( A - B ) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG - PM - mAG1 ( green ) and incubated for two days in vitro . Immunocytochemical analyses with anti - caveolin - 1 ( red ) antibody were performed . The images are obtained with high - resolution microscopy ( Nikon ) . The graph in ( B ) indicates the ratio of the caveolin - 1 staining signals in the plasma membrane to total fluorescence intensities of caveolin - 1 in each immature neuron . Each score represents the mean of ratios with the individual points . Control : n = 20 cells , Rab21 - sh115 : n = 22 cells , Rab21 - sh115 + wt - Rab21 : n = 23 cells . Overexpression of wt - Rab21 resulted in neuronal cell death , which making it difficult to determine the accurate DNA concentration of wt - Rab21 in the rescue experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-PM-mAG1 (green) and incubated for two days in vitro. Immunocytochemical analyses with anti-caveolin-1 (red) antibody were performed. The images are obtained with high-resolution microscopy (Nikon). The graph in (B) indicates the ratio of the caveolin-1 staining signals in the plasma membrane to total fluorescence intensities of caveolin-1 in each immature neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells, Rab21-sh115: n = 22 cells, Rab21-sh115 + wt-Rab21: n = 23 cells. Overexpression of wt-Rab21 resulted in neuronal cell death, which making it difficult to determine the accurate DNA concentration of wt-Rab21 in the rescue experiments.


( H - I ) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids , incubated for two days in vitro , treated with Bafilomycin A1 ( Baf A1 ) for 6 h and stained with the indicated antibodies . The images are obtained with high - resolution microscopy ( Nikon ) and the lower panels are high magnification images of the lysosomes , indicated by white or blue rectangles . Blue alone channels are shown in black and white images . The graph in ( I ) shows the Pearson ' s correlation coefficient between caveolin - 1 and Lamp1 , a lysosomal marker , in control and Rab21 - sh115 - transfected neurons . Each score represents the mean of ratios with the individual points . Control : n = 31 cells , Rab21 - sh115 : n = 22 cells .

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(H-I) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro, treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Lamp1, a lysosomal marker, in control and Rab21-sh115-transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21-sh115: n = 22 cells.


( A ) Locomoting neurons in the upper IZ of the cerebral cortices at E17 , electroporated with the indicated plasmids plus pCAG - EGFP at E14 . ( B ) The ratio of locomoting neurons with more than three primary neurites . Each score represents the mean of ratios with the individual points . Control : n = 4 brains , Rab21 - sh115 : n = 5 brains , Rab21 - sh115 + Caveolin - 1 : n = 6 brains . ( C ) The box - and - whisker plot shows average leading process length of the locomoting neurons in the IZ . Control : n = 115 cells , Rab21 - sh115 : n = 188 cells , Rab21 - sh115 + Caveolin - 1 : n = 185 cells . (

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(A) Locomoting neurons in the upper IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. (B) The ratio of locomoting neurons with more than three primary neurites. Each score represents the mean of ratios with the individual points. Control: n = 4 brains, Rab21-sh115: n = 5 brains, Rab21-sh115+Caveolin-1: n = 6 brains. (C) The box-and-whisker plot shows average leading process length of the locomoting neurons in the IZ. Control: n = 115 cells, Rab21-sh115: n = 188 cells, Rab21-sh115+Caveolin-1: n = 185 cells. (


( D - E ) Cerebral cortices at P0 , electroporated with the indicated plasmids plus pCAG - EGFP at E14 . The lower graphs in ( D ) and the graph in ( E ) show the estimation of cell migration , as measured by EGFP fluorescence intensities in distinct regions of the cerebral cortices using Leica SP5 software . Each bar in the graph in ( E ) represents the mean percentage of relative intensities . Rab21 - sh115 : n = 4 brains , Rab21 - sh115 + CAG - wt - caveolin - 1 : n = 6 brains . II - IV : layers II - IV of the cortical plate , V - VI : layers V - VI of the cortical plate , IZ : intermediate zone , WM : white matter , SVZ / VZ : subventricular zone / ventricular zone .

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(D-E) Cerebral cortices at P0, electroporated with the indicated plasmids plus pCAG-EGFP at E14. The lower graphs in (D) and the graph in (E) show the estimation of cell migration, as measured by EGFP fluorescence intensities in distinct regions of the cerebral cortices using Leica SP5 software. Each bar in the graph in (E) represents the mean percentage of relative intensities. Rab21-sh115: n = 4 brains, Rab21-sh115+CAG-wt-caveolin-1: n = 6 brains. II-IV: layers II-IV of the cortical plate, V-VI: layers V-VI of the cortical plate, IZ: intermediate zone, WM: white matter, SVZ/VZ: subventricular zone/ventricular zone.


( F ) Examples of expression levels of Treg signature genes Foxp3 , Il10ra , Nrp1 , Ikzf4 and Ccr6 ; and examples of expression levels of genes upregulated in Tet2 / 3 DKO Treg cells Eomes , Ccl4 , Chek1 and Fn1 . The Y - axis shows FPKM ( Fragment Per Kilobase per Million reads ) , the X - axis shows the different cell types . Error bars show mean ± S . D . from two ( CD4 + naïve T cells ) or four ( WT and Tet2 / 3 DKO Treg ) biological replicates .

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(F) Examples of expression levels of Treg signature genes Foxp3, Il10ra, Nrp1, Ikzf4 and Ccr6; and examples of expression levels of genes upregulated in Tet2/3 DKO Treg cells Eomes, Ccl4, Chek1 and Fn1. The Y-axis shows FPKM (Fragment Per Kilobase per Million reads), the X-axis shows the different cell types. Error bars show mean ± S.D. from two (CD4+ naïve T cells) or four (WT and Tet2/3 DKO Treg) biological replicates.


( D ) Genome browser view of CMS - IP data showing CMS ( 5hmC ) distribution at Il2ra and Foxp3 locus for CD4 + naïve T cells and WT Treg cells . Hydroxymethylated regions ( hmRs ) and DhmRs are shown on the top ; conservation ( cons ) is shown at the bottom .

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

(D) Genome browser view of CMS-IP data showing CMS (5hmC) distribution at Il2ra and Foxp3 locus for CD4+ naïve T cells and WT Treg cells. Hydroxymethylated regions (hmRs) and DhmRs are shown on the top; conservation (cons) is shown at the bottom.


( E ) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4 + naïve T cells , WT Treg cells and Tet2 / 3 DKO Treg cells . Differentially hydroxymethylated regions ( DhmRs ) and differentially methylated CpGs ( DM - CpGs ) are shown on top of the tracks . Conservation track ( Cons ) and denotation of all CpGs are shown at the bottom . WGBS data are from two biological replicate samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. Differentially hydroxymethylated regions (DhmRs) and differentially methylated CpGs (DM-CpGs) are shown on top of the tracks. Conservation track (Cons) and denotation of all CpGs are shown at the bottom. WGBS data are from two biological replicate samples.


( E ) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4 + naïve T cells , WT Treg cells and Tet2 / 3 DKO Treg cells . DhmRs , DM - CpGs and DARs ( shaded ) are shown on top of the tracks . Conservation track ( Cons ) is shown at the bottom .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. DhmRs, DM-CpGs and DARs (shaded) are shown on top of the tracks. Conservation track (Cons) is shown at the bottom.


( H - J ) Box - and - whisker plots of DNA methylation levels for DM - CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs ( H ) , DM - CpGs less methylated in WT Treg cells vs CD4 + naïve T cells ( I ) and DM - CpGs less methylated in WT Treg cells vs Tet2 / 3 DKO Treg cells ( J ) . DM - CpGs denotes the CpGs in the respective cell types used for the comparison . Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range . P - values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey ' s post - hoc test . Data are from two biological replicates .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Box-and-whisker plots of DNA methylation levels for DM-CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs (H), DM-CpGs less methylated in WT Treg cells vs CD4+ naïve T cells (I) and DM-CpGs less methylated in WT Treg cells vs Tet2/3 DKO Treg cells (J). DM-CpGs denotes the CpGs in the respective cell types used for the comparison. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. P-values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey's post-hoc test. Data are from two biological replicates.


( D ) Graphs depicting the percentage of viable cells for TGFβ iTregs ( left ) and TGFβ + RA + VitC iTregs ( right ) differentiated with 0 U / ml , 1 U / ml , 5 U / ml and 10 U / ml IL - 2 . Error bars show mean ± S . D . from three biological replicates .

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(D) Graphs depicting the percentage of viable cells for TGFβ iTregs (left) and TGFβ+RA+VitC iTregs (right) differentiated with 0 U/ml, 1 U/ml, 5 U/ml and 10 U/ml IL-2. Error bars show mean ± S.D. from three biological replicates.


( C ) ChIP - qPCR showing STAT5 occupancy at Foxp3 CNS2 and IL2Rα IN1b regions in TGFβ and TGFβ + RA + VitC iTregs without or with 3 U / ml IL - 2 restimulation . GMPR is a negative control region . Data are from two biological replicates .

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(C) ChIP-qPCR showing STAT5 occupancy at Foxp3 CNS2 and IL2Rα IN1b regions in TGFβ and TGFβ + RA + VitC iTregs without or with 3 U/ml IL-2 restimulation. GMPR is a negative control region. Data are from two biological replicates.


A . Expression of Dot1L in different B - cell populations ( Shi et al . , 2015 ) . B1 : B1 cells , MZB : marginal zone B , FOB : follicular B , GCB : germinal center B , SPLPB : spleen plasma blast , SPLPC : spleen plasma cells , and BMPC : Bone marrow plasma cells . Expression is shown as transcript per million ( TPM ) . Results represent the data from two biological replicates except SPLPB and BMPC which lack replicates ( WT ; n = 2 ) . . Bars indicate mean values .

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A. Expression of Dot1L in different B-cell populations (Shi et al., 2015). B1: B1 cells, MZB: marginal zone B, FOB: follicular B, GCB: germinal center B, SPLPB: spleen plasma blast, SPLPC: spleen plasma cells, and BMPC: Bone marrow plasma cells. Expression is shown as transcript per million (TPM). Results represent the data from two biological replicates except SPLPB and BMPC which lack replicates (WT; n=2)..Bars indicate mean values.


A - G . Statistical analysis of absolute number of total nucleated splenocytes , splenic B - and T - cells , and indicated mature B cell subsets in WT and KO mice . Results represent the data pooled from at least two independent experiments and numbers represent biological replicates for each group ( A : ( WT ) n = 16 , ( KO ) n = 17 ; B ( B - cells ) : ( WT ) n = 16 , ( KO ) n = 17 ; B ( T - cells ) : ( WT ) n = 8 , ( KO ) n = 9 ; C - E : ( WT ) n = 9 , ( KO ) n = 9 ; F : ( WT ) n = 6 , ( KO ) n = 6 ; G : ( WT ) n = 9 , ( KO ) n = 9 ) .

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A-G. Statistical analysis of absolute number of total nucleated splenocytes, splenic B- and T-cells, and indicated mature B cell subsets in WT and KO mice. Results represent the data pooled from at least two independent experiments and numbers represent biological replicates for each group (A: (WT) n = 16, (KO) n = 17; B (B-cells): (WT) n = 16, (KO) n = 17; B (T-cells): (WT) n = 8, (KO) n = 9; C-E: (WT) n = 9, (KO) n = 9; F: (WT) n = 6, (KO) n = 6; G: (WT) n = 9, (KO) n = 9).


D - E . Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone ( IgG3 switching ) ( D ) or with LPS + IL - 4 ( IgG1 switching ) ( E ) . Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group ( D : ( WT ) n = 8 , ( KO ) n = 9 ; E : ( WT ) n = 11 , ( KO ) n = 13 ) . F . Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti - CD40 + IL - 4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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 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 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

D-E. Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone (IgG3 switching) (D) or with LPS + IL-4 (IgG1 switching) (E). Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group (D: (WT) n = 8, (KO) n = 9; E: (WT) n = 11, (KO) n = 13). F. Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti-CD40 + IL-4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


C . Statistical analysis of serum titers of LCMV - specific IgM and IgG of WT and KO mice before ( D0 ) and 14 days after ( D14 ) LCMV Armstrong infection . Results represent the data from two experiments and numbers represent biological replicates for each group ( WT ; n = 7 , KO ; n = 8 ) .

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C. Statistical analysis of serum titers of LCMV-specific IgM and IgG of WT and KO mice before (D0) and 14 days after (D14) LCMV Armstrong infection. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8).


D . Statistical analysis of serum titers of NP - specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge . Adjusted p - value were calculated using two - way ANOVA . Error bars indicate mean ± standard error of mean ( SEM ) . Results represent the data from one experiment and numbers represent biological replicates for each group ( WT ; n = 6 , KO ; n = 6 ) . * * * denotes p - value < 0 . 0001 and * * denotes p - value < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Statistical analysis of serum titers of NP-specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge. Adjusted p-value were calculated using two-way ANOVA. Error bars indicate mean ± standard error of mean (SEM). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 6, KO; n = 6).*** denotes p- value < 0.0001 and ** denotes p- value < 0.001.


A . Representative flow cytometry plots showing gating strategy to identify and compare plasma cells ( Sca - I + CD138 + ) after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analyses of their relative numbers ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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

A. Representative flow cytometry plots showing gating strategy to identify and compare plasma cells (Sca-I+CD138+) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


B . Representative flow cytometry plots showing gating strategy to identify and compare activated B cells ( CD138 + Blimp1 + ) , Pre - PB cells ( CD138 - Blimp1 + ) and plasma blast ( PB ) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analyses of their relative numbers ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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

B. Representative flow cytometry plots showing gating strategy to identify and compare activated B cells (CD138+ Blimp1+), Pre-PB cells (CD138- Blimp1+) and plasma blast (PB) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


C . Representative flow cytometry plots showing the relative surface density for B220 ( CD45R ) after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analysis of MFI of B220 for each treatment ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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

C. Representative flow cytometry plots showing the relative surface density for B220 (CD45R) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analysis of MFI of B220 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


B . Differential ( FDR < 0 . 05 ) expression of Bach2 , Prdm1 ( encoding BLIMP - 1 ) , Cd138 ( a plasma - cell marker ) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO . Data was generated from three independent biological replicates for each genotype . Bars and error bars indicate mean ± SD . Statistical significance is indicated by FDR after the Benjamini - Hochberg multiple testing correction performed by edgeR package using R language .

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B. Differential (FDR < 0.05) expression of Bach2, Prdm1 (encoding BLIMP-1), Cd138 (a plasma-cell marker) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO. Data was generated from three independent biological replicates for each genotype. Bars and error bars indicate mean ± SD. Statistical significance is indicated by FDR after the Benjamini-Hochberg multiple testing correction performed by edgeR package using R language.


D . H3K79me2 methylation at the Ezh2 locus from WT activated and naïve B cells , as determined by reads per genomic content ( RPGC ) . Data represent three independent biological replicates .

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D. H3K79me2 methylation at the Ezh2 locus from WT activated and naïve B cells, as determined by reads per genomic content (RPGC). Data represent three independent biological 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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

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

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

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

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

O O O O O O O O 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) 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 .

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

(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 ) .

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

(c) Representative 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 ) .

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

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

(c) Representative 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 O O O 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 B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes ( CellROX green , Molecular Probes , USA ) in HC ( n = 4 ) and DM ( n = 4 ) platelets . ( * * p = 0 . 032 vs . HC ) .

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A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC).


B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC ( # 1 - 3 ) and DM ( # 1 - 8 ) patient platelets . Quantification analysis on HC ( n = 5 ) and DM ( n = 18 ) individuals . ( * * p = 0 . 0025 vs . HC ) . GAPDH served as the loading control .

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B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control.


C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets . Graph indicates HC and DM platelets positive for phosphorylated p53 signal . The y - axis indicates percentage of phosphorylated p53 positive cell from total cells ( * * p = 0 . 0064 vs . HC ) . Representative figure from n = 5 .

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C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets. Graph indicates HC and DM platelets positive for phosphorylated p53 signal. The y-axis indicates percentage of phosphorylated p53 positive cell from total cells (**p=0.0064 vs. HC). Representative figure from n=5.


D Mitochondrial membrane potential ( ΔΨm ) was measured by MitoTracker staining ( 50nM for 15 min ) in HC and DM patient platelets . Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets . The y - axis indicates fold of signal intensity in HC and DM platelet ( * * p = 1 . 1608E - 05 vs HC ) . Signal intensity of each group was converted to fold change compared with HC values . Representative figure from HC ( n = 6 ) and DM ( n = 4 ) .

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D Mitochondrial membrane potential (ΔΨm) was measured by MitoTracker staining (50nM for 15 min) in HC and DM patient platelets. Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets. The y-axis indicates fold of signal intensity in HC and DM platelet (**p=1.1608E-05 vs HC). Signal intensity of each group was converted to fold change compared with HC values. Representative figure from HC (n=6) and DM (n=4).


E ΔΨm and platelet apoptosis were measured by flow cytometry analysis . ΔΨm was detected using TMRM and apoptosis was assessed with Annexin - V ( PS externalization ) . Representative figure from n = 3 .

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E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3.


F Western blot analysis of apoptosis - related pp53 , p53 , cytochrome c , and active caspase 3 in HC and DM platelets . Tubulin was used as the loading control . Representative figure from n = 3 .

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F Western blot analysis of apoptosis-related pp53, p53, cytochrome c, and active caspase 3 in HC and DM platelets. Tubulin was used as the loading control. Representative figure from n=3.


G Western blot analysis showing release of cytochrome c ( Cyto c ) into the cytosol in DM . Representative figure from n = 3 .

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G Western blot analysis showing release of cytochrome c (Cyto c) into the cytosol in DM. Representative figure from n=3.


H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets ( DM ) compared to a typical healthy mitochondria ( HC ) . Arrows indicate disrupted outer membranes . I Quantification of mitochondria in HC ( n = 33 ) and DM ( n = 17 ) patient platelets . The y - axis indicates average number of mitochondria in HC and DM single platelet EM view ( * * p = 5 . 50811E - 05 vs . HC ) . J Quantification of healthy and damaged mitochondria in HC and DM platelets .

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H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).J Quantification of healthy and damaged mitochondria in HC and DM platelets.


A Western blot analysis of autophagy markers , Beclin1 , ATG3 , ATG7 , ATG12 - 5 , and LC3I / II , and the mitophagy - related proteins Parkin and PINK1 in HC ( # 1 - 3 ) and DM platelets ( # 1 - 8 ) . B Quantification of autophagy markers and mitophagy - related proteins from HC ( # 1 - 5 ) and DM ( # 1 - 18 ) individuals . ( Beclin1 ; * * p = 0 . 0003 , ATG12 ; * p = 0 . 0388 , ATG3 ; * p = 0 . 0213 , ATG7 ; p = 0 . 4051 , LC3 ; * p = 0 . 0298 , PINK1 ; * p = 0 . 0155 , Parkin ; * p = 0 . 0103 vs . HC ) . GAPDH served as a loading control .

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A Western blot analysis of autophagy markers, Beclin1, ATG3, ATG7, ATG12-5, and LC3I/II, and the mitophagy-related proteins Parkin and PINK1 in HC (#1-3) and DM platelets (#1-8).B Quantification of autophagy markers and mitophagy-related proteins from HC (#1-5) and DM (#1-18) individuals. (Beclin1; **p=0.0003, ATG12; *p=0.0388, ATG3; *p=0.0213, ATG7; p=0.4051, LC3; *p=0.0298, PINK1; *p=0.0155, Parkin; *p=0.0103 vs. HC). GAPDH served as a loading control.


C The three panels represent autophagy stages found in DM platelets . Autophagosome structures containing / enclosing organelles are indicated by the black box . Shown for each panel is an enlargement of the autophagosome ( DM1 and 2 ) or the autolysosome ( DM3 ) . Dashed structures are an outline of the autophagosome membranes .

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C The three panels represent autophagy stages found in DM platelets. Autophagosome structures containing/enclosing organelles are indicated by the black box. Shown for each panel is an enlargement of the autophagosome (DM1 and 2) or the autolysosome (DM3). Dashed structures are an outline of the autophagosome membranes.


D LC3 immuno - EM analysis of HC and DM platelets . Arrows indicate immunogold - labeled LC3 clusters . No clusters were found in HC platelets . Representative areas of clusters of gold labeling in DM patients ( DM1 - 3 ) are presented . Shown in the insets are enlargements of the gold clusters adjacent to mitochondria - like structures .

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D LC3 immuno-EM analysis of HC and DM platelets. Arrows indicate immunogold-labeled LC3 clusters. No clusters were found in HC platelets. Representative areas of clusters of gold labeling in DM patients (DM1-3) are presented. Shown in the insets are enlargements of the gold clusters adjacent to mitochondria-like structures.


E Double staining for MitoTracker ( mitochondria membrane potential ) ( 50nM for 15 min ) and LC3 ( autophagy ) in HC and DM platelets . Boxed platelets are enlarged in Panel F . F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3 . G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV ( mitochondria ) and LC3 ( autophagy marker ) . The y - axis indicates percentage of LC3 positive cells in total cells ( * * p = 0 . 006 vs . HC ) .

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E Double staining for MitoTracker (mitochondria membrane potential) (50nM for 15 min) and LC3 (autophagy) in HC and DM platelets. Boxed platelets are enlarged in Panel F.F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3.G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV (mitochondria) and LC3 (autophagy marker). The y-axis indicates percentage of LC3 positive cells in total cells (**p=0.006 vs. HC).


A Representative western blot analysis of recognized autophagy signaling molecules ( pJNK , JNK , pAKT , and AKT , pmTOR , and mTOR ) in HC ( # 1 - 3 ) and DM ( # 1 - 8 ) patient platelets . Quantification analysis of autophagy upstream signaling molecules on HC ( # 1 - 5 ) and DM ( # 1 - 18 ) individuals . ( pAKT / AKT ; p = 0 . 0747 , pJNK / JNK ; * p = 0 . 001 , pmTOR / mTOR ; p = 0 . 3356 vs . HC ) . GAPDH was used as the loading control .

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A Representative western blot analysis of recognized autophagy signaling molecules (pJNK, JNK, pAKT, and AKT, pmTOR, and mTOR) in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis of autophagy upstream signaling molecules on HC (#1-5) and DM (#1-18) individuals. (pAKT/AKT; p=0.0747, pJNK/JNK; *p=0.001, pmTOR/mTOR; p=0.3356 vs. HC). GAPDH was used as the loading control.


B Representative western blot analysis using HC and HG platelets with or without H2O2 . And assessing for LC3I / II . GAPDH was used as the loading control .

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B Representative western blot analysis using HC and HG platelets with or without H2O2. And assessing for LC3I/II. GAPDH was used as the loading control.


C Platelet suspensions were incubated in HG for 2 hours and with H2O2 ( 1mM ) . ROS levels were evaluated using a detection kit ( Enzo Life Science ) for 60 mins at 37 ºC .

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C Platelet suspensions were incubated in HG for 2 hours and with H2O2 (1mM). ROS levels were evaluated using a detection kit (Enzo Life Science) for 60 mins at 37 ºC.


D Western blot analysis of p53 , JNK and LC3I / II in HC platelets treated with H2O2 ( 1mM for 1 hour ) alone or with NAC ( 100 μM for 30 min ) . GAPDH was used as the loading control .

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D Western blot analysis of p53, JNK and LC3I/II in HC platelets treated with H2O2 (1mM for 1 hour) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control.


E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker , CoxIV , and LC3 in HC or H2O2 . The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2 - treated platelets .

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E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker, CoxIV, and LC3 in HC or H2O2. The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2-treated platelets.


F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 ( JNK inhibitor ) at 3 concentrations ( 1 , 5 and 10 µM ) to assess for inhibition of autophagy . This Western blot is representative of three independent experiments . Quantification of the three independent experiments is provided at right . ( pJNK / JNK in H2O2 / SP600125 ; * * p = 0 . 0286 vs . H2O2 group , LC3II in H2O2 ; * p = 0 . 0241 vs . HC group , LC3II in H2O2 / SP600125 ; * * p = 0 . 0044 vs . H2O2 group , n = 3 for each group ) .

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F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 (JNK inhibitor) at 3 concentrations (1, 5 and 10 µM) to assess for inhibition of autophagy. This Western blot is representative of three independent experiments. Quantification of the three independent experiments is provided at right. (pJNK/JNK in H2O2/SP600125; **p=0.0286 vs. H2O2 group, LC3II in H2O2; *p=0.0241 vs. HC group, LC3II in H2O2/SP600125; **p=0.0044 vs. H2O2 group, n=3 for each group).


G Triple staining of CoxIV , LC3 , and LAMP1 in HC and H2O2 with / without SP600125 - treated platelets using confocal microscopy . Arrow indicates localization of LC3 and LAMP1 . Graph indicates colocalization between LC3 , LAMP1 and CoxIV signal . The y - axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area . Signal intensity of each group was converted to fold and compared with HC values ( H2O2 ; * p = 0 . 023 vs . HC , H2O2 / SP600125 ; * p = 0 . 04 vs . H2O2 group ) .

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G Triple staining of CoxIV, LC3, and LAMP1 in HC and H2O2 with/without SP600125-treated platelets using confocal microscopy. Arrow indicates localization of LC3 and LAMP1. Graph indicates colocalization between LC3, LAMP1 and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area. Signal intensity of each group was converted to fold and compared with HC values (H2O2;*p=0.023 vs. HC, H2O2/SP600125;*p=0.04 vs. H2O2 group).


A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53 , p53 , and LAMP1 in HC platelets . Platelets were treated with high glucose ( 25mM ) , with or without CCCP ( 10µM ) . This Western blot is representative of three independent experiments . B Quantification analysis of phosphorylated p53 and LAMP1 ( HG ; * p = 0 . 018 vs DMSO group , HG / CCCP ; * * p = 0 . 001 vs HG group , n = 4 for each group ) .

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A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53, p53, and LAMP1 in HC platelets. Platelets were treated with high glucose (25mM), with or without CCCP (10µM). This Western blot is representative of three independent experiments.B Quantification analysis of phosphorylated p53 and LAMP1 (HG; *p=0.018 vs DMSO group, HG/CCCP; **p=0.001 vs HG group, n=4 for each group).


C The recognized autophagy inhibitor ( 2mM 3 - Methyadenine ; 3MA for 1h ) was used to treat DM platelets compared to HC assessing for phosphorylated p53 , p53 , and LC3I / IID Quantification analysis of phosphorylated p53 / p53 was performed . Each value indicated the average band density from a total of three independent samples ( DM / 3MA ; * p = 0 . 026 vs . DM group ) .

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C The recognized autophagy inhibitor (2mM 3-Methyadenine; 3MA for 1h) was used to treat DM platelets compared to HC assessing for phosphorylated p53, p53, and LC3I/IID Quantification analysis of phosphorylated p53/p53 was performed. Each value indicated the average band density from a total of three independent samples (DM/3MA; *p=0.026 vs. DM group).


E Western blot analysis of PLPs transfected with control or ATG3 siRNA , with or without treatment of 1 mM H2O2 . Each lane displayed a different transfected group . F Quantification analysis of ATG3 , phosphorylated p53 ( ser15 ) , p53 , and GAPDH in each group . ( ATG3 in ATG3 siRNA ; * * p = 0 . 0003 vs control group , pp53 in ATG3 siRNA / H2O2 ; * p = 0 . 0145 vs . H2O2 group ) .

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E Western blot analysis of PLPs transfected with control or ATG3 siRNA, with or without treatment of 1 mM H2O2. Each lane displayed a different transfected group.F Quantification analysis of ATG3, phosphorylated p53 (ser15), p53, and GAPDH in each group. (ATG3 in ATG3 siRNA; **p=0.0003 vs control group, pp53 in ATG3 siRNA/ H2O2; *p=0.0145 vs. H2O2 group).


A Western blot analysis of phosphorylated p53 ( ser15 ) , p53 , LC3I / II , and GAPDH in WT mouse platelets . Mouse platelets were treated high glucose ( 25mM ) with or without CCCP ( 10µM ) . B Quantification analysis of phosphorylated p53 ( ser15 ) and LC3 II ( pp53 in HG ; * * p = 0 . 009 vs WT group , pp53 in HG / CCCP ; * p = 0 . 022 vs HG group , LC3 II in HG / CCCP ; * p = 0 . 028 vs HG group , n = 3 for each group ) .

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A Western blot analysis of phosphorylated p53 (ser15), p53, LC3I/II, and GAPDH in WT mouse platelets. Mouse platelets were treated high glucose (25mM) with or without CCCP (10µM).B Quantification analysis of phosphorylated p53 (ser15) and LC3 II (pp53 in HG; **p=0.009 vs WT group, pp53 in HG/CCCP ; *p=0.022 vs HG group, LC3 II in HG/CCCP ; *p=0.028 vs HG group, n=3 for each group).


C ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT _ H2O2 or WT _ H2O2 / 3MA in mouse platelets . ΔΨm was detected using TMRM and apoptosis levels were assessed with Annexin - V ( PS externalization ) . Representative of n = 3 . D Graph indicates the percentage of TMRM positive cells in total cell population ( H2O2 ; * * p = 0 . 002 vs . not treated group , H2O2 / 3MA ; * * p = 0 . 008 vs . not treated group , NS means no significance , n = 3 ) . E Graph indicates the percentage of Annexin V positive cell in total cell population ( H2O2 ; NS , p = 0 . 058 vs . not treated group , H2O2 / 3MA ; * * p = 0 . 004 vs . not treated group , * * p = 0 . 008 vs . H2O2 group , NS means no significance , n = 3 ) .

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C ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 or WT_ H2O2/3MA in mouse platelets. ΔΨm was detected using TMRM and apoptosis levels were assessed with Annexin-V (PS externalization). Representative of n=3.D Graph indicates the percentage of TMRM positive cells in total cell population (H2O2; **p=0.002 vs. not treated group, H2O2/3MA; **p=0.008 vs. not treated group, NS means no significance, n=3).E Graph indicates the percentage of Annexin V positive cell in total cell population (H2O2; NS, p=0.058 vs. not treated group, H2O2/3MA; **p=0.004 vs. not treated group, **p=0.008 vs. H2O2 group, NS means no significance, n=3).


F Representative western blot analysis of LC3 I / II in analysis in WT or WT / H2O2 or WT / H2O2 / 3MA in mouse platelets ( n = 3 ) .

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F Representative western blot analysis of LC3 I/II in analysis in WT or WT/H2O2 or WT/H2O2/3MA in mouse platelets (n=3).


G Representative western blot analysis of phosphorylated p53 , and Parkin in Parkin - / - mice compared to WT . Quantification analysis of phosphorylated p53 and Parkin ( Parkin ; * * p = 0 . 0001 vs . WT , pp53 ; * p = 0 . 019 vs . WT , n = 4 ) .

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G Representative western blot analysis of phosphorylated p53, and Parkin in Parkin -/- mice compared to WT. Quantification analysis of phosphorylated p53 and Parkin (Parkin; **p=0.0001 vs. WT, pp53; *p=0.019 vs. WT, n=4).


H ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT _ H2O2 and Parkin - / - or Parkin - / - _ H2O2 mouse platelets . ΔΨm was detected using TMRM ( WT _ H2O2 ; * * p = 0 . 002 vs . WT group , Parkin - / - _ H2O2 ; * * p = 1 . 61525E - 06 vs . Parkin - / - group , n = 3 ) I Apoptosis level was assessed with Annexin - V ( PS externalization ) . Graph indicates the percentage of Annexin V positive cell in total cell population ( Parkin - / - : * p = 0 . 010 vs . WT group , Parkin - / - _ H2O2 ; * * p = 0 . 008 vs . WT _ H2O2 ; group , Parkin - / - _ H2O2 ; * * p = 0 . 001 vs . Parkin - / - group , NS means no significance n = 3 )

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H ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 and Parkin -/- or Parkin -/-_H2O2 mouse platelets. ΔΨm was detected using TMRM (WT_ H2O2; **p= 0.002 vs. WT group, Parkin-/-_ H2O2; **p= 1.61525E-06 vs. Parkin-/- group, n=3)I Apoptosis level was assessed with Annexin-V (PS externalization). Graph indicates the percentage of Annexin V positive cell in total cell population (Parkin-/- : *p=0.010 vs. WT group, Parkin-/-_ H2O2 ;**p= 0.008 vs. WT_ H2O2; group, Parkin-/-_ H2O2; **p=0.001 vs. Parkin-/- group, NS means no significance n=3)


J Western blot analysis in WT and Parkin - / - demonstrating the inability to induce LC3 despite the addition of H2O2 . Shown graphically are the LC3II to LC3I ratios ( WT _ H2O2 ; * p = 0 . 028 vs . WT group , n = 3 )

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J Western blot analysis in WT and Parkin -/- demonstrating the inability to induce LC3 despite the addition of H2O2. Shown graphically are the LC3II to LC3I ratios (WT_ H2O2;*p=0.028 vs. WT group, n=3)


A Representative western blot analysis of pp53 in WT ( n = 6 ) , DM ( n = 6 ) and PINK1 - / - DM mice ( n = 3 ) . GAPDH served as the loading control ( DM ; * * p = 0 . 006 vs . WT group , PINK1 - / - DM ; * * p = 0 . 004 vs . DM group ) .

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A Representative western blot analysis of pp53 in WT (n=6), DM (n=6) and PINK1-/-DM mice (n=3). GAPDH served as the loading control (DM ; **p=0.006 vs. WT group, PINK1-/-DM; **p=0.004 vs. DM group).


B Measurement of p - selectin translocation to the cell membrane by flow cytometry analysis in WT ( n = 3 ) , DM ( n = 3 ) and PINK1 - / - DM ( n = 3 ) miceplatelets . Graphs indicate mean fluorescence intensity ( MFI ) in each group . ( * p < 0 . 05 vs . WT or DM ) .

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B Measurement of p-selectin translocation to the cell membrane by flow cytometry analysis in WT (n=3), DM (n=3) and PINK1 -/- DM (n=3) miceplatelets. Graphs indicate mean fluorescence intensity (MFI) in each group. (*p<0.05 vs. WT or DM).


C Quantification of vessel occlusion time after carotid arterial flow measurement ( WT and DM ; and PINK1 - / - : * * p < 0 . 01 vs . WT or DM ) .

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C Quantification of vessel occlusion time after carotid arterial flow measurement (WT and DM; and PINK1 -/- : **p<0.01 vs. WT or DM).


D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury , indicative of occlusive thrombosis . Flow was plotted as percentage of baseline flow before injury ( DM : n = 3 , PINK1 - / - DM ; n = 3 ) .

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D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury, indicative of occlusive thrombosis. Flow was plotted as percentage of baseline flow before injury (DM: n=3, PINK1-/- DM; n=3).


A , SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 ( SB ) or LGK974 ( LGK ) for 16 h . Total cell extracts were analyzed by WB . Protein quantification was analyzed by densitometry of three different experiments and represented B , SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB . Quantification of the WBs from three biological replicates are presented in the right panel . Values correspond to fold variation in shRor2 versus shCtl cells ( mean ± SD ) .

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A, SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 (SB) or LGK974 (LGK) for 16 h. Total cell extracts were analyzed by WB. Protein quantification was analyzed by densitometry of three different experiments and represented B, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs from three biological replicates are presented in the right panel. Values correspond to fold variation in shRor2 versus shCtl cells (mean ± SD).


C , RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT - PCR . Values correspond to fold variation in shRor2 versus shCtl cells . Results are the mean ± SD of at least three biological replicates performed in triplicate .

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C, RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT-PCR. Values correspond to fold variation in shRor2 versus shCtl cells. Results are the mean ± SD of at least three biological replicates performed in triplicate.


D , SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB . Quantification of the WBs are presented in Appendix Fig S3D as in B .

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D, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs are presented in Appendix Fig S3D as in B.


E , HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a - producing cells for 16 h and expression of the indicated genes assessed by qRT - PCR ( E ) ( mean ± SD of three biological replicates performed in triplicate in E ) .

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E, HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a-producing cells for 16 h and expression of the indicated genes assessed by qRT-PCR (E) (mean ± SD of three biological replicates performed in triplicate in E).


G , WB analysis of HT29 M6 and SW480 overexpressing Snail1 . Quantification is presented in Appendix Fig S5I .

G, WB analysis of HT29 M6 and SW480 overexpressing Snail1. Quantification is presented in Appendix Fig S5I.


I , SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by qRT - PCR ( I , mean ± SD of three biological replicates performed in triplicate ) .

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I, SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by qRT-PCR (I, mean ± SD of three biological replicates performed in triplicate).


A , Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells . The quantification is presented in the right panel ( mean ± SD or three biological replicates ) .

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A, Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells. The quantification is presented in the right panel (mean ± SD or three biological replicates).


D , MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D ) for the indicated times . TGFβ receptor inhibitor SB505124 ( SB , 5 μM ) was added when indicated in D . Cells were lysed and proteins analyzed by WB . Quantification of the protein levels from D is represented at the lower panel ( mean ± SD of three biological replicates )

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D, MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D) for the indicated times. TGFβ receptor inhibitor SB505124 (SB, 5 μM) was added when indicated in D. Cells were lysed and proteins analyzed by WB. Quantification of the protein levels from D is represented at the lower panel (mean ± SD of three biological replicates)


E , GFP - labelled MSC were seeded in Matrigel - coated transwells with SW620 and HCT116 cells , and supplemented with SB when indicated . After 48 h non - invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed . GFP - labelled cells were counted in five different fields per filter by ImageJ software and represented ( bottom ) . A representative picture of SW620 cells in each condition is shown ( top ) . The bar corresponds to 500 μm . The figure shows the mean ± SD of three experiments .

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E, GFP-labelled MSC were seeded in Matrigel-coated transwells with SW620 and HCT116 cells, and supplemented with SB when indicated. After 48 h non-invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed. GFP-labelled cells were counted in five different fields per filter by ImageJ software and represented (bottom). A representative picture of SW620 cells in each condition is shown (top). The bar corresponds to 500 μm. The figure shows the mean ± SD of three experiments.


C , Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft - agar colony formation ( 21 days ) ( left ) . Representative micrographs of SW620 cells are shown in Appendix Fig S7B . Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments ( right ) .

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C, Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft-agar colony formation (21 days) (left). Representative micrographs of SW620 cells are shown in Appendix Fig S7B. Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments (right).


Cells were seeded for tumor - sphere formation in tumor organoid complete medium . In E , SW620 cells were supplemented with LGK ( 10 μM ) , starting the day of seeding . After four days , the wells were stained ( top ) and the number of colonies counted and represented ( bottom ) ( mean ± SD of three biological replicates ) . A more detailed micrograph of the colonies is shown in Appendix Fig S7D .

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Cells were seeded for tumor-sphere formation in tumor organoid complete medium. In E, SW620 cells were supplemented with LGK (10 μM), starting the day of seeding. After four days, the wells were stained (top) and the number of colonies counted and represented (bottom) (mean ± SD of three biological replicates). A more detailed micrograph of the colonies is shown in Appendix Fig S7D.


F , RNA was isolated from SW620 control , stably - depleted of Ror2 , treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5 , ASCL2 , SMOC2 and the differentiation marker KRT20 was assessed by qRT - PCR . The figure shows the mean ± SD of at least three biological replicates ( performed in triplicate ) represented with respect to the corresponding control ( dashed line ) .

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F, RNA was isolated from SW620 control, stably-depleted of Ror2, treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5, ASCL2, SMOC2 and the differentiation marker KRT20 was assessed by qRT-PCR. The figure shows the mean ± SD of at least three biological replicates (performed in triplicate) represented with respect to the corresponding control (dashed line).


B , C , Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin - embedded tumors obtained when tumors were initially detected ( days 18 or 22 ) . Staining with an anti - human antibody is also enclosed . The scale bar corresponds to 10 μm ( B ) . Fibronectin and Vimentin staining were quantified as described in Methods ( six tumors , three areas per tumor ) and represented in C ( mean ± SD ) .

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B, C, Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin-embedded tumors obtained when tumors were initially detected (days 18 or 22). Staining with an anti-human antibody is also enclosed. The scale bar corresponds to 10 μm (B). Fibronectin and Vimentin staining were quantified as described in Methods (six tumors, three areas per tumor) and represented in C (mean ± SD).