under the curve in F.


G , H MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 1 , 2 or 5 µM YM201636 or DMSO as a vehicle control for 4 hours . Cells were lysed and immunoblotted with LC3B antibodies , and GAPDH antibodies as a loading control ( G ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to DMSO - treated GFP - vector cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( H ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in H ,

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 1, 2 or 5 µM YM201636 or DMSO as a vehicle control for 4 hours. Cells were lysed and immunoblotted with LC3B antibodies, and GAPDH antibodies as a loading control (G). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (H). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in H,


A , B MCF - 7 cells expressing GFP - INPP4B or GFP - vector were transfected with NT , SNX1 or SNX2 siRNA . After 24 hours , cells were fixed and immunostained with LAMP2 antibodies , and co - stained with DAPI and phalloidin ( A ) . Data represent the number of LAMP2 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 40 cells per experiment ) ( B ) . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in A , p values determined by one - way ANOVA in with Tukey post hoc test in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 MCF-7 cells expressing GFP-INPP4B or GFP-vector were transfected with NT, SNX1 or SNX2 siRNA. After 24 hours, cells were fixed and immunostained with LAMP2 antibodies, and co-stained with DAPI and phalloidin (A). Data represent the number of LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (B). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by one-way ANOVA in with Tukey post hoc test in B


C Timelapse snapshots of LAMP1 - mCherry and GFP - SNX2 signals from MCF - 7 cells captured by spinning disk microscopy . Maximum intensity projections of 2 z - planes taken 0 . 27 µm apart are shown . Data information : The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in C ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Timelapse snapshots of LAMP1-mCherry and GFP-SNX2 signals from MCF-7 cells captured by spinning disk microscopy. Maximum intensity projections of 2 z-planes taken 0.27 µm apart are shown. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C,


D , E MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 5 µM YM201636 for 2 hours , then YM201636 was washed out for 1 hour . Cells were fixed and immunostained with SNX2 and LAMP1 antibodies , and co - stained with DAPI ( D ) . Data represent the proportion of SNX2 + LAMP2 puncta ( n = 3 experiments , > 40 cells / experiment ) ( E ) . Arrows show co - localization between SNX2 and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in D , p values determined by one - way ANOVA in with Tukey post hoc test in E ,

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and immunostained with SNX2 and LAMP1 antibodies, and co-stained with DAPI (D). Data represent the proportion of SNX2+ LAMP2 puncta (n=3 experiments, >40 cells/experiment) (E). Arrows show co-localization between SNX2 and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D, p values determined by one-way ANOVA in with Tukey post hoc test in E,


F HeLa cells were transfected with GFP - SNX2 . After 24 hours , cells were treated with 5 µM YM201636 for 2 hours , then YM201636 was washed out for 1 hour . Cells were fixed and subjected to immuno - electron microscopy analysis using GFP ( 10 nm ) antibodies . Arrows show GFP - SNX2 localization to lysosome . Data information : Scale bar is 100 nm in F .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with GFP-SNX2. After 24 hours, cells were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and subjected to immuno-electron microscopy analysis using GFP (10 nm) antibodies. Arrows show GFP-SNX2 localization to lysosome. Data information: Scale bar is 100 nm in F.


G HeLa cells were transfected with GFP - PIKfyve . After 24 hours , cells were were treated with 5 µM YM201636 for 2 hours , then YM201636 was washed out for 1 hour . Cells were fixed and immunostained with SNX2 , GFP and LAMP2 antibodies , and imaged using super resolution microscopy . Arrows show co - localization between GFP - PIKfyve , SNX2 and LAMP2 . Data information : The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with GFP-PIKfyve. After 24 hours, cells were were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and immunostained with SNX2, GFP and LAMP2 antibodies, and imaged using super resolution microscopy. Arrows show co-localization between GFP-PIKfyve, SNX2 and LAMP2. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in


H MCF - 7 cells expressing LAMP1 - mCherry were transfected with NT or SNX2 siRNA . After 24 hours , cells were imaged by spinning disk microscopy for 20 minutes . Representative timelapse snapshots of LAMP1 - mCherry from a maximum projection of 3 z - planes taken 0 . 27 µm apart are shown . Arrow indicates lysosome fission . I Data represent the number of LAMP1 + reformation events per cell per minute ( n = 21 NT siRNA cells , n = 27 SNX2 siRNA cells ) . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in H , p values determined by two - tailed unpaired t test in I .

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H MCF-7 cells expressing LAMP1-mCherry were transfected with NT or SNX2 siRNA. After 24 hours, cells were imaged by spinning disk microscopy for 20 minutes. Representative timelapse snapshots of LAMP1-mCherry from a maximum projection of 3 z-planes taken 0.27 µm apart are shown. Arrow indicates lysosome fission. I Data represent the number of LAMP1+ reformation events per cell per minute (n=21 NT siRNA cells, n=27 SNX2 siRNA cells). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in H, p values determined by two-tailed unpaired t test in I.


A , B HeLa cells transfected with INPP4B or NT siRNA were treated for 1 hour with 5 µg / mL puromycin . Cells were fixed and immunostained with ubiquitin antibodies , and co - stained with DAPI and phalloidin ( A ) . Data represent the percentage of cells with ubiquitinated protein aggregates ( n = 3 experiments , > 200 cells / experiment ) ( B ) . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in A p values determined by one - way ANOVA in with Tukey post hoc test in

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells transfected with INPP4B or NT siRNA were treated for 1 hour with 5 µg/mL puromycin. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (A). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (B). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A p values determined by one-way ANOVA in with Tukey post hoc test in B


C , D HeLa cells were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours , then 5 µg / mL puromycin for 1 hour . Cells were fixed and immunostained with ubiquitin antibodies , and co - stained with DAPI and phalloidin ( C ) . Data represent the percentage of cells with ubiquitinated protein aggregates ( n = 3 experiments , > 200 cells / experiment ) ( D ) . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in C , p values determined by one - way ANOVA in with Tukey post hoc test in D ,

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D HeLa cells were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours, then 5 µg/mL puromycin for 1 hour. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (C). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (D). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C, p values determined by one-way ANOVA in with Tukey post hoc test in D,


E , F MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated for 4 hours with 10 µg / mL puromycin , 5 µm MG132 and either 5 µM YM201636 or DMSO as a vehicle control . Cells were fixed and immunostained with ubiquitin antibodies , and co - stained with DAPI and phalloidin ( E ) . Data represent the percentage of cells with ubiquitinated protein aggregates ( n = 3 experiments , > 200 cells / experiment ) ( F ) . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in E . p values determined by one - way ANOVA in with Tukey post hoc test

O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated for 4 hours with 10 µg/mL puromycin, 5 µm MG132 and either 5 µM YM201636 or DMSO as a vehicle control. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (E). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (F). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in E. p values determined by one-way ANOVA in with Tukey post hoc test in


G HeLa cells transfected with INPP4B or NT siRNA were treated with 10 µg / mL puromycin for 2 - 6 hours , then cell viability was assessed using CellTiter - Glo ® assays . Data represent the relative cell viability normalized to untreated NT siRNA cells ( n = 3 experiments ) . Data information : p values determined by two - tailed unpaired t test of the area under the curve in

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells transfected with INPP4B or NT siRNA were treated with 10 µg/mL puromycin for 2-6 hours, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated NT siRNA cells (n=3 experiments). Data information: p values determined by two-tailed unpaired t test of the area under the curve in G


H HeLa cells were treated 10 µg / mL puromycin ± 5 µM YM201636 or DMSO as a vehicle control for 2 - 6 hours , then cell viability was assessed using CellTiter - Glo ® assays . Data represent the relative cell viability normalized to DMSO - treated cells ( n = 3 experiments ) . Data information : p values determined by one - way ANOVA with Tukey post hoc test of the area under the curve in H .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were treated 10 µg/mL puromycin ± 5 µM YM201636 or DMSO as a vehicle control for 2-6 hours, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to DMSO-treated cells (n=3 experiments). Data information: p values determined by one-way ANOVA with Tukey post hoc test of the area under the curve in H.


( a ) HACE1 mRNA expression is markedly increased in ischaemic and dilated cardiomyopathy ( ICM and DCM ) heart failure ( HF ) patient heart tissue compared with non - failing hearts ( NF ) . Expression levels were normalized to HPRT and GAPDH mRNAs . n = 15 per group . Control , non - failing heart .

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(a) HACE1 mRNA expression is markedly increased in ischaemic and dilated cardiomyopathy (ICM and DCM) heart failure (HF) patient heart tissue compared with non-failing hearts (NF). Expression levels were normalized to HPRT and GAPDH mRNAs. n=15 per group. Control, non-failing heart.


( b ) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls , expression levels were normalized to Hprt and Gapdh mRNAs . n = 6 - 9 per group , P = 0 . 004 ( unpaired two - tailed Student ' s t - tests ) .

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(b) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls, expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group, P=0.004 (unpaired two-tailed Student's t-tests).


( c - e ) Hace1 − / − and WT mice were subjected to sTAC or sham surgery and analysed 4 days after the operation . Echocardiography measurement of ( c ) LV fractional shortening ( FS = ( LVEDD − LVESD ) / LVEDD × 100 % ) ; ( d ) LV end - diastolic dimension ( LVEDD ) ( mm ) and ( e ) LV end - systolic dimension ( LVESD ) ( mm ) are shown . n = 6 - 8 for each group .

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(c-e) Hace1−/− and WT mice were subjected to sTAC or sham surgery and analysed 4 days after the operation. Echocardiography measurement of (c) LV fractional shortening (FS=(LVEDD−LVESD)/LVEDD × 100%); (d) LV end-diastolic dimension (LVEDD) (mm) and (e) LV end-systolic dimension (LVESD) (mm) are shown. n=6-8 for each group.


( f ) Mortality rate of WT and Hace1 − / − mice after sTAC is shown by Kaplan - Meier plots as the survival proportion of each cohort of mice . Survival incidence in mice over the indicated follow - up period is shown as the ratio of the number of mice survived / total mice analysed for each group . This represents 60 % ( 12 / 20 ) for Hace1 − / − sTAC mice , 84 % ( 16 / 19 ) for Hace1 + / + sTAC mice and 100 % ( 12 / 12 ) for both Hace1 − / − and Hace1 + / + sham mice on day 4 ; P value as indicated ( log - rank χ2 - test ) .

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(f) Mortality rate of WT and Hace1−/− mice after sTAC is shown by Kaplan-Meier plots as the survival proportion of each cohort of mice. Survival incidence in mice over the indicated follow-up period is shown as the ratio of the number of mice survived/total mice analysed for each group. This represents 60% (12/20) for Hace1−/− sTAC mice, 84% (16/19) for Hace1+/+ sTAC mice and 100% (12/12) for both Hace1−/− and Hace1+/+ sham mice on day 4; P value as indicated (log-rank χ2-test).


( g ) Heart weight / body weight ratios ( HW / BW ) and ( h ) lung weight / body weight ratio ( LW / BW ) of WT and Hace1 − / − mice 4 days after sTAC or sham surgery . n = 15 - 21 for each group .

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(g) Heart weight/body weight ratios (HW/BW) and (h) lung weight/body weight ratio (LW/BW) of WT and Hace1−/− mice 4 days after sTAC or sham surgery. n=15-21 for each group.


( i ) Representative haematoxylin and eosin staining transverse heart cross - sections showing myocyte cross - sectional area ( scale bars , 20 μm ) .

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(i) Representative haematoxylin and eosin staining transverse heart cross-sections showing myocyte cross-sectional area (scale bars, 20 μm).


( j ) Real - time RT - PCR analyses reveal a significant increase in the expression of the atrial natriuretic peptide gene ( Nppa ) , a clinical biomarker for heart hypertrophy , in Hace1 − / − sTAC heart ; expression levels were normalized to Hprt and Gapdh mRNAs . n = 6 - 9 per group . In all panels , error bars represent s . e . m . , one - way analysis of variance were used to calculate P - values .

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(j) Real-time RT-PCR analyses reveal a significant increase in the expression of the atrial natriuretic peptide gene (Nppa), a clinical biomarker for heart hypertrophy, in Hace1−/− sTAC heart; expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group. In all panels, error bars represent s.e.m., one-way analysis of variance were used to calculate P-values.


Representative immunohistochemistry staining for ( a ) ubiquitinated protein ( brown ) ; ( b ) p62 ( brown ) and ( c ) GFP - LC3 ( brown ) in paraffin sections of heart revealing increased accumulation of p62 , ubiquitinated protein and LC3 in Hace1 − / − sTAChearts . Hearts from three mice in each group were analysed ( scale bars , 10 μm ) .

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Representative immunohistochemistry staining for (a) ubiquitinated protein (brown); (b) p62 (brown) and (c) GFP-LC3 (brown) in paraffin sections of heart revealing increased accumulation of p62, ubiquitinated protein and LC3 in Hace1−/− sTAChearts. Hearts from three mice in each group were analysed (scale bars, 10 μm).


Representative immunoblot and quantification for ( d , e ) Ub - conjugated proteins , ( d , f ) p62 and ( g , h ) LC3 in heart extracts ( n = 3 per group ) , Gapdh were used as a loading control .

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Representative immunoblot and quantification for (d,e) Ub-conjugated proteins, (d,f) p62 and (g,h) LC3 in heart extracts (n=3 per group), Gapdh were used as a loading control.


( i ) Increased Ub proteasome activity in Hace1 − / − sTAC hearts as measured by 20S proteasome activity assay ( n = 4 - 6 per group ) . In all panels , error bars represent s . e . m . , P value between KO and WT sTAC as indicated ( one - way analysis of variance ) .

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(i) Increased Ub proteasome activity in Hace1−/− sTAC hearts as measured by 20S proteasome activity assay (n=4-6 per group). In all panels, error bars represent s.e.m., P value between KO and WT sTAC as indicated (one-way analysis of variance).


( a ) Representative confocal microscopy images showing HACE1 ( green ) localizing to the Ub + protein aggregates ( red ) induced by puromycin treatment in HA - HACE1 MEF . Arrows indicate co - localized puncta . Scale bar , 10 μm .

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(a) Representative confocal microscopy images showing HACE1 (green) localizing to the Ub+ protein aggregates (red) induced by puromycin treatment in HA-HACE1 MEF. Arrows indicate co-localized puncta. Scale bar, 10 μm.


( b ) Representative confocal micrographs of Hace1 − / − and Hace1 + / + NCM treated with puromycin for 4 h ( puromycin ) , puromycin followed by 8 h recover ( puromycin + recover ) . Vehicle ( PBS ) - treated cells were used as control . Hace1 deficiency in cardiomyocytes ( Hace1 − / − NCM ) blocks ubiquitinated protein aggregate degradation . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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) Representative confocal micrographs of Hace1−/− and Hace1+/+ NCM treated with puromycin for 4 h (puromycin), puromycin followed by 8 h recover (puromycin+recover). Vehicle (PBS)-treated cells were used as control. Hace1 deficiency in cardiomyocytes (Hace1−/− NCM) blocks ubiquitinated protein aggregate degradation. Scale bars, 10 μm.


( c , d ) WT and Hace1 − / − MEF as well as Hace1 − / − MEF stably transfected with WT HACE1 and C876S - mutant HACE1were subjected to protein aggregates clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8 - h chase to monitor for aggregate clearance . Cells were fixed and stained for ubiquitinated proteins to visualize aggregates . Vehicle ( PBS ) - treated MEFs were used as control . ( c ) Representative confocal micrographs of images used for quantification of the efficiency of aggregates clearance in d ( percentage of cells that cleared all aggregates by 8 h after puromycin removal ) . At least 10 images from each group were used for the calculation , error bars represent s . e . m . , P0 . 001 ( one - way analysis of variance ) . Scale bars , 10 μm .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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,d) WT and Hace1−/− MEF as well as Hace1−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1were subjected to protein aggregates clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Cells were fixed and stained for ubiquitinated proteins to visualize aggregates. Vehicle (PBS)-treated MEFs were used as control. (c) Representative confocal micrographs of images used for quantification of the efficiency of aggregates clearance in d (percentage of cells that cleared all aggregates by 8 h after puromycin removal). At least 10 images from each group were used for the calculation, error bars represent s.e.m., P0.001 (one-way analysis of variance). Scale bars, 10 μm.


( a ) Diagrams representing GFP - tagged WT HACE1 and mutant HACE1 ( HACE1Δ HECT and HACE1Δ ANK ) constructs transiently transfected into Hace1 − / − MEF for protein aggregate clearance assay . ( b - d ) Representative confocal micrographs of ( b ) WT , ( c ) ΔHECT and ( d ) ΔANK MEF subjected to protein aggregate clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8 - h chase to monitor for aggregate clearance . Vehicle - treated MEFs were used as control . Scale bars , 10 μm .

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(a) Diagrams representing GFP-tagged WT HACE1 and mutant HACE1 (HACE1Δ HECT and HACE1Δ ANK) constructs transiently transfected into Hace1−/− MEF for protein aggregate clearance assay. (b-d) Representative confocal micrographs of (b) WT, (c) ΔHECT and (d) ΔANK MEF subjected to protein aggregate clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Vehicle-treated MEFs were used as control. Scale bars, 10 μm.


( a ) Fluorescence microscopy images showing HACE1 ( green ) co - aggregating with p62 ( red ) in the perinuclear space on puromycin treatment ( 8 h ) in Hace1 − / − NCM transiently expressing HACE1 - GFP . p62 was visualized by immunofluorescent staining . Arrows indicate co - localized puncta .

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(a) Fluorescence microscopy images showing HACE1 (green) co-aggregating with p62 (red) in the perinuclear space on puromycin treatment (8 h) in Hace1−/− NCM transiently expressing HACE1-GFP. p62 was visualized by immunofluorescent staining. Arrows indicate co-localized puncta.


( b ) Representative confocal images showing HACE1 ( red ) localizes to LC3 + puncta ( green ) induced by MG132 treatment ( 8 h ) in Hace1 − / − GFP - Lc3Tg NCM cells transiently expressing HACE1 - RFP . Arrows indicate co - localized puncta .

(b) Representative confocal images showing HACE1 (red) localizes to LC3+ puncta (green) induced by MG132 treatment (8 h) in Hace1−/−GFP-Lc3Tg NCM cells transiently expressing HACE1-RFP. Arrows indicate co-localized puncta.


( c ) Primary NCMs from Hace1 + / + Lc3Tg and Hace1 − / − Lc3Tg newborn pups were treated with or without MG132 for 8 h and the cell lysates were subjected to western blot with anti - GFP antibodies . Gapdh was used as a loading control .

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(c) Primary NCMs from Hace1+/+Lc3Tg and Hace1−/−Lc3Tg newborn pups were treated with or without MG132 for 8 h and the cell lysates were subjected to western blot with anti-GFP antibodies. Gapdh was used as a loading control.


( d ) Fluorescence microscopy images showing co - localization of HACE1 ( green ) with Lamp1 ( red ) on MG132 treatment ( 8 h ) in Hace1 − / − NCM cells transiently expressing HACE1 - GFP . Lamp1 was visualized by immunofluorescent staining . Scale bar , 10 μm in all images .

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(d) Fluorescence microscopy images showing co-localization of HACE1 (green) with Lamp1 (red) on MG132 treatment (8 h) in Hace1−/−NCM cells transiently expressing HACE1-GFP. Lamp1 was visualized by immunofluorescent staining. Scale bar, 10 μm in all images.


( a , b ) Primary NCMs from WT and Hace1 − / − newborn pups stably transfected with WT HACE1 and C876S - mutant HACE1 , were transiently transfected with a tandem tagged mRFP - GFP - LC3 reporter ( tfLC3 ) plasmid , followed by 8 h treatment with MG132 or MG132 plus Baf in the last 4 h , and were subjected to confocal microscopy . DMSO treatment only was used in the control group . ( a , c ) Representative confocal micrographs of images used for quantification of the percentage of yellow dots in b and d . At least 10 images from each group were used for the calculation ; error bars represent s . e . m . , P - value as indicated ( one - way ANAVO ) . Scale bars , 10 μm . Autophagosomes show both GFP and mRFP - LC3 signals ( yellow ) while autolysosomes exhibit mRFP - L3 signals only ( red ) .

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(a,b) Primary NCMs from WT and Hace1−/− newborn pups stably transfected with WT HACE1 and C876S-mutant HACE1, were transiently transfected with a tandem tagged mRFP-GFP-LC3 reporter (tfLC3) plasmid, followed by 8 h treatment with MG132 or MG132 plus Baf in the last 4 h, and were subjected to confocal microscopy. DMSO treatment only was used in the control group. (a,c) Representative confocal micrographs of images used for quantification of the percentage of yellow dots in b and d. At least 10 images from each group were used for the calculation; error bars represent s.e.m., P-value as indicated (one-way ANAVO). Scale bars, 10 μm. Autophagosomes show both GFP and mRFP-LC3 signals (yellow) while autolysosomes exhibit mRFP-L3 signals only (red).


( c , d ) WT and Hace1 − / − MEF , as well as Hace1 − / − MEF stably transfected with WT HACE1 and C876S - mutant HACE1 , were transiently transfected with a tandem tagged mRFP - GFP - LC3 reporter ( tfLC3 ) plasmid , followed by 8 h treatment with MG132 or MG132 plus Baf in the last 4 h , and were subjected to confocal microscopy . DMSO treatment only was used in the control group . ( a , c ) Representative confocal micrographs of images used for quantification of the percentage of yellow dots in b and d . At least 10 images from each group were used for the calculation ; error bars represent s . e . m . , P - value as indicated ( one - way ANAVO ) . Scale bars , 10 μm . Autophagosomes show both GFP and mRFP - LC3 signals ( yellow ) while autolysosomes exhibit mRFP - L3 signals only ( red ) .

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(c,d) WT and Hace1−/− MEF, as well as Hace1−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1, were transiently transfected with a tandem tagged mRFP-GFP-LC3 reporter (tfLC3) plasmid, followed by 8 h treatment with MG132 or MG132 plus Baf in the last 4 h, and were subjected to confocal microscopy. DMSO treatment only was used in the control group. (a,c) Representative confocal micrographs of images used for quantification of the percentage of yellow dots in b and d. At least 10 images from each group were used for the calculation; error bars represent s.e.m., P-value as indicated (one-way ANAVO). Scale bars, 10 μm. Autophagosomes show both GFP and mRFP-LC3 signals (yellow) while autolysosomes exhibit mRFP-L3 signals only (red).


( a , b ) Hace1 − / − MEF cells were stably transfected with HA - tagged WT HACE1 ( HA - HACE1 ) and empty vector MSCV . ( a ) Immunoblot for HA shows stable expression of HA - tagged HACE1 in HA - HACE1MEF cell lysate . ( b ) HA - HACE1MEF and control MSCVMEF cell lysates were subjected to Co - IP with anti - HA antibody and coomassie blue staining shows a couple of distinct bands only detected in HA - HACE1MEFIP pull - down as indicated by red arrows .

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(a,b) Hace1−/− MEF cells were stably transfected with HA-tagged WT HACE1 (HA-HACE1) and empty vector MSCV. (a) Immunoblot for HA shows stable expression of HA-tagged HACE1 in HA-HACE1MEF cell lysate. (b) HA-HACE1MEF and control MSCVMEF cell lysates were subjected to Co-IP with anti-HA antibody and coomassie blue staining shows a couple of distinct bands only detected in HA-HACE1MEFIP pull-down as indicated by red arrows.


( c - f ) Identification of HACE1 interacting proteins by MS analysis of Co - IP products . ( c ) Hierarchical clustering ( heat ) map demonstrating the clustering of proteins based on their expression intensities in HA - HACE1MEF and control MSCVMEFHA - IP pull - down products . To the left of the heat map , selected annotations found to be enriched in the protein clusters are shown . n = 5 for HA - HACE1 group and n = 4 for control MSCV group . ( d ) Pie chart shows enriched representative HACE1 - interacting proteins according to molecular function by GO term . ( e ) Selected HACE1 - interacting proteins identified by HA - IP MS were evaluated by western blot with the indicated antibodies ( IB ) . HA - HACE1 denotes HA - IP pull - down from HA - HACE1MEF ; MSCV denotes HA - IP pull - down from control MSCVMEF ; and input indicates HA - HACE1MEF lysate before IP . ( f ) HA - HACE1MEF and control MSCVMEF lysates were subjected to co - IP with an anti - Unc - 45b antibody and the pull - down were analysed by western blot with an anti - HA antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f) Identification of HACE1 interacting proteins by MS analysis of Co-IP products. (c) Hierarchical clustering (heat) map demonstrating the clustering of proteins based on their expression intensities in HA-HACE1MEF and control MSCVMEFHA-IP pull-down products. To the left of the heat map, selected annotations found to be enriched in the protein clusters are shown. n=5 for HA-HACE1 group and n=4 for control MSCV group. (d) Pie chart shows enriched representative HACE1-interacting proteins according to molecular function by GO term. (e) Selected HACE1-interacting proteins identified by HA-IP MS were evaluated by western blot with the indicated antibodies (IB). HA-HACE1 denotes HA-IP pull-down from HA-HACE1MEF; MSCV denotes HA-IP pull-down from control MSCVMEF; and input indicates HA-HACE1MEF lysate before IP. (f) HA-HACE1MEF and control MSCVMEF lysates were subjected to co-IP with an anti-Unc-45b antibody and the pull-down were analysed by western blot with an anti-HA antibody.


( g , h ) HA - IP pull - down products from HA - HACE1MEF ( HA - HACE1 ) and Hace1 KO MSCVMEF ( MSCV ) immortalized in HA beads were further subjected to Co - Co - IP with WT mouseheart lysates , and the pull - down products were analysed by western blot using the indicated antibodies . HACE1 and those proteins with which it interacts were still detectable ( g ) . There were also increased ubiquitinated proteins in the Co - Co - IPheart tissues ( h ) .

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(g,h) HA-IP pull-down products from HA-HACE1MEF (HA-HACE1) and Hace1 KO MSCVMEF (MSCV) immortalized in HA beads were further subjected to Co-Co-IP with WT mouseheart lysates, and the pull-down products were analysed by western blot using the indicated antibodies. HACE1 and those proteins with which it interacts were still detectable (g). There were also increased ubiquitinated proteins in the Co-Co-IPheart tissues (h).


( a - c ) Representative western blots and ( d ) quantification show increased accumulation of some of the HACE1 - interacting proteins in the Hace1 − / − sTAC myocardium . n = 3 mice per group , error bars represent s . e . m . , P - value between KO and WT sTAC as indicated ( one - way analysis of variance ) . Gapdh was used as a loading control .

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(a-c) Representative western blots and (d) quantification show increased accumulation of some of the HACE1-interacting proteins in the Hace1−/− sTAC myocardium. n=3 mice per group, error bars represent s.e.m., P-value between KO and WT sTAC as indicated (one-way analysis of variance). Gapdh was used as a loading control.


g - h ) Representative β - catenin ChIP - qPCR of indicated target genes in wt and TCF1 / LEF1 long isoform double KO ( DLKO ) clones ( g ) and Kaiso KO clones ( h ) in RPMI8402 cells . Enrichment of each ChIP is normalized to its respective negative IgG control , indicated by a red - dashed line . DLKO1 , DLKO2 , KKO1 and KKO2 designate independent single - cell derived clones . Graph represents the mean and sd of 3 technical replicates .

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g-h) Representative β-catenin ChIP-qPCR of indicated target genes in wt and TCF1/LEF1 long isoform double KO (DLKO) clones (g) and Kaiso KO clones (h) in RPMI8402 cells. Enrichment of each ChIP is normalized to its respective negative IgG control, indicated by a red-dashed line. DLKO1, DLKO2, KKO1 and KKO2 designate independent single-cell derived clones. Graph represents the mean and sd of 3 technical replicates.


e ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell line ( right panel ) after knockdown of β - catenin ( left panel ) compared to control . Graph represents the mean and sd of 6 independent experiments . f ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell line after treatment with the β - catenin inhibitor ICG - 001 ( 10µM , overnight ) . Graph represents the mean and sd of 3 independent experiments . g ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell lines ( right panel ) after knockdown of ZBTB33 / Kaiso ( left panel ) compared to control . Graph represents the mean and sd of 3 independent experiments . Data information : Statistical significance was determined by two - sided Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line (right panel) after knockdown of β-catenin (left panel) compared to control. Graph represents the mean and sd of 6 independent experiments. f) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line after treatment with the β-catenin inhibitor ICG-001 (10µM, overnight). Graph represents the mean and sd of 3 independent experiments. g) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell lines (right panel) after knockdown of ZBTB33/Kaiso (left panel) compared to control. Graph represents the mean and sd of 3 independent experiments. Data information: Statistical significance was determined by two-sided Student's t test.


c ) Kaplan - Meier curves representing disease free ( DSF , top panel ) and overall survival ( OS , bottom panel ) probability for groups of patients P1 ( N = 7 ) , P2 ( N = 5 ) , P3 ( N = 4 ) , P4 ( N = 9 ) and P5 ( N = 15 ) obtained in a ) . Time is represented in years . Statistical significance among groups was determined by log - rank test .

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c) Kaplan-Meier curves representing disease free (DSF, top panel) and overall survival (OS, bottom panel) probability for groups of patients P1 (N = 7), P2 (N = 5), P3 (N = 4), P4 (N = 9) and P5 (N = 15) obtained in a). Time is represented in years. Statistical significance among groups was determined by log-rank test.


a ) Effect of Vincristine ( VCR ) treatment on cell viability of RPMI8402 cells transduced with sh β - catenin or sh - control . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * p ≤ 0 . 001 of IC50 comparison with respect to sh control Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for a ( at day 0 )

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of Vincristine (VCR) treatment on cell viability of RPMI8402 cells transduced with sh β-catenin or sh-control. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. *** p ≤ 0.001 of IC50 comparison with respect to sh control Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for a (at day 0)


Effect of VCR ( b ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 comparison with respect VCR - treated cells . Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for , b , ( at day 0 )

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of VCR (b) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 comparison with respect VCR-treated cells. Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for , b, (at day 0)


Methotrexate ( MTX ) ( c left ) , L - Asparaginase ( L - ASP ) ( c central ) or Cytarabine ( Ara - C ) ( c right ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for c , ( at day 0 )

B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Methotrexate (MTX) (c left), L-Asparaginase (L-ASP) (c central) or Cytarabine (Ara-C) (c right) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for c, (at day 0)


c ) Percentage of RPMI8402 cells PB , BM , spleen and liver at sacrifice . RPMI8402 cells identified as human CD45 + HLA - ABC + CD5 + by flow cytometry and gated on DAPI negative cells . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by two - sided Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

c) Percentage of RPMI8402 cells PB, BM, spleen and liver at sacrifice. RPMI8402 cells identified as human CD45+HLA-ABC+CD5+ by flow cytometry and gated on DAPI negative cells. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


g ) H & E staining of representative images per each mouse treated as indicated . h ) Percentage of liver area covered by cellular colonies per 10X micrograph . 4 different images were quantified per animal and mean values per animal are represented by dots . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by two - sided Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

g) H&E staining of representative images per each mouse treated as indicated. h) Percentage of liver area covered by cellular colonies per 10X micrograph. 4 different images were quantified per animal and mean values per animal are represented by dots. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


A - C 10x106 ZsGreen - and luciferase - expressing U - 2932 , RC - K8 and RIVA cells were intravenously injected into six - week - old male ( M ) and female ( F ) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks . The color scales on the right indicate the radiance , i . e . the sum of the photons per second from each pixel inside the ROI / number of pixels ( photons / sec / cm2 / sr ) .

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A-C 10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. The color scales on the right indicate the radiance, i.e. the sum of the photons per second from each pixel inside the ROI/number of pixels (photons/sec/cm2/sr).


10x106 ZsGreen - and luciferase - expressing U - 2932 , RC - K8 and RIVA cells were intravenously injected into six - week - old male ( M ) and female ( F ) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks . D The frequency of involvement of the indicated tissues is shown for one cohort of mice and is representative of two independently analyzed cohorts per cell line .

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10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. D The frequency of involvement of the indicated tissues is shown for one cohort of mice and is representative of two independently analyzed cohorts per cell line.


E Gating strategy for the identification of U - 2932 cells residing in spleen and bone marrow , among all leukocytes in these organs . Cells were stained for human and mouse CD45 . Very few hCD45 + U - 2932 cells were detectable in the spleen , whereas their frequencies were much higher in the bone marrow ( BM ) . In BM , > 90 % of U - 2932 cells were positive for ZsGreen . The green curve shows U - 2932 cells recovered from BM , overlayed with unlabelled U - 2932 cells grown in vitro ( grey curve ) .

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E Gating strategy for the identification of U-2932 cells residing in spleen and bone marrow, among all leukocytes in these organs. Cells were stained for human and mouse CD45. Very few hCD45+ U-2932 cells were detectable in the spleen, whereas their frequencies were much higher in the bone marrow (BM). In BM, >90% of U-2932 cells were positive for ZsGreen. The green curve shows U-2932 cells recovered from BM, overlayed with unlabelled U-2932 cells grown in vitro (grey curve).


F , G Frequencies of human lymphoma cells , as judged by hCD45 expression , among all ( human and mouse , h + m ) CD45 + leukocytes in the spleen ( S ) , femoral bone marrow ( BM ) and jaw bone marrow ( JB ) at the study endpoint of the mice shown in D ( four weeks post injection for U - 2932 , five weeks p . i . for RIVA and six weeks p . i . for RC - K8 ) .

F, G Frequencies of human lymphoma cells, as judged by hCD45 expression, among all (human and mouse, h+m) CD45+ leukocytes in the spleen (S), femoral bone marrow (BM) and jaw bone marrow (JB) at the study endpoint of the mice shown in D (four weeks post injection for U-2932, five weeks p.i. for RIVA and six weeks p.i. for RC-K8).


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . The reconstitution efficiency ( frequency of human among all human and mouse CD45 + leukocytes ) is shown in

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution efficiency (frequency of human among all human and mouse CD45+ leukocytes) is shown in A


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . the average human leukocyte composition in the bone marrow of the mice is shown in B .

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. the average human leukocyte composition in the bone marrow of the mice is shown in B.


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . Tumor volumes over time are presented in

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. Tumor volumes over time are presented in C


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . tumor weights and volumes at the study endpoint in D , where each data point represents one tumor .

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. tumor weights and volumes at the study endpoint in D, where each data point represents one tumor.


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . The reconstitution level directly correlates with the tumor weight at the endpoint ( E ) .

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution level directly correlates with the tumor weight at the endpoint (E).


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , and intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age . The lymphoma burden at the study endpoint was quantified in the spleen ( F ) and bone marrow ( G ) by staining for hCD45 ( as well as hCD19 and hCD20 for U - 2932 and RC - K8 , respectively )

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the spleen (F) and bone marrow (G) by staining for hCD45 (as well as hCD19 and hCD20 for U-2932 and RC-K8, respectively)


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , and intravenously injected with RC - K8 cells at six weeks of age . The lymphoma burden at the study endpoint was quantified by ZsGreen expression and in the case of RC - K8 cells , also over time by IVIS ( H ) .

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified by ZsGreen expression and in the case of RC-K8 cells, also over time by IVIS (H).


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , and intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age . The frequency of tissue involvement was assessed by ex vivo IVIS and macroscopic evaluation ( I ) ,

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The frequency of tissue involvement was assessed by ex vivo IVIS and macroscopic evaluation (I),


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , and intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age . the occurrence of clinical symptoms ( weight loss , hind leg paralysis ) was recorded for each mouse ( J ) .

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. the occurrence of clinical symptoms (weight loss, hind leg paralysis) was recorded for each mouse (J).


MISTRG and MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint ( A ) .

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MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A).


MISTRG and MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint ( A ) . The lymphoma burden at the study endpoint ( six weeks p . i . for RC - K8 and four weeks p . i . for U - 2932 ) was quantified in the spleen ( B ) and bone marrow ( C ) by flow cytometric staining for hCD45 and by ZsGreen expression .

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MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A). The lymphoma burden at the study endpoint (six weeks p.i. for RC-K8 and four weeks p.i. for U-2932) was quantified in the spleen (B) and bone marrow (C) by flow cytometric staining for hCD45 and by ZsGreen expression.


MISTRG and MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint The involvement of the indicated tissues as assessed by ex vivo IVIS is shown in D ( solid bars , MISTRG ; hatched bars , MISTRG6 ) ,

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MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint The involvement of the indicated tissues as assessed by ex vivo IVIS is shown in D (solid bars, MISTRG; hatched bars, MISTRG6),


MISTRG and MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint mice that have developed clinical symptoms ( paralysis of hind legs ) are plotted in E .

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MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint mice that have developed clinical symptoms (paralysis of hind legs) are plotted in E.


F Surface expression of the IL - 6 receptor α - chain ( CD126 ) as assessed by flow cytometry of U - 2932 and RC - K8 cells . Data is representative of two stainings .

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F Surface expression of the IL-6 receptor α-chain (CD126) as assessed by flow cytometry of U-2932 and RC-K8 cells. Data is representative of two stainings.


1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . The spleen weights ( B , representative pictures in A ) are

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1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. The spleen weights (B, representative pictures in A) are shown


1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . with representative H & E - stained spleen ( MISTRG6 ) and lymph node ( patient ) sections in

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1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. with representative H&E-stained spleen (MISTRG6) and lymph node (patient) sections in C


1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . the lymphoma burden at the study endpoint eight weeks p . i . , as quantified in the spleen and bone marrow in D Representative flow cytometry plots are shown in D for bone marrow and spleen ( MISTRG , MISTRG6 ) .

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1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrow in D Representative flow cytometry plots are shown in D for bone marrow and spleen (MISTRG, MISTRG6).


x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . the lymphoma burden at the study endpoint eight weeks p . i . , as quantified in the spleen and bone

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x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrow


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPC , and intravenously injected with 1x106 primary DLBCL cells at six weeks of age . The lymphoma burden at the study endpoint was quantified in the bone marrow of reconstituted and un - reconstituted animals by flow cytometric staining for the indicated surface markers .

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPC, and intravenously injected with 1x106 primary DLBCL cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the bone marrow of reconstituted and un-reconstituted animals by flow cytometric staining for the indicated surface markers.


Primary lymphoma cells harvested at the study endpoint express the IL - 6R α chain ( CD126 , as assessed by flow cytometry , G ) and are positive for phosphorylated STAT3 ( G ) .

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Primary lymphoma cells harvested at the study endpoint express the IL-6R α chain (CD126, as assessed by flow cytometry, G) and are positive for phosphorylated STAT3 (G).


Primary lymphoma cells harvested and the signaling chain gp130 ( as assessed by immunohistochemistry , H )

Primary lymphoma cells harvested and the signaling chain gp130 (as assessed by immunohistochemistry, H)


The expression of the IL - 6Rα ( CD126 ) and signaling chains ( gp130 ) was assessed by flow cytometry ( A ) on a panel of DLBCL cell lines . ABC - and GCB - DLBCL cell lines are color - coded in red and green ; RC - K8 are depicted in grey , as their subtype is a matter of

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The expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by flow cytometry (A) on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debate


The expression of the IL - 6Rα ( CD126 ) and signaling chains ( gp130 ) was assessed by SYBR or Taqman qRT - PCR ( B , C ) respectively , on a panel of DLBCL cell lines . ABC - and GCB - DLBCL cell lines are color - coded in red and green ; RC - K8 are depicted in grey , as their subtype is a matter of

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The expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by SYBR or Taqman qRT-PCR (B, C) respectively, on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debate


D and E . The expression of gp130 in 114 tumor biopsies of DLBCL patients was assessed by immunohistochemistry , and correlated with the ABC / non - GCB vs . GCB subtype , as assessed via Hans classification . Representative micrographs are shown in D , alongside the quantification of gp130 + cases among all samples , and among the two subtypes , in E . Scale bar , 100 μm .

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D and E. The expression of gp130 in 114 tumor biopsies of DLBCL patients was assessed by immunohistochemistry, and correlated with the ABC/non-GCB vs. GCB subtype, as assessed via Hans classification. Representative micrographs are shown in D, alongside the quantification of gp130+ cases among all samples, and among the two subtypes, in E. Scale bar, 100 μm.


A IL - 6 secretion of the indicated cell lines ( ABC - DLBCL in red , GCB - DLBCL in green ) and primary patient material , as quantified by ELISA and normalized to cell number ( shown per 1000 cells ) . n . d . , not detectable .

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A IL-6 secretion of the indicated cell lines (ABC-DLBCL in red, GCB-DLBCL in green) and primary patient material, as quantified by ELISA and normalized to cell number (shown per 1000 cells). n.d., not detectable.


STAT3 phosphorylation as assessed by Western blotting ( B , D ) of the indicated ABC - DLBCL and GCB - DLBCL cell lines , with and without 1h of exposure to 50ng / ml hIL - 6 ( B , D ) . STAT3 expression levels are shown as Western blot control .

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STAT3 phosphorylation as assessed by Western blotting (B, D) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, with and without 1h of exposure to 50ng/ml hIL-6 (B,D). STAT3 expression levels are shown as Western blot control.


STAT3 phosphorylation as assessed by flow cytometry ( C ) of the indicated ABC - DLBCL and GCB - DLBCL cell lines , Histograms in C represent STAT3 phosphorylation in the absence of exogenously added IL - 6 .

STAT3 phosphorylation as assessed by flow cytometry (C) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, Histograms in C represent STAT3 phosphorylation in the absence of exogenously added IL-6.


E STAT3 phosphorylation as assessed by Western blotting of CD126 - proficient ( WT ) or - deficient ( ko ) RC - K8 cells , with and without 1h of exposure to 50ng / ml hIL - 6 .

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E STAT3 phosphorylation as assessed by Western blotting of CD126-proficient (WT) or -deficient (ko) RC-K8 cells, with and without 1h of exposure to 50ng/ml hIL-6.


F Kinetics of STAT3 phosphorylation as assessed by Western blotting , of the IL - 6 - expressing SOCS1wt and SOCS1mut cell lines OCI - Ly3 and SU - DHL - 2 , under 1 , 6 and 24h exposure to an IL - 6 - neutralizing antibody ( α - IL - 6 , anti - IL - 6 neutralizing antibody ) .

F Kinetics of STAT3 phosphorylation as assessed by Western blotting, of the IL-6-expressing SOCS1wt and SOCS1mut cell lines OCI-Ly3 and SU-DHL-2, under 1, 6 and 24h exposure to an IL-6-neutralizing antibody (α-IL-6, anti-IL-6 neutralizing antibody).


STAT3 phosphorylation and gp130 expression of two representative cases ( G ) assessed by immunohistochemistry . The specific pSTAT3 and gp130 signal is seen as brown ( DAB staining ) , and counterstaining was performed using hematoxylin ( blue nuclei ) . STAT3 phosphorylation is more common in biopsies with gp130 expression ; the p - value was calculated by chi - square test . Scale bar , 100 μm .

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STAT3 phosphorylation and gp130 expression of two representative cases (G) assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression; the p-value was calculated by chi-square test. Scale bar, 100 μm.


STAT3 phosphorylation and gp130 expression of two representative cases ( G ) and summarized for the entire cohort of 114 patients ( H ) , as assessed by immunohistochemistry . The specific pSTAT3 and gp130 signal is seen as brown ( DAB staining ) , and counterstaining was performed using hematoxylin ( blue nuclei ) . STAT3 phosphorylation is more common in biopsies with gp130

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STAT3 phosphorylation and gp130 expression of two representative cases (G) and summarized for the entire cohort of 114 patients (H), as assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression


Disease - specific mortality ( A ) of cohorts of 9 - 12 mice each ( n indicated in figure ) of the indicated genotypes .

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Disease-specific mortality (A) of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.


lymphoma burden as determined by lymph node weight ( B ) , of cohorts of 9 - 12 mice each ( n indicated in figure ) of the indicated genotypes .

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lymphoma burden as determined by lymph node weight (B), of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.


Mice of the indicated genotypes were immunized once with sheep red blood cells and assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B - cells ( C ) , centroblasts ( D ) and centrocytes ( E ) .

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Mice of the indicated genotypes were immunized once with sheep red blood cells and assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B-cells (C), centroblasts (D) and centrocytes (E).


Spleen sections were further stained for Ki67 to reveal GCs ( F ) , which were quantified both in number ( G ) and area ( H ) . Arrows in F point to GCs ; scale bar , 1000 μm .

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Spleen sections were further stained for Ki67 to reveal GCs (F), which were quantified both in number (G) and area (H). Arrows in F point to GCs; scale bar, 1000 μm.


Frequency of STAT3 phosphorylation ( A ) , of DLBCL patients stratified according to subtype and disease stage Phospho - STAT3 positivity was defined as expression in ≥ 17 % of tumor cells and more common found in ABC - DLBCL tumors , and in more advanced ( stage 3 and 4 , relative to stage 1 and 2 ) disease ( A ) .

Frequency of STAT3 phosphorylation (A), of DLBCL patients stratified according to subtype and disease stage Phospho-STAT3 positivity was defined as expression in ≥17% of tumor cells and more common found in ABC-DLBCL tumors, and in more advanced (stage 3 and 4, relative to stage 1 and 2) disease (A).


overall as well as disease - specific survival probability ( B ) of DLBCL patients pSTAT3 positivity was a strong negative prognostic factor in the entire cohort ( B )

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overall as well as disease-specific survival probability (B) of DLBCL patients pSTAT3 positivity was a strong negative prognostic factor in the entire cohort (B)


overall mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall - as well as disease - specific mortality in both ABC - and GCB - DLBCL

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overall mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypes


disease - specific mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall - as well as disease - specific mortality in both ABC - and GCB - DLBCL

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disease-specific mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypes


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two weeks post tumor cell injection , on ( E and F ) the spleen weights of MISTRG6 mice transplanted with 1x106 primary DLBCL cells ( expanded by one serial passage through MISTRG6 mice ) ,

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, on (E and F) the spleen weights of MISTRG6 mice transplanted with 1x106 primary DLBCL cells (expanded by one serial passage through MISTRG6 mice),