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

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

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


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

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

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


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

O O O O O 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

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


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

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

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

O O 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 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 patient - derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the 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 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 , 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 .

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 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 . 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 .

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

O O O O O O O O O O O O O O O O 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 . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 ) .

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

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

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 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 ) .

O O O O O O O O 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 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) 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 .

O O O O O O 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

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 . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

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 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 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 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

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 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 .

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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 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 ) .

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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 - 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 .

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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 - 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 .

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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 - 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 .

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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 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 -

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal - 3 , Gal - 3 CRD and 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 the presence of 10 nM CXCL12 alone and with 10 nM Gal - 3 , Gal - 3 CRD and Gal -

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(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 the presence of 10 nM CCL26 alone and with 10 nM Gal - 3 , Gal - 3 CRD , and 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 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 ) .

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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 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 ) .

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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 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 .

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

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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

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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 ) 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

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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 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 .

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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 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 .

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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 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 ) .

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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

(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 / + 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 .

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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 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 ) .

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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 / + 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 .

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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 ) 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 ) .

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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).


( c ) Tumour growth curves based on mean tumour - to - lung area ratios ( ± s . e . m . ) determined in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates at the indicated time points . At least three different planes from each lung were analysed . n ≥ 6 per cohort for each time point . * P0 . 05 , * * P0 . 01 ( unpaired , two - sided Wicoxon test ) .

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(c) Tumour growth curves based on mean tumour-to-lung area ratios (±s.e.m.) determined in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates at the indicated time points. At least three different planes from each lung were analysed. n≥6 per cohort for each time point. *P0.05, **P0.01 (unpaired, two-sided Wicoxon test).


( d ) MicroCT analysis of lung tumours of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice 18 weeks after AdCre inhalation . Representative data from individual mice are shown . Scale bars , 5

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(d) MicroCT analysis of lung tumours of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice 18 weeks after AdCre inhalation. Representative data from individual mice are shown. Scale bars, 5 mm


( a ) Distribution and ( b ) absolute numbers of hyperplastic lesions , adenomas and adenocarcinomas in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice6 , 12 and 18 weeks post AdCre infection . Data are shown as means ± s . e . m . ; n = 6 per group . Combining the tumour progression data from 6 , 12 and 18 weeks , a cumulative linked mixed model ( clmm ) analysis showed also a significant dependency of tumour progression on the genotype ; P0 . 05 ( χ2 - test of clmms assessing the genotype effect ) . Applying generalized linear - mixed effects ( glmer ) models with logit link to individual time points , shows a significant dependency of the transition from hyperplastic lesions to adenomas ( 12 weeks , P0 . 05 ) and from adenomas to adenocarcinomas ( 12 weeks , P0 . 01 and 18 weeks , P0 . 01 ) on the mouse genotype ( the last three P - values are from χ2 - tests of a generalized linear models with logit link assessing the genotype effect ) .

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(a) Distribution and (b) absolute numbers of hyperplastic lesions, adenomas and adenocarcinomas in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice6, 12 and 18 weeks post AdCre infection. Data are shown as means±s.e.m.; n=6 per group. Combining the tumour progression data from 6, 12 and 18 weeks, a cumulative linked mixed model (clmm) analysis showed also a significant dependency of tumour progression on the genotype; P0.05 (χ2-test of clmms assessing the genotype effect). Applying generalized linear-mixed effects (glmer) models with logit link to individual time points, shows a significant dependency of the transition from hyperplastic lesions to adenomas (12 weeks, P0.05) and from adenomas to adenocarcinomas (12 weeks, P0.01 and 18 weeks, P0.01) on the mouse genotype (the last three P-values are from χ2-tests of a generalized linear models with logit link assessing the genotype effect).


( c , d ) Histological analyses ( haematoxylin and eosin staining ) of lung tumours in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . ( c ) Nuclear and cellular polymorphy , more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas ; Atg5fl / + mice as compared with KRas ; Atg5fl / fl tumours . Sections were analysed 18 weeks after AdCre inhalation . Scale bars , 50 μm . ( d ) KRas ; Atg5fl / fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours ( asterisk ) .

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(c,d) Histological analyses (haematoxylin and eosin staining) of lung tumours in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. (c) Nuclear and cellular polymorphy, more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas;Atg5fl/+ mice as compared with KRas;Atg5fl/fl tumours. Sections were analysed 18 weeks after AdCre inhalation. Scale bars, 50 μm. (d) KRas;Atg5fl/fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours (asterisk).


( c , d ) Histological analyses ( haematoxylin and eosin staining ) of lungtumours in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . ( c ) Nuclear and cellular polymorphy , more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas ; Atg5fl / + mice as compared with KRas ; Atg5fl / fl tumours . Sections were analysed 18 weeks after AdCre inhalation . Scale bars , 50 μm . ( d ) KRas ; Atg5fl / fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours ( asterisk ) .

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(c,d) Histological analyses (haematoxylin and eosin staining) of lungtumours in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. (c) Nuclear and cellular polymorphy, more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas;Atg5fl/+ mice as compared with KRas;Atg5fl/fl tumours. Sections were analysed 18 weeks after AdCre inhalation. Scale bars, 50 μm. (d) KRas;Atg5fl/fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours (asterisk).


( a ) Primary pneumocytes were infected with AdCre for 96 h and Atg5 protein expression determined by western blot . β - actin is shown as a loading control .

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(a) Primary pneumocytes were infected with AdCre for 96 h and Atg5 protein expression determined by western blot. β-actin is shown as a loading control.


( b ) Atg5 mRNA expression was determined by quantitative PCR . Data are shown as mean relative mRNA expression ± s . e . m . in KRas ; Atg5fl / fl tumours as compared with tumours from KRas ; Atg5fl / + mice ( set to 1 ) . Values were normalized to β - actin mRNA expression . n = 3 per group .

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(b) Atg5 mRNA expression was determined by quantitative PCR. Data are shown as mean relative mRNA expression±s.e.m. in KRas;Atg5fl/fl tumours as compared with tumours from KRas;Atg5fl/+ mice (set to 1). Values were normalized to β-actin mRNA expression. n=3 per group.


( b ) Representative immunohistochemistry and quantitative assessment of γH2AX and phospho - Chk2 in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . For γH2AX and phospho - Chk2 staining , sections were scored independently in a double - blinded manner . Data are shown as mean percentages ( ± s . e . m . ) of positive cells per total cell numbers . n = 6 mice per genotype . Scale bars , 50 μm . * * * P0 . 001 ( χ2 - test of a generalized linear model with logit link assessing the genotype effect ) .

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

(b) Representative immunohistochemistry and quantitative assessment of γH2AX and phospho-Chk2 in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. For γH2AX and phospho-Chk2 staining, sections were scored independently in a double-blinded manner. Data are shown as mean percentages (±s.e.m.) of positive cells per total cell numbers. n=6 mice per genotype. Scale bars, 50 μm. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect).


( c ) Representative histological lung sections of KRas ; Atg5fl / + ; p53fl / fl and KRas ; Atg5fl / fl ; p53fl / fl littermate mice16 weeks after AdCre inhalation . Scale bars , 2 mm . ( d ) Distribution of hyperplastic lesions , adenomas and adenocarcinomas ( ± s . e . m . ) in KRas ; Atg5fl / + ; p53fl / fl and KRas ; Atg5fl / fl ; p53fl / fl littermates 16 weeks post AdCre infection ( n = 4 per genotype ) .

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(c) Representative histological lung sections of KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermate mice16 weeks after AdCre inhalation. Scale bars, 2 mm. (d) Distribution of hyperplastic lesions, adenomas and adenocarcinomas (±s.e.m.) in KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermates 16 weeks post AdCre infection (n=4 per genotype).


( e ) The Kaplan - Meier survival curves for KRas ; Atg5fl / + ; p53fl / fl ( n = 8 ) and KRas ; Atg5fl / fl ; p53fl / fl ( n = 9 ) littermate mice injected intranasally with AdCre ( 2 . 5 × 107 p . f . u . ) . There was no statistical difference between the cohorts ( log - rank test ) .

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(e) The Kaplan-Meier survival curves for KRas;Atg5fl/+;p53fl/fl (n=8) and KRas;Atg5fl/fl;p53fl/fl (n=9) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). There was no statistical difference between the cohorts (log-rank test).


( a , b ) Representative immunohistochemistry and quantitative assessment of ( a ) tumour - infiltrating CD3ε - positive T cells and ( b ) FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 6 weeks after AdCre inhalation . Data are shown as mean percentages ( ± s . e . m . ) of the indicated cell types per total cell numbers in the tumour areas . Only cells within tumours were scored . At least six mice per genotype were analysed . Scale bars , 50 μm . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) .

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

(a,b) Representative immunohistochemistry and quantitative assessment of (a) tumour-infiltrating CD3ε-positive T cells and (b) FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 6 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of the indicated cell types per total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. ***P0.001(χ2-test assessing the genotype effect in a generalized linear model with logit link).


( c ) Representative immunohistochemistry and quantitative assessment of FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 2 weeks after AdCre inhalation . Data are shown as mean percentages ( ± s . e . m . ) of FoxP3 + cells among total cell numbers in the tumour areas . Only cells within tumours were scored . At least six mice per genotype were analysed . Scale bars , 50 μm . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) .

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(c) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. **P0.01(χ2-test assessing the genotype effect in a generalized linear model with logit link).


( d ) FACS blots to detect CD25 + FoxP3 + Treg cells in lungs 2 weeks after AdCre inhalation . Cells were gated on CD4 + populations . Numbers indicate percentages among total CD4 + cells in the lungs .

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(d) FACS blots to detect CD25+FoxP3+ Treg cells in lungs 2 weeks after AdCre inhalation. Cells were gated on CD4+ populations. Numbers indicate percentages among total CD4+ cells in the lungs.


( e ) Mediastinal lymph node and lung cells were isolated from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 2 weeks after AdCre inhalation and stained for flow cytometric analysis as described in Methods . The left panel shows the percentage of CD4 + T cells among all lung - infiltrating CD45 + haematopoietic cells ( ± s . e . m . ) . The right panel shows the percentage of FoxP3 + cells among CD4 + T cells in mediastinal lymph nodes and lungs ( ± s . e . m . ) . n = 4 per genotype ; * P0 . 05 ( unpaired , two - sided t - test ) .

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

(e) Mediastinal lymph node and lung cells were isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation and stained for flow cytometric analysis as described in Methods. The left panel shows the percentage of CD4+ T cells among all lung-infiltrating CD45+ haematopoietic cells (±s.e.m.). The right panel shows the percentage of FoxP3+ cells among CD4+ T cells in mediastinal lymph nodes and lungs (±s.e.m.). n=4 per genotype; *P0.05 (unpaired, two-sided t-test).


( a ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) in KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice2 weeks after AdCre inhalation treated either with an isotype control Ab or a depleting anti - CD25 Ab . n = 4 per group . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . ( b ) Histological lung sections of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice treated with an isotype control Ab or anti - CD25 - immunodepleting Abs . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology . Representative lung images ( haematoxylin and eosin ( HE ) staining ) are shown for each genotype and treatment . Scale bars , 2 mm .

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

(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated either with an isotype control Ab or a depleting anti-CD25 Ab. n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). (b) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with an isotype control Ab or anti-CD25-immunodepleting Abs. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (haematoxylin and eosin (HE) staining) are shown for each genotype and treatment. Scale bars, 2 mm.


( c ) 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 treated either with an isotype control Ab or an Ab specific to FR4 . n = 4 per group . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . ( d ) Histological lung sections of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice treated with isotype control Abs or Abs specific to FR4 . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology . Representative lung images ( HE staining ) are shown for each genotype and treatment . Scale bars , 2 mm .

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

(c) 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 treated either with an isotype control Ab or an Ab specific to FR4. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). (d) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with isotype control Abs or Abs specific to FR4. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (HE staining) are shown for each genotype and treatment. Scale bars, 2 mm.


( b ) Protein expression of Hif1α , Atg5 and glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) in isolated KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes ( analysed at 4 days after AdCre infection ) and tumours purified from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice at week 2 after AdCre inhalation . Quantifications of the hypoxia - inducible factor ( HIF ) 1α / GAPDH ratios are shown for each lane ; two independent blots are quantified .

O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-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 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) Protein expression of Hif1α, Atg5 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes (analysed at 4 days after AdCre infection) and tumours purified from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice at week 2 after AdCre inhalation. Quantifications of the hypoxia-inducible factor (HIF)1α/GAPDH ratios are shown for each lane; two independent blots are quantified.


( c ) CD39 , Atg5 and as a control β - actin protein expression in isolated Atg5fl / + , Atg5fl / fl , KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes . Protein lysated were analysed at 4 days after AdCre infections by western blot . Quantifications of the CD39 / β - actin ratios are shown for each lane , normalized to the Atg5fl / + samples . Two independent blots are quantified .

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

(c) CD39, Atg5 and as a control β-actin protein expression in isolated Atg5fl/+, Atg5fl/fl, KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes. Protein lysated were analysed at 4 days after AdCre infections by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane, normalized to the Atg5fl/+ samples. Two independent blots are quantified.


( d , e ) CD39 , Atg5 and as a control β - actin protein expression in isolated KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes treated with the ( d ) HIF1α inhibitor 3 - ( 2 - ( 4 - adamantan - 1 - yl - phenoxy ) - acetylamino ) - 4 - hydroxybenzoic acid methyl ester or left untreated and ( e ) the Nrf2 inhibitor Brusatol ( 100 nM ) or the ROS scavenger N - acetyl - L - cysteine ( 5 mM ) . Protein lysates were analysed on day 4 by western blot . Quantifications of the CD39 / β - actin ratios are shown for each lane . The inhibitors were present during the entire 4 day culture period .

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

(d,e) CD39, Atg5 and as a control β-actin protein expression in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes treated with the (d) HIF1α inhibitor 3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester or left untreated and (e) the Nrf2 inhibitor Brusatol (100 nM) or the ROS scavenger N-acetyl-L-cysteine (5 mM). Protein lysates were analysed on day 4 by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane. The inhibitors were present during the entire 4 day culture period.


( a ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) and ( b ) representative histological lung sections ( haematoxylin and eosin ( HE ) staining ) of KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 ( intraperitoneally ; i . p . 10 mg kg − 1 daily ) to inhibit CD39 activity . n = 4 per group . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . Scale bars , 2 mm .

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

(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (b) representative histological lung sections (haematoxylin and eosin (HE) staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 (intraperitoneally; i.p. 10 mg kg−1 daily) to inhibit CD39 activity. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm.


( c ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) and ( d ) representative histological lung sections ( HE staining ) of KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 ( i . p . 10 mg kg − 1 daily ) . n = 4 per group . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . Scale bars , 2 mm .

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

(c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (d) representative histological lung sections (HE staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 (i.p. 10 mg kg−1 daily). n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm.


( e , f ) Representative immunohistochemistry and quantitative assessment of FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates treated with vehicle control or the CD39 blocker polyoxometalate - 1 ( POM1 ) and treated with vehicle or the adenosine receptor inhibitor PSB1115 ( 4 - ( 2 , 3 , 6 , 7 - tetrahydro - 2 , 6 - dioxo - 1 - propyl - 1H - purin - 8 - yl ) - benzenesulphonic acid potassium salt ) . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology . Data are shown as mean percentages ( ± s . e . m . ) of FoxP3 + cells among total cell numbers in the tumour areas . Only cells within tumours were scored . At least four mice per genotype were analysed . Scale bars , 50 μm . * * P0 . 01 . NS , not significant ; ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) .

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 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 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

(e,f) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates treated with vehicle control or the CD39 blocker polyoxometalate-1 (POM1) and treated with vehicle or the adenosine receptor inhibitor PSB1115 (4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulphonic acid potassium salt). Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least four mice per genotype were analysed. Scale bars, 50 μm. **P0.01. NS, not significant; (χ2-test assessing the genotype effect in a generalized linear model with logit link).


( A ) Immunoblot with anti - TG2 or anti - β - actin as loading control in in whole lysates from small intestine homogenates of CftrF508del / F508del and wild - type ( CftrWT ) mice ( n = 5 per group ) and densitometric analysis of immunoblots . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 ( Student ' s t test ) . Data information : The blots are representative of one experiment for group of treatment .

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

(A) Immunoblot with anti-TG2 or anti-β-actin as loading control in in whole lysates from small intestine homogenates of CftrF508del/F508del and wild-type (CftrWT) mice (n=5 per group) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **p<0.01 (Student's t test). Data information: The blots are representative of one experiment for group of treatment.


( B ) Detection of NLRP3 expression ( top ) and caspase - 1 cleavage ( bottom ) by immunoblot of whole lysates from small intestine homogenates of CftrWT and CftrF508del / F508del mice ( n = 5 ) and densitometric analysis of immunoblots . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 CftrWT vs CftrF508del / F508del ( Student ' s t test ) . Data information : The blots are representative of one experiment for group of treatment .

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

(B) Detection of NLRP3 expression (top) and caspase-1 cleavage (bottom) by immunoblot of whole lysates from small intestine homogenates of CftrWT and CftrF508del/F508del mice (n= 5) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **p<0.01 CftrWT vs CftrF508del/F508del (Student's t test). Data information: The blots are representative of one experiment for group of treatment.


( C ) Protein levels of IL - 17A and IFN - γ from small intestine homogenates of CftrWT and CftrF508del / F508del ( n = 10 ) . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 CftrWT vs CftrF508del / F508del ( Student ' s t test ) .

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

(C) Protein levels of IL-17A and IFN-γ from small intestine homogenates of CftrWT and CftrF508del/F508del (n= 10). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 CftrWT vs CftrF508del/F508del (Student's t test).


( D ) IL - 15 mRNA ( left ) and protein ( right ) levels in small intestine homogenates from CftrF508del / F508del , Cftr - / - or their CftrWT littermates ( n = 10 per group ) . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 CftrF508del / F508del vs CftrWT ( FVB / 129 ) , or ° ° p < 0 . 01 Cftr - / - vs CftrWT ( B6 . 129P2 ) ( ANOVA , Bonferroni post hoc test ) .

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

(D) IL-15 mRNA (left) and protein (right) levels in small intestine homogenates from CftrF508del/F508del, Cftr-/- or their CftrWT littermates (n= 10 per group). Mean ± SD of triplicates of independent pooled samples. **p<0.01 CftrF508del/F508del vs CftrWT(FVB/129), or °°p<0.01 Cftr-/- vs CftrWT(B6.129P2) (ANOVA, Bonferroni post hoc test).


( E ) IL - 15 mRNA levels in small intestine homogenates from CftrWT mice or CftrF508del / F508del or TG - / - / CftrF508del / F508del or TG - / - mice ( n = 10 per group ) . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 vs CftrWT , ° ° p < 0 . 01 , # # p < 0 . 01 vs CftrF508del / F508del ( ANOVA , Bonferroni post hoc test ) .

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

(E) IL-15 mRNA levels in small intestine homogenates from CftrWT mice or CftrF508del/F508del or TG-/-/CftrF508del/F508del or TG-/- mice (n= 10 per group). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 vs CftrWT, °°p<0.01, ##p<0.01 vs CftrF508del/F508del (ANOVA, Bonferroni post hoc test).


( F ) Effects of 4 weeks of oral administration of gliadin on IL - 15 , IL - 17A and IFN - γ protein levels in small intestine homogenates from CftrF508del / F508del and CftrWT mice ( n = 10 mice per group of treatment ) . Mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 , * * * p < 0 . 001 ( CftrF508del / F508del vs CftrWT mice prior gliadin challenge ) , ° ° ° p < 0 . 001 ( CftrF508del / F508del mice vs CftrF508del / F508del mice after gliadin challenge ) , ( ANOVA , Bonferroni post hoc test ) .

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

(F) Effects of 4 weeks of oral administration of gliadin on IL-15, IL-17A and IFN-γ protein levels in small intestine homogenates from CftrF508del/F508del and CftrWT mice (n =10 mice per group of treatment). Mean ± SD of triplicates of independent pooled samples. **p<0.01, ***p<0.001 (CftrF508del/F508del vs CftrWT mice prior gliadin challenge), °°°p<0.001 (CftrF508del/F508del mice vs CftrF508del/F508del mice after gliadin challenge), (ANOVA, Bonferroni post hoc test).


( G - I ) BALB / c mice ( G ) fed with a gluten - free diet for at least 3 generations , or ( H ) NOD or ( I ) NOD - DQ8 mice orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) . Representative traces of CFTR - dependent Cl - secretion measured by forskolin ( Fsk ) - induced increase of chloride current ( Isc ( μA / cm2 ) ) in small intestines mounted in Ussing chambers ; quantification of the peak CFTR inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in tissue samples ( n = 3 independent experiments ) . Mean ± SD of samples assayed ; * * p < 0 . 01 , * * * p < 0 . 001 vs challenged with gliadin ( Student ' s t test ) .

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

(G-I) BALB/c mice (G) fed with a gluten-free diet for at least 3 generations, or (H) NOD or (I) NOD-DQ8 mice orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks). Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n=3 independent experiments). Mean±SD of samples assayed; **p<0.01, ***p<0.001 vs challenged with gliadin (Student's t test).


( A ) Representative traces of CFTR - dependent Cl - secretion measured by forskolin ( Fsk ) - inducible chloride current ( Isc ( μA / cm2 ) ) in Caco - 2 cells mounted in Ussing chambers after 3 h of incubation with P57 - 68 or P33 - mer or P31 - 43 peptides ( 20 µg / ml ) and of P31 - 43 in the presence of up to 30 minutes of pre - treatment of VX - 770 ( 10 µM ) ; quantification of the peak CFTR Inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in Caco - 2 cells ( n = 3 independent experiments ) . Mean ± SD of samples assayed . * * * p < 0 . 001 vs P31 - 43 challenge , # # p < 0 . 01 vs Caco - 2 challenged with P31 - 43 in the presence of VX - 770 ( ANOVA , Bonferroni post hoc test ) .

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

(A) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-inducible chloride current (Isc (μA/cm2)) in Caco-2 cells mounted in Ussing chambers after 3 h of incubation with P57-68 or P33-mer or P31-43 peptides (20 µg/ml) and of P31-43 in the presence of up to 30 minutes of pre-treatment of VX-770 (10 µM); quantification of the peak CFTR Inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in Caco-2 cells (n= 3 independent experiments). Mean ± SD of samples assayed. ***p<0.001 vs P31-43 challenge, ##p<0.01 vs Caco-2 challenged with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test).


( B ) Incubation of Caco - 2 cells with P31 - 43 or control peptides ( p57 - 68 or PGAV ) for 1 h that were preceded or not by 20 min of pre - treatment with VX - 770 . Immunoprecipitation in non - reducing and non - denaturing conditions of CFTR protein and immunblot with streptavidin - HRP or CFTR antibody . Data information : The blots are representative of one experiment for group of treatment .

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 B-MEASURED_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 O O

(B) Incubation of Caco-2 cells with P31-43 or control peptides (p57-68 or PGAV) for 1 h that were preceded or not by 20 min of pre-treatment with VX-770. Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunblot with streptavidin-HRP or CFTR antibody. Data information: The blots are representative of one experiment for group of treatment.


( E ) Surface plasmon resonance ( SPR ) analysis of increasing concentrations of P31 - 43 and P57 - 68 peptides on rhNBD1 covalently bound to the CM5 sensor chip .

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(E) Surface plasmon resonance (SPR) analysis of increasing concentrations of P31-43 and P57-68 peptides on rhNBD1 covalently bound to the CM5 sensor chip.


( F ) Surface plasmon resonance ( SPR ) analysis of rhNBD1 binding to P31 - 43 and P57 - 68 biotinylated peptides immobilized on SA sensor chip .

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

(F) Surface plasmon resonance (SPR) analysis of rhNBD1 binding to P31-43 and P57-68 biotinylated peptides immobilized on SA sensor chip.