Relative to TaSDN1 - DD , TaSDN1 was decreased in TaSDN1 - Dd TaSDN1 - dd mutants , with a stronger reduction found for TaSDN1 - dd . reactive TaSDN1 detected in TaSDN1 - dd was caused by un - mutated TaSDN1 - A - B homoeologs .

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

Relative to TaSDN1-DD line, TaSDN1 protein was decreased in TaSDN1-Dd and TaSDN1-dd mutants, with a stronger reduction found for TaSDN1-dd. The reactive TaSDN1 protein detected in TaSDN1-dd was caused by the un-mutated TaSDN1-A and -B homoeologs.


( M ) of BYDV CP gene in TaSDN1 - DD , TaSDN1 - Dd TaSDN1 - dd at 21 dpi .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

protein (M) levels of BYDV CP gene in TaSDN1-DD, TaSDN1-Dd and TaSDN1-dd plants at 21 dpi.


BYDV vsiRNA abundance in TaSDN1 - DD , TaSDN1 - Dd , TaSDN1 - dd detected by sRNA blotting at 21 dpi of BYDV .

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

BYDV vsiRNA abundance in TaSDN1-DD, TaSDN1-Dd, and TaSDN1-dd plants detected by sRNA blotting at 21 dpi of BYDV.


graphs depict recovery of CAR1 - mcherry ( left ) tPH - CynA - KikGR ( right ) . Mean ± is shown for n = 2 .

O O O O B-MEASURED_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

The graphs depict the recovery of CAR1-mcherry (left) and tPH-CynA-KikGR (right). Mean ± SEM is shown for n = 2 cells.


Time - lapse confocal of tPH - CynA - KikGR expressing AX3 showing docking events at back of a migrating Dictyostelium . Front back of are shown .

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

Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell. Front and back of the cell are shown.


Time - lapse confocal of AX3 co - expressing PH - Crac - GFP ( green ) another established PI ( 3 , 4 ) P2 biosensor tPH - TAPP1 - RFP ( red ) . Box shows vesicle .

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 B-MEASURED_VAR O O O O O O O O O O

Time-lapse confocal images of AX3 cell co-expressing PH-Crac-GFP (green) and another established PI(3,4)P2 biosensor tPH-TAPP1-RFP (red). Box shows the vesicle.


Time - lapse confocal of tPH - CynA - KikGR ( green ) expressing wild - type AX3 . FM4 - 64 ( red ) was added at time 0 during live imaging . Front back of are shown . Box shows vesicle .

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

Time-lapse confocal images of tPH-CynA-KikGR (green) expressing wild-type AX3 cells. FM4-64 (red) was added at time 0 during live cell imaging. Front and back of the cell are shown. Box shows the vesicle.


Time - lapse confocal of wild - type AX3 expressing tPH - CynA - KikGR ( green ) PH - crac ( red ) before after PI3K inhibition by 5 µM Wortmannin .

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

Time-lapse confocal images of wild-type AX3 cells expressing tPH-CynA-KikGR (green) and PH-crac (red) before and after PI3K inhibition by 5 µM Wortmannin.


Confocal of tPH - CynA - KikGR ( green ) PH - Crac ( red ) expressing wild - type Dictyostelium AX3 ( left ) PI3K1 - 2 - Dictyostelium ( right ) treated with 5 µM Latrunculin A .

O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR 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

Confocal images of tPH-CynA-KikGR (green) and PH-Crac (red) expressing wild-type Dictyostelium AX3 cells (left) and PI3K1-2- Dictyostelium cells (right) treated with 5 µM Latrunculin A.


Time - lapse confocal showing dynamic localization of PI ( 3 , 4 ) P2 . Nocodazole was added at time 0 during live imaging . of vesicle number , vesicle number / min , vesicle lifetime ( s ) from experiment in E . T - was carried out by GraphPad Prism , * * P ≤ 0 . 01 versus After Nocodazole group ; mean ± ( n = 10 ) .

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

Time-lapse confocal images showing dynamic localization of PI(3,4)P2. Nocodazole was added at time 0 during the live cell imaging.   Quantification of vesicle number, vesicle number/min, and vesicle lifetime (s) from experiment in E. T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus After Nocodazole group; mean±SEM (n = 10).


( L ) Representative confocal of tPH - CynA - KikGR in wild - type Dictyostelium AX2 or RacH − treated with 5 μM latrunculin A Scale bar : 5 μm . ( M ) of to cytosol of tPH - CynA - KikGR in wild - type Dictyostelium AX2 RacH − . T - was carried out by GraphPad Prism , * * * * P ≤ 0 . 0001 versus AX2 group ; mean ± ( n = 33 ) .

O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_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 O O O O O O O O O O O O O O O O O O O

(L) Representative confocal images of tPH-CynA-KikGR in wild-type Dictyostelium AX2 or RacH− cells treated with 5 μM latrunculin A Scale bar: 5 μm. (M) Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wild-type Dictyostelium AX2 and RacH− cells. T-test was carried out by GraphPad Prism, ****P ≤ 0.0001 versus AX2 group; mean ± SEM (n = 33).


Time - lapse confocal of tPH - CynA - KikGR expressing Dictyostelium AX3 . macropinosome at front of was photoconverted at time 0 . arrow points to PI ( 3 , 4 ) P2 localization at back of . front back are shown , box showing photoconverted region .

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

Time-lapse confocal images of tPH-CynA-KikGR expressing Dictyostelium AX3 cells. The macropinosome at the front of the cell was photoconverted at time 0. White arrow points to PI(3,4)P2 localization at the back of the membrane. Cell front and back are shown, box showing the photoconverted region.


B , C MCF - 7 expressing GFP - INPP4B or GFP - vector were cultured in growth media or EBSS for indicated times , then lysed immunblotted with LC3B antibodies GAPDH antibodies as a loading control ( B ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to growth media - treated GFP - vector which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( C ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc in C ,

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 B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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

B, C MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (B). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (C). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in C,


D , E MCF - 7 expressing NT , INPP4B # 1 or INPP4B # 2 shRNA were cultured in growth media or EBSS for indicated times , then lysed immunblotted with LC3B antibodies GAPDH antibodies as a loading control ( D ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to growth media - treated NT shRNA which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( E ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in E .

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 B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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

D, E MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (D). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (E). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in E.


F , G MCF - 7 expressing GFP - INPP4B or GFP - vector were cultured in growth media or EBSS for 4 hours , then fixed immunostained with p62 antibodies , co - stained with DAPI phalloidin ( F ) . Data represent number of p62 + relative to ( µm2 ) ( n = 3 experiments , > 50 / experiment ) ( G ) . Data information : Data is presented as mean ± SD . insets at lower right of each image are higher power regions of boxed areas . Scale bar is 10 µm in F p values determined by one - way ANOVA with Tukey post hoc in G ,

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 B-MEASURED_VAR O O O 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

F, G MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (F). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (G). 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 F p values determined by one-way ANOVA with Tukey post hoc test in G,


H , I MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours , then lysed immunblotted with LC3B antibodies GAPDH antibodies as a loading control ( H ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to DMSO - treated GFP - vector which were assigned an arbitrary value of 1 ( I ) ( n = 3 experiments ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc in , I ,

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 B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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

H, I MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours, then lysed and immunblotted 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 DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (I) (n=3 experiments). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in , I,


J , K MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours , then fixed immunostained with p62 antibodies , co - stained with DAPI phalloidin ( J ) . Data represent number of p62 + relative to ( µm2 ) ( n = 3 experiments , > 50 / experiment ) ( K ) . Data information : Data is presented as mean ± SD . insets at lower right of each image are higher power regions of boxed areas . Scale bar is 10 µm in J . p values determined by one - way ANOVA with Tukey post hoc in K ,

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 B-MEASURED_VAR O O O 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

J, K MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (J). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (K). 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 J. p values determined by one-way ANOVA with Tukey post hoc test in K,


L , M MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 2 µM BYL719 or DMSO as a vehicle control for indicated times . were lysed immunblotted with LC3B , pAKTS473 , AKT ( pan ) , pS6KT389 or S6K antibodies GAPDH antibodies as a loading control ( L ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to DMSO - treated GFP - vector 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 in M ,

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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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

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 expressing GFP - INPP4B or GFP - vector were treated with 1 µM or 10 µM SAR405 or DMSO as a vehicle control for 4 hours . were lysed immunblotted with LC3B antibodies GAPDH antibodies as a loading control ( N ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to DMSO - treated GFP - vector 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 in 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 B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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

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

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 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 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

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 were treated with 0 . 02 % ( w / v ) saponin for 30 seconds , then fixed immunostained with INPP4B either LAMP1 or CD63 antibodies , co - stained with DAPI . Arrows indicate co - localization between INPP4B CD63 . Data information : insets at lower right or bottom of each image are higher power regions of 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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-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

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

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 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 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

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

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 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

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 co - expressing GFP - INPP4B or GFP - vector , Hrs or NT shRNA , were lysed immunoblotted with LC3B antibodies , GAPDH antibodies as a loading control ( H ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to GFP - vector ; NT shRNA 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-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

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

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

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

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-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

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 expressing NT or INPP4B # 1 shRNA were cultured in growth media or EBSS for indicated times , then fixed immunostained with LAMP1 antibodies co - stained with DAPI phalloidin ( E ) . Data represent number of LAMP1 + relative to ( µm2 ) ( n = 3 experiments , > 30 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 in

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 B-MEASURED_VAR O O 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

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 expressing GFP - INPP4B or GFP - vector were transfected with NT , PIK3CA # 1 or PIK3CA # 2 siRNA . After 24 hours , were fixed immunostained with LAMP1 antibodies , co - stained with DAPI phalloidin ( G ) . Data represent number of LAMP1 + relative to ( µm2 ) ( n = 3 experiments , > 40 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 B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O 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

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

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

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

O O O O B-MEASURED_VAR 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

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

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 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

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

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 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 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

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 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 . were fixed immunostained with LAMP1 antibodies , co - stained with DAPI phalloidin ( D ) . Data represent number of LAMP1 + relative to ( µm2 ) ( E ) , lysosome fold change relative to YM201636 - treated condition for each ( F ) ( n = 3 experiments , > 30 per experiment ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in D . p values determined by one - way ANOVA in E , or by two - unpaired t of under curve in F .

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 B-MEASURED_VAR O O O 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 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 expressing GFP - INPP4B or GFP - vector were treated with 1 , 2 or 5 µM YM201636 or DMSO as a vehicle control for 4 hours . were lysed immunoblotted with LC3B antibodies , GAPDH antibodies as a loading control ( G ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to DMSO - treated GFP - vector 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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

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

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

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

O O O O B-MEASURED_VAR 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

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

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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR I-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

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 were transfected with GFP - SNX2 . After 24 hours , were treated with 5 µM YM201636 for 2 hours , then YM201636 was washed out for 1 hour . were fixed subjected to immuno - electron microscopy 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 O O O O O O O O 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-MEASURED_VAR 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 were transfected with GFP - PIKfyve . After 24 hours , were were treated with 5 µM YM201636 for 2 hours , then YM201636 was washed out for 1 hour . were fixed immunostained with SNX2 , GFP LAMP2 antibodies , imaged using super resolution microscopy . Arrows show co - localization between GFP - PIKfyve , SNX2 LAMP2 . Data information : insets at lower right or bottom of each image are higher power regions of 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 B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-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

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 expressing LAMP1 - mCherry were transfected with NT or SNX2 siRNA . After 24 hours , 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 number of LAMP1 + reformation events per per minute ( n = 21 NT siRNA , n = 27 SNX2 siRNA ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in H , p values determined by two - unpaired t in I .

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 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 B-MEASURED_VAR 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

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

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

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 were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours , then 5 µg / mL puromycin for 1 hour . were fixed immunostained with ubiquitin antibodies , co - stained with DAPI phalloidin ( C ) . Data represent of with ubiquitinated ( n = 3 experiments , > 200 / experiment ) ( D ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in C , p values determined by one - way ANOVA in with Tukey post hoc 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 O O 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 expressing GFP - INPP4B or GFP - vector were treated for 4 hours with 10 µg / mL puromycin , 5 µm MG132 either 5 µM YM201636 or DMSO as a vehicle control . were fixed immunostained with ubiquitin antibodies , co - stained with DAPI phalloidin ( E ) . Data represent of with ubiquitinated ( n = 3 experiments , > 200 / experiment ) ( F ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in E . p values determined by one - way ANOVA in with Tukey post hoc

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

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

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

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.


C . HDo - TRaMs were engineered with vectors relevant to treatment of Fabry , Gaucher , Farber Pompe diseases . Intracellular lysosomal enzyme specific activities were measured in transduced T - Rapa controls . Transductions varied across vectors / donors , as indicated . D , E . Enzyme activity was assessed in transduced T - Rapa supernatants compared to controls in activated / dividing ( D ) quiescent / resting ( E ) states .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HDo-TRaMs were engineered with vectors relevant to treatment of Fabry, Gaucher, Farber and Pompe diseases. Intracellular lysosomal enzyme specific activities were measured in transduced T-Rapa and controls. Transductions varied across vectors/donors, as indicated. D, E. Enzyme activity was assessed in transduced T-Rapa supernatants compared to controls in activated/dividing (D) and quiescent/resting (E) states.


F . from FDo were stably transduced , as indicated . Intracellular α - galactosidase A ( α - gal A ) specific activities were determined . G , H . Secreted α - gal A activity was measured from FDo - derived TRaMs in activated ( G ) , quiescent / resting ( H ) states .

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

F. Cells from all FDo were stably transduced, as indicated. Intracellular α-galactosidase A (α-gal A) specific activities were determined. G, H. Secreted α-gal A activity was measured from FDo-derived TRaMs in activated (G), and quiescent/resting (H) states. I


I . α - Gal A specific activity in Fabry - derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in presence or absence of 1mM M6P .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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

I. α-Gal A specific activity in Fabry patient-derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the presence or absence of 1mM M6P.


B . Engraftment was evaluated by flow cytometry for human CD3 expression .

B. Engraftment was evaluated by flow cytometry for human CD3 expression.


C , D . α - Gal A activity in plasma , specific activities in liver , spleen , heart kidneys were measured after transplant of transduced healthy donor ( HDo ) - derived ( C ) or Fabry donor ( FDo ) - derived ( D ) TRaMs , non - transduced ( NT ) , or in sham - treated NOD / SCID ( NS ) NSF mice .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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

C, D. α-Gal A activity in plasma, and specific activities in liver, spleen, heart and kidneys were measured after transplant of transduced healthy donor (HDo)-derived (C) or Fabry donor (FDo)-derived (D) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.


A , B . of globotriaosylceramide ( Gb3 ) were measured by LC / MS in plasma extracts of liver , spleen , heart , kidneys of mice engrafted with healthy donor ( HDo ) - derived ( A ) or Fabry donor ( FDo ) - derived ( B ) TRaMs , non - transduced ( NT ) , or in sham - treated NOD / SCID ( NS ) NSF mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Levels of globotriaosylceramide (Gb3) were measured by LC/MS in plasma and extracts of liver, spleen, heart, and kidneys of mice engrafted with healthy donor (HDo)-derived (A) or Fabry donor (FDo)-derived (B) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.


C , D . of lyso - Gb3 were measured by LC / MS in plasma extracts of tissues of mice engrafted with HDo - derived ( C ) or FDo - derived ( D ) TRaMs .

O O O O O O O O O O O O O O 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. Levels of lyso-Gb3 were measured by LC/MS in plasma and extracts of tissues of mice engrafted with HDo-derived (C) or FDo-derived (D) TRaMs.


A , B . of oryzalin root growth assays in single ( A ) double ( B ) mutants . DAG = days after germination . Graphs show mean ± SD of three biological replicates with at least 10 per genotype per replicate . Asterisks indicate a significant difference in root in a two - way ANOVA followed by Tukey ' s multiple comparisons ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 ) .

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A, B. Quantification of oryzalin root growth assays in single (A) and double (B) mutants. DAG = days after germination. Graphs show mean ± SD of three biological replicates with at least 10 plants per genotype per replicate. Asterisks indicate a significant difference in root length in a two-way ANOVA followed by Tukey's multiple comparisons test (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001).


E . Silique pictures of cycb1 double mutant combinations . arrowheads indicate aborted ovules seeds . Scale bars : 500 μm . F , G . of aborted seeds in single ( F ) double ( G ) mutants . Graphs represents average seed abortion rate per ± SD of three biological replicates , n = 550 - 1029 seeds analyzed per genotype . Asterisks indicate significant differences in seed abortion rate in an ordinary one - way ANOVA , followed by a Dunnett ' s multiple comparisons ( * * * * P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Silique pictures of cycb1 double mutant combinations. White arrowheads indicate aborted ovules and seeds. Scale bars: 500 μm. F, G. Quantification of aborted seeds in single (F) and double (G) mutants. Graphs represents the average seed abortion rate per plant ± SD of three biological replicates, n = 550-1029 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).


C , D . of endosperm nuclei in cycb1 single ( C ) double ( D ) mutants . Boxes whiskers represent min to max values with median indicated as a central horizontal , n = 26 - 30 seeds per genotype . Asterisks show significant differences in number of endosperm nuclei per seed in a Kruskal - Wallis followed by Dunn ' s multiple comparisons ( * * * * P < 0 . 0001 ) .

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C, D. Quantification of endosperm nuclei in cycb1 single (C) and double (D) mutants. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 26-30 seeds per genotype. Asterisks show significant differences in the number of endosperm nuclei per seed in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (**** P < 0.0001).


A . DIC of abnormal sacs in cycb1 mutant combinations . Red arrowheads indicate visible nuclei in Col - 0 sacs ( central egg ) corresponding structures in quadruple cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cyb1 ; 4 - mutant . Scale bars : 20 μm . B . of different abnormal sac structures in cycb1 mutant combinations ( n = 202 - 459 sacs per genotype ) . Different letters indicate significant differences in proportion of abnormal sacs in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons . WT = wildtype .

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

A. DIC images of abnormal embryo sacs in cycb1 mutant combinations. Red arrowheads indicate the visible nuclei in Col-0 embryo sacs (central and egg cells) and the corresponding structures in the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant. Scale bars: 20 μm. B. Quantification of the different abnormal embryo sac structures in cycb1 mutant combinations (n = 202-459 embryo sacs per genotype). Different letters indicate significant differences in the proportion of abnormal embryo sacs in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. WT = wildtype.


C . DIC of sacs 3 DAP with wildtype pollen ( x ) . Red arrowheads indicate visible sac nuclei in crosses with quadruple cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cyb1 ; 4 - mutant as a donor , while control Col - 0 x Col - 0 cross exhibits a developing . Scale bars : 20 μm . D . of seed abortion in different cycb1 mutant combinations . Graph represents average seed abortion rate per ± SD of three biological replicates , n = 463 - 579 seeds analyzed per genotype . Asterisks indicate significant differences in seed abortion rate in an ordinary one - way ANOVA , followed by a Dunnett ' s multiple comparisons ( * * * * P < 0 . 0001 ) .

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

C. DIC images of embryo sacs 3 DAP with wildtype pollen (female x male). Red arrowheads indicate the visible embryo sac nuclei in the crosses with the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant as a female donor, while the control Col-0 x Col-0 cross exhibits a developing embryo. Scale bars: 20 μm. D. Quantification of seed abortion in different cycb1 mutant combinations. Graph represents the average seed abortion rate per plant ± SD of three biological replicates, n = 463-579 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).


A , B . DAPI of pollen in cycb1 mutants , including pollen configurations found in cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cycb1 ; 4 - / - mutants ( B ) . Scale bars : 5 μm . C . of DAPI - stained pollen configurations in different cycb1 mutant combinations , n = 420 - 616 pollen grains per genotype . Different letters indicate significant differences in proportion of abnormal pollen ( uni - bicellular ) in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons .

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

A, B. DAPI staining of pollen in cycb1 mutants, including pollen configurations found in cycb1;1-/- cycb1;2+/- cycb1;3+/- cycb1;4-/- mutants (B). Scale bars: 5 μm. C. Quantification of DAPI-stained pollen configurations in different cycb1 mutant combinations, n = 420-616 pollen grains per genotype. Different letters indicate significant differences in the proportion of abnormal pollen (uni- and bicellular) in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.


D . Alexander of mature pollen indicating pollen viability . Scale bars : 5 μm . E . of Alexander - stained pollen viability , n = 403 - 498 pollen grains per genotype . Different letters indicate significant differences in proportion of dead pollen in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons .

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D. Alexander staining of mature pollen indicating pollen viability. Scale bars: 5 μm. E. Quantification of Alexander-stained pollen viability, n = 403-498 pollen grains per genotype. Different letters indicate significant differences in the proportion of dead pollen in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.


A , B , C . Co - immunolocalization against tubulin ( magenta ) KNOLLE ( green ) in root meristematic . Nuclei were counterstained with DAPI for ( cyan ) . arrowheads indicate laggards in metaphase stage . Scale bars : 5 μm .

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A, B, C. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). White arrowheads indicate laggards in the metaphase stage. Scale bars: 5 μm.


D . of wildtype ( WT ) , double misplaced PPBs . Different letters indicate significant differences in proportions of different arrays per category in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype . E . of PPBs with prominent perinuclear microtubules ( MTs ) . Boxes whiskers represent min to max values with median indicated as a central horizontal , n = 10 roots per genotype . Asterisks show significant differences in of PPBs with prominent perinuclear microtubules per root in an ANOVA followed by Dunnett ' s multiple comparisons ( * * P < 0 . 01 * * * * P < 0 . 0001 ) . F . of spindles with lagging chromosomes . Boxes whiskers represent min to max values with median indicated as a central horizontal , n = 10 roots per genotype . Asterisks show significant differences in of spindles with lagging chromosomes per root in a Kruskal - Wallis followed by Dunn ' s multiple comparisons ( * * P < 0 . 01 ns = non - significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of wildtype (WT), double and misplaced PPBs. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. E. Quantification of PPBs with prominent perinuclear microtubules (MTs). Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of PPBs with prominent perinuclear microtubules per root in an ANOVA test followed by Dunnett's multiple comparisons test (** P < 0.01 and **** P < 0.0001). F. Quantification of spindles with lagging chromosomes. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of spindles with lagging chromosomes per root in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (** P < 0.01 and ns = non-significant).


A . Co - immunolocalization against tubulin ( magenta ) KNOLLE ( green ) in root meristematic . Nuclei were counterstained with DAPI for ( cyan ) . Scale bars : 5 μm . B . of wildtype ( WT ) abnormal phragmoplasts . Different letters indicate significant differences in proportions of different arrays per category in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype . C . of different mitotic stages in roots of different genotypes . Different letters indicate significant differences in proportions of different arrays per category in a Chi - squared followed by Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype .

O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O

A. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). Scale bars: 5 μm. B. Quantification of wildtype (WT) and abnormal phragmoplasts. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. C. Quantification of the different mitotic stages in roots of the different genotypes. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype.


A . Kinase assays against Histone H1 . Top middle panels indicate shorter longer exposures respectively of same kinase assays . Bottom panel is a CBB of Histone H1 showing equal loading of . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . B . Kinase assays against GIP1 . Top middle panels indicate shorter longer exposures respectively of same kinase assays . Bottom panel is a CBB of GIP1 showing equal loading of . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . C . Western blot against StrepIII - tagged to show loaded amounts of CDKs . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . Double CDKB2 ; 2 bands are likely due to a truncation of expressed .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-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 B-MEASURED_VAR O O O O O O O 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

A. Kinase assays against Histone H1. Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. B. Kinase assays against GIP1. Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of GIP1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. C. Western blot against StrepIII-tagged proteins to show loaded amounts of the CDKs. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. Double CDKB2;2 bands are likely due to a truncation of the expressed protein.


D . Time - lapse of confocal microscope pictures of root meristematic tagged with GFP - GIP1 in Col - 0 ( top panel ) cycb1 ; 1 cycb1 ; 2 ( bottom panel ) . GIP1 localizes at polar caps , followed by co - localization with microtubules at spindle phragmoplast arrays . In cycb1 ; 1 cycb1 ; 2 double mutants , GIP1 exhibited an abnormal localization , being found at spindle ( magenta arrowheads ) phragmoplast ( arrowheads ) midzones , which are normally devoid of . Scale bars : 5 μm .

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

D. Time-lapse of confocal microscope pictures of root meristematic cells tagged with GFP-GIP1 in Col-0 (top panel) and cycb1;1 cycb1;2 (bottom panel). GIP1 localizes at the nuclear polar caps, followed by co-localization with microtubules at the spindle and phragmoplast arrays. In cycb1;1 cycb1;2 double mutants, GIP1 exhibited an abnormal localization, being found at the spindle (magenta arrowheads) and phragmoplast (white arrowheads) midzones, which are normally devoid of the protein. Scale bars: 5 μm.


A - E Chemokine - galectin interactions were detected by using a solid - phase immunoassay . For this , 46 human CC CXC chemokines were adsorbed on nitrocellulose membranes stripes incubated in parallel with ( A ) TBS or ( B ) TBS containing biotinylated galectins ( representative image shows a processed tested with labeled Gal - 3 ) . Signals had been generated by using SA - HRP chemiluminescence reagents . ( C - D ) blots were subjected to densitometric , ( E ) independent experiments were combined ( binding chemokines in blue , Gal - 3 : n = 5 , Gal - 3 CRD : n = 5 , Gal - 1 n = 4 ) .

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A-E Chemokine-galectin interactions were detected by using a solid-phase immunoassay. For this, 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with (A) TBS or (B) TBS containing biotinylated galectins (the representative image shows a processed membrane tested with labeled Gal-3). Signals had been generated by using SA-HRP and chemiluminescence reagents. (C-D) The blots were subjected to densitometric analysis, and (E) all independent experiments were combined (binding chemokines in light blue, Gal-3: n = 5, Gal-3 CRD: n = 5, Gal-1 n = 4).


For SPR - experiments , ( F ) Gal - 3 ( density 650 RU ) , were immobilized increasing concentrations of CXCL12 were passed over flow . red curve represents a single - site fit to data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR O O O O O

For SPR-based experiments, (F) Gal-3 (density 650 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


For SPR - experiments , ( G ) Gal - 3 CRD ( density 1180 RU ) , were immobilized increasing concentrations of CXCL12 were passed over flow . red curve represents a single - site fit to data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR O O O O O

For SPR-based experiments, (G) Gal-3 CRD (density 1180 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


For SPR - experiments , ( H ) Gal - 1 ( density 130 RU ) were immobilized increasing concentrations of CXCL12 were passed over flow . red curve represents a single - site fit to data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR O O O O O

For SPR-based experiments, (H) Gal-1 (density 130 RU) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


a - b Expansions of 1H - 15N HSQC spectra are overlaid Δδ values are plotted vs . acid sequences , along with secondary structures : ( a ) 10 µM 15N - enriched CXCL12 alone ( black peaks ) in presence of 330 µM Gal - 3 CRD ( red peaks ; assignments reported by Murphy et al . [ 74 ] ) ( b ) 30 µM 15N - enriched Gal - 3 CRD alone ( black peaks ) in presence of 500 µM CXCL12 ( red peaks ; assignments for Gal -

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a-b Expansions of 1H-15N HSQC spectra are overlaid and Δδ values are plotted vs. the amino acid sequences, along with secondary structures: (a) 10 µM 15N-enriched CXCL12 alone (black peaks) and in the presence of 330 µM Gal-3 CRD (red peaks; assignments reported by Murphy et al.[74]) and (b) 30 µM 15N-enriched Gal-3 CRD alone (black peaks) and in the presence of 500 µM CXCL12 (red peaks; assignments for Gal-3


a - c Jurkat T migrated in presence of 10 nM CXCL12 with increasing concentrations of ( a ) Gal - 3 , ( b ) Gal - 3 CRD , ( c ) Gal - 1 ( A - C : n = 4 ) .

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a-c Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of (a) Gal-3, (b) Gal-3 CRD, and (c) Gal-1 (A-C: n = 4).


D Human CD4 + T migrated in presence of 10 nM CXCL12 alone with 10 nM Gal - 3 ( green ) , Gal - 3 CRD ( green ) , Gal - 1 ( blue , n = 3 ) .

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D Human CD4+ T cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3 (dark green), Gal-3 CRD (light green), and Gal-1 (blue, n = 3).


E - F ( E ) Jurkat T migrated to increasing concentrations of CXCL12 alone ( black ) CXCL12 in presence of 0 . 1 nM of Gal - 3 CRD ( green ) . ( F ) inhibitory effect of Gal - 3 CRD is shown as of chemotactic effect of CXCL12 ( E - F : n = 3 ) .

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E-F (E) Jurkat T cells migrated to increasing concentrations of CXCL12 alone (black) and CXCL12 in the presence of 0.1 nM of Gal-3 CRD (light green). (F) The inhibitory effect of Gal-3 CRD is shown as percentage of the chemotactic effect of CXCL12 (E-F: n = 3).


G Jurkat T were allowed to migrate in presence of CXCL12 at increasing concentrations alone with 0 . 1 nM Gal - 1 or 100 nM Gal - 1 ( n = 3 ) .

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G Jurkat T cells were allowed to migrate in the presence of CXCL12 at increasing concentrations alone and with 0.1 nM Gal-1 or 100 nM Gal-1 (n=3).


H Jurkat T did not migrate in sole presence of 1 nM to 1 µM Gal - 3 , Gal - 3 CRD ( both n = 5 ) , or Gal - 1 ( n = 4 ) .

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H Jurkat T cells did not migrate in the sole presence of 1 nM to 1 µM Gal-3, Gal-3 CRD (both n = 5), or Gal-1 (n = 4).


I viability of Jurkat T was assessed after incubation with 1 µM CXCL12 alone in presence of Gal - 3 , Gal - 3 CRD Gal - 1 ( n = 2 ) .

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I The viability of Jurkat T cells was assessed after incubation with 1 µM CXCL12 alone and in the presence of Gal-3, Gal-3 CRD and Gal-1 (n = 2).


( J ) THP - 1 migrated in presence of 10 nM CXCL12 alone with 10 nM Gal - 3 , Gal - 3 CRD Gal -

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(J) THP-1 cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1


( K ) neutrophils migrated in presence of 10 nM CXCL12 alone with 10 nM Gal - 3 , Gal - 3 CRD Gal -

(K) neutrophils migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1


L Human eosinophils migrated in presence of 10 nM CCL26 alone with 10 nM Gal - 3 , Gal - 3 CRD , Gal - 1 ( n = 3 ) .

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L Human eosinophils migrated in the presence of 10 nM CCL26 alone and with 10 nM Gal-3, Gal-3 CRD, and Gal-1 (n = 3).


M CD4 + T migrated in presence of 1 nM CCL17 alone with equimolar concentrations of Gal - 3 , Gal - 3 CRD , Gal - 1 ( n = 4 ) .

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M CD4+ T cells migrated in the presence of 1 nM CCL17 alone and with equimolar concentrations of Gal-3, Gal-3 CRD, and Gal-1 (n = 4).


A - B peritoneal recruitment of ( A ) CD45 + / CD115 - / Ly6G + neutrophils ( B ) CD45 + / CD115 + / Ly6Chi classical monocytes in C57BL / 6J mice was assessed 4 hours after intraperitoneal ( IP ) injection of 500 nM CXCL12 alone ( black ) in presence of 50 nM Gal - 3 ( green ; A - B : n = 7 mice ) .

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A-B The peritoneal recruitment of (A) CD45+/CD115-/Ly6G+ neutrophils and (B) CD45+/CD115+/Ly6Chi classical monocytes in C57BL/6J mice was assessed 4 hours after intraperitoneal (IP) injection of 500 nM CXCL12 alone (black) and in the presence of 50 nM Gal-3 (dark green; A-B: n = 7 mice).


C peritoneal recruitment of classical monocytes after IP injection of PBS ( n = 6 ) or TG in C57BL / 6J WT ( green , n = 10 mice ) Gal - 3 - / - ( black , n = 5 mice ) mice was assessed after 18 h . Where indicated , mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to experiment .

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C The peritoneal recruitment of classical monocytes after IP injection of PBS (n = 6) or TG in C57BL/6J WT (dark green, n = 10 mice) and Gal-3-/- (black, n = 5 mice) mice was assessed after 18 h. Where indicated, the mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to the experiment.


D of CXCL12 was determined by ELISA on peritoneal lavage normalized with from mesenterium ( n = 4 mice ) .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

D The concentration of CXCL12 concentration was determined by ELISA on the peritoneal lavage normalized with levels from the mesenterium (n = 4 mice).


E CXCR4 expression on Ly6Chi monocytes of blood peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry indicated as mean ( MFI ) ( n = 4 mice ) .

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E CXCR4 expression levels on Ly6Chi monocytes of the blood and the peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry and indicated as mean fluorescence intensity (MFI) (n = 4 mice).


A - B Jurkat T migrated in presence of 10 nM CXCL12 with increasing concentrations of E185A ( orange , n = 4 ) N222A ( red , n = 5 ) mutants of ( A ) Gal - 3 ( B ) Gal - 3 CRD .

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A-B Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of E185A (orange, n = 4) and N222A (red, n = 5) mutants of (A) Gal-3 and (B) Gal-3 CRD.


C - D Jurkat T migrated in presence of 10 nM CXCL12 with 10 nM ( C ) Gal - 3 ( D ) Gal - 3 CRD mutants as indicated ( C - D : n = 3 ) .

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C-D Jurkat T cells migrated in the presence of 10 nM CXCL12 with 10 nM (C) Gal-3 and (D) Gal-3 CRD mutants as indicated (C-D: n = 3).


E Aggregation of Jurkat T in presence of 1 nM to 1 µM Gal - 3 Gal - 1 alone with 70 mM lactose , Gal - 3 CRD , was determined by flow cytometry ( n = 4 ) .

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E Aggregation of Jurkat T cells in the presence of 1 nM to 1 µM Gal-3 and Gal-1 alone and with 70 mM lactose, and Gal-3 CRD, was determined by flow cytometry (n = 4).


Jurkat T migrated with 10 nM CXCL12 alone in presence of ( F ) 1 µM Gal - 3 , ( G ) Gal - 1 , ( H ) Gal - 3 CRD in presence of 70 mM lactose or cellobiose or after treatment of with 150 µM DMJ .

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Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (F) 1 µM Gal-3, (G) Gal-1, (H) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.


Jurkat T migrated with 10 nM CXCL12 alone in presence of ( I ) 0 . 1 nM Gal - 3 ( J ) Gal - 3 CRD in presence of 70 mM lactose or cellobiose or after treatment of with 150 µM DMJ .

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Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (I) 0.1 nM Gal-3 and (J) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.


a HEK transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone in presence of 10 nM Gal - 3 CRD followed by stimulation with forskolin , an activator of adenylate cyclase . Results are shown as luminescence relative to baseline ( RLU , example of n = 3 ) .

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a HEK cells transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD followed by stimulation with forskolin, an activator of adenylate cyclase. Results are shown as luminescence relative to baseline (RLU, example of n = 3).


b effect of 10 nM Gal - 3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation , was tested . Control experiments were performed as indicated ( n = 3 , four technical replicates ) .

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b The effect of 10 nM Gal-3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation, was tested. Control experiments were performed as indicated (n = 3, four technical replicates).


c HEK transfected with a RlucII - conjugated CXCR4 an eYFP - β - arrestin 2 construct were stimulated with 10 nM CXCL12 alone in presence of 10 nM Gal - 3 CRD . Control experiments were performed as indicated . Results are given as net BRET ( i . e . of emissions at 535 / 485 nm minus of mock , n = 5 , three technical replicates ) .

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c HEK cells transfected with a RlucII-conjugated CXCR4 and an eYFP-β-arrestin 2 construct were stimulated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD. Control experiments were performed as indicated. Results are given as the net BRET ratio (i.e. ratio of emissions at 535/485 nm minus the ratio of mock cells, n = 5, three technical replicates).


D Internalization of CXCR4 with 10 nM CXCL12 alone in presence of 10 nM Gal - 3 CRD was assessed by incubation with an APC - conjugated anti - CXCR4 antibody ( baseline signal = 100 % , n = 8 , two technical replicates ) .

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D Internalization of CXCR4 with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD was assessed by incubation with an APC-conjugated anti-CXCR4 antibody (baseline signal = 100%, n = 8, two technical replicates).


E 100 nM CXCL12 Gal - 3 were added to Jurkat T , their co - localization was assessed on surface by PLA . Control experiments were performed as indicated ( representative example of n = 3 ) . scale bar : 10 µm .

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E 100 nM CXCL12 and Gal-3 were added to Jurkat T cells, and their co-localization was assessed on the cell surface by PLA. Control experiments were performed as indicated (representative example of n = 3). White scale bar: 10 µm.


F - G Jurkat T were incubated with 100 nM of Gal - 3 CRD Alexa Fluor 555 alone , with 10 µM AMD 3100 , with 100 nM CXCL12 or with CXCL12 AMD 3100 . Experiments were performed ( f ) without ( g ) in presence of 70 mM lactose ( F - G : n = 3 , two technical replicates ) .

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F-G Jurkat T cells were incubated with 100 nM of Gal-3 CRD Alexa Fluor 555 alone, with 10 µM AMD 3100, with 100 nM CXCL12 or with CXCL12 and AMD 3100. Experiments were performed (f) without and (g) in the presence of 70 mM lactose (F-G: n = 3, two technical replicates).


H Jurkat T were incubated with 100 nM CXCL12 FITC alone with increasing concentrations of Gal - 3 CRD as indicated ( n = 4 , two technical replicates ) .

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

H Jurkat T cells were incubated with 100 nM CXCL12 FITC alone and with increasing concentrations of Gal-3 CRD as indicated (n = 4, two technical replicates).


( a ) Kaplan - Meier survival curves for KRas ; Atg5 + / + ( n = 12 ) , KRas ; Atg5fl / + ( n = 19 ) KRas ; Atg5fl / fl ( n = 18 ) littermate mice injected intranasally with AdCre ( 2 . 5 × 107 p . f . u . ) . P0 . 01 ( log - rank ) between KRas ; Atg5fl / fl cohort

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(a) The Kaplan-Meier survival curves for KRas;Atg5+/+ (n=12), KRas;Atg5fl/+ (n=19) and KRas;Atg5fl/fl (n=18) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). P0.01 (log-rank test) between the KRas;Atg5fl/fl cohort and controls


( b ) Western blot for Atg5 , LC3I LC3II p62 in purified lung tumour . Representative data are shown for tumour isolated from KRas ; Atg5fl / + KRas ; Atg5fl / fl mice 18 after AdCre inhalation . β - actin is shown as a loading control .

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(b) Western blot analysis for Atg5, LC3I and LC3II and p62 in purified primary lung tumour cells. Representative data are shown for tumour cells isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. β-actin is shown as a loading control.


( c ) Immunohistochemistry to detect LC3I , p62 Atg5 in tumours from KRas ; Atg5fl / + KRas ; Atg5fl / fl mice 18 after AdCre inhalation . Note highly elevated of LC3I p62 , both indicative of defective autophagy , in KRas ; Atg5fl / fl tumours . Scale bars , 50 μm . ( d , e ) Impaired formation of autophagosomes ( arrows ) in KRas ; Atg5fl / fl tumour .

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(c) Immunohistochemistry to detect LC3I, p62 and Atg5 in tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. Note highly elevated levels of LC3I and p62, both indicative of defective autophagy, in KRas;Atg5fl/fl tumours. Scale bars, 50 μm. (d,e) Impaired formation of autophagosomes (arrows) in KRas;Atg5fl/fl tumour cells.


( d , e ) Impaired formation of autophagosomes ( arrows ) in KRas ; Atg5fl / fl tumour . ( d ) Representative electron microscopy are shown for KRas ; Atg5fl / + KRas ; Atg5fl / fl tumours18 after AdCre inhalation . Scale bars , 5 μm for upper 2 μm for lower panels . M , mitochondria ; * , lamellar bodies ( Corpuscula lamellariae ) , rare organelles containing surfactant lipoproteins characteristic for type II pneumocytes . ( e ) Mean percentages ( ± s . e . m . ) of tumour with detectable autophagosomes or autolysosomes , counted on electron microscopy . n ≥ 8 mice per genotype at least 20 intact for each electron microscopy section were counted . * * * P0 . 001 ( χ2 - of a generalized linear model with logit link assessing genotype effect ) .

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 B-CONTROLLED_VAR 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

(d,e) Impaired formation of autophagosomes (arrows) in KRas;Atg5fl/fl tumour cells. (d) Representative electron microscopy images are shown for KRas;Atg5fl/+ and KRas;Atg5fl/fl tumours18 weeks after AdCre inhalation. Scale bars, 5 μm for upper and 2 μm for lower panels. M, mitochondria; *, lamellar bodies (Corpuscula lamellariae), rare cell organelles containing surfactant lipoproteins characteristic for type II pneumocytes. (e) Mean percentages (±s.e.m.) of tumour cells with detectable autophagosomes or autolysosomes, counted on electron microscopy images. n≥8 mice per genotype and at least 20 intact cells for each electron microscopy section were counted. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect).


( a ) Representative electron microscopy of autolysosomes ( left panels ) , autophagosomes ( middle panels ) mitophagy ( right panels ) , indicated by arrows , in lungtumour from control KRas ; Atg5fl / + mice18 after AdCre inhalation . Note near - to - complete absence of such structures in lungtumour from KRas ; Atg5fl / fl . Very rarely , we found with one autolysosome ( left panels ) or one single autophagosome ( middle panels ; arrows ) in KRas ; Atg5fl / fl tumours , whereas such structures can frequently be seen within single KRas ; Atg5fl / + tumour . Of note , presence of a single autophagosome or a single autolysosome was scored as a positive event in Fig . 1e . Also note aberrant mitochondrial morphologies increased mitochondrial numbers in KRas ; Atg5fl / fl tumour . * indicates lamellar bodies . Scale bars , 2 μm . ( b ) Mean numbers ( ± s . e . m . ) of mitochondria per lungtumour from KRas ; Atg5fl / + KRas ; Atg5fl / fl mice18 after tumour induction . Numbers of mitochondria were determined by electron microscopy . n ≥ 8 mice per genotype a minimum of 20 intact were counted per sample . * * * P0 . 001 ( χ2 - assessing genotype effect in a generalized linear model with log link ) .

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

(a) Representative electron microscopy images of autolysosomes (left panels), autophagosomes (middle panels) and mitophagy (right panels), all indicated by arrows, in lungtumour cells from control KRas;Atg5fl/+ mice18 weeks after AdCre inhalation. Note the near-to-complete absence of such structures in the lungtumour cells from KRas;Atg5fl/fl littermates. Very rarely, we found cells with one autolysosome (left panels) or one single autophagosome (middle panels; arrows) in KRas;Atg5fl/fl tumours, whereas such structures can frequently be seen within single KRas;Atg5fl/+ tumour cells. Of note, the presence of a single autophagosome or a single autolysosome was scored as a positive event in Fig. 1e. Also note aberrant mitochondrial morphologies and increased mitochondrial numbers in KRas;Atg5fl/fl tumour cells. * indicates lamellar bodies. Scale bars, 2 μm. (b) Mean numbers (±s.e.m.) of mitochondria per lungtumour cell from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice18 weeks after tumour induction. Numbers of mitochondria were determined by electron microscopy. n≥8 mice per genotype and a minimum of 20 intact cells were counted per sample. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link).


( c ) Mitochondria ( arrows ) in KRas ; Atg5fl / + KRas ; Atg5fl / fl lung tumour 18 after AdCre inhalation . Note markedly altered mitochondrial , that is , disorganized matrix structures , rounding enlargement in autophagy - defective KRas ; Atg5fl / fl . Au , autophagosomes ; Au - L , autolysosome ; * , lamellar bodies . Scale bars , 2 μm .

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(c) Mitochondria (arrows) in KRas;Atg5fl/+ and KRas;Atg5fl/fl lung tumour cells 18 weeks after AdCre inhalation. Note markedly altered mitochondrial morphology, that is, disorganized matrix structures, rounding and enlargement in the autophagy-defective KRas;Atg5fl/fl cells. Au, autophagosomes; Au-L, autolysosome; *, lamellar bodies. Scale bars, 2 μm.


( d - g ) Bioenergetics profiling of purified tumour from KRas ; Atg5fl / + KRas ; Atg5fl / fl mice ( e ) 6 ( g ) 18 after tumour induction . Representative experimental data are shown . Oxygen consumption rates ( OCR ) were used to detect bioenergetics profiles of purified tumour using a Seahorse Bioscience 24XF extracellular flux analyser . OCR ± s . e . m . were recorded to construct bioenergetics profiles for basal respiration ( BR ) , ATP turnover ( AT ) , respiratory capacity ( RC ) , spare respiratory capacity ( SRC ) proton leakage ( PL ) . A minimum of seven different samples were analysed for each group . * P0 . 05 ; * * P0 . 01 ( unpaired , two - sided t - ) .

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(d-g) Bioenergetics profiling of purified tumour cells from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice (e) 6 weeks and (g) 18 weeks after tumour induction. Representative experimental data are shown. Oxygen consumption rates (OCR) were used to detect the bioenergetics profiles of purified tumour cells using a Seahorse Bioscience 24XF extracellular flux analyser. OCR±s.e.m. were recorded to construct bioenergetics profiles for basal respiration (BR), ATP turnover (AT), respiratory capacity (RC), spare respiratory capacity (SRC) and proton leakage (PL). A minimum of seven different samples were analysed for each group. *P0.05; **P0.01(unpaired, two-sided t-test).


( a ) Increased tumour initiation in KRas ; Atg5fl / fl mice as compared with KRas ; Atg5fl / + . Representative histological sections at 2 after AdCre inhalation are shown . Insets show typical lung lesions . Haematoxylin eosin . Scale bars , 2 mm for whole lung section ; 50 μm for insets .

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(a) Increased tumour initiation in KRas;Atg5fl/fl mice as compared with KRas;Atg5fl/+ littermates. Representative histological sections at 2 weeks after AdCre inhalation are shown. Insets show typical lung lesions. Haematoxylin and eosin staining. Scale bars, 2 mm for whole lung section; 50 μm for insets.


( b ) of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) in KRas ; Atg5fl / + KRas ; Atg5fl / fl mice 2 after AdCre inhalation . n = 6 per group . * * P0 . 01 ( χ2 - assessing genotype effect in a generalized linear model with log link ) .

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(b) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation. n=6 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link).