Matrix pH was measured with Sypher3smito in WT HeLa cells transiently expressing TMBIM5 or TMBIM5D294K D325K for 24 h . The ratio of absorbance at 488 nm and 405 nm is shown in arbitrary units ( a . u . ) . Each measurement was performed in at least 20 cells from 3 different preparations . Representative immunoblot of WT HeLa cells transfected with TMBIM5 or TMBIM5D294K D325K for 24 h ( right panel ) . Mean ± SD ; two - tailed t - test . A p - value of < 0 . 05 was considered statistically 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

Matrix pH was measured with Sypher3smito in WT HeLa cells transiently expressing TMBIM5 or TMBIM5D294K D325K for 24 h. The ratio of absorbance at 488 nm and 405 nm is shown in arbitrary units (a.u.). Each measurement was performed in at least 20 cells from 3 different preparations. Representative immunoblot of WT HeLa cells transfected with TMBIM5 or TMBIM5D294K D325K for 24 h (right panel). Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Liposome assay . Recombinant TMBIM5 was reconstituted in liposomes containing 100 µM Fura - 2 . CaCl2 was added later at the concentration of 100 µM . Rmax was obtained at the end of the experiment after the addition of ionomycin ( 10 µM ) . Data are expressed as mean of R / Rmax , where R is the peak after CaCl2 addition . Where indicated GdCl3 ( 100 µM ) was added 10 min before the experiment . n = 3 independent experiments , shown as black , white and grey dots . Mean ± SD ; two - tailed t - test . A p - value of < 0 . 05 was considered statistically significant . Liposome assay ( as in E ) . Slope was calculated in the 100 s after 100 µM CaCl2 addition as expressed as mean ± SD . n = 3 independent experiments , shown as black , white and grey dots . Two - tailed t - test . A p - value of < 0 . 05 was considered statistically 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 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

Liposome assay. Recombinant TMBIM5 was reconstituted in liposomes containing 100 µM Fura-2. CaCl2 was added later at the concentration of 100 µM. Rmax was obtained at the end of the experiment after the addition of ionomycin (10 µM). Data are expressed as mean of R/Rmax, where R is the peak after CaCl2 addition. Where indicated GdCl3 (100 µM) was added 10 min before the experiment. n=3 independent experiments, shown as black, white and grey dots. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant. Liposome assay (as in E). Slope was calculated in the 100 s after 100 µM CaCl2 addition as expressed as mean ±SD. n=3 independent experiments, shown as black, white and grey dots. Two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Alterations in mitochondrial proteome in WT and TMBIM5 - / - HeLa cells upon depletion of AFG3L2 . WT and TMBIM5 - / - HeLa cells were transfected with scrambled siRNA ( control ) or siRNA targeting AFG3L2 for 48 h . Proteins , which were downregulated in the absence of TMBIM5 and whose steady state levels was altered upon depletion of AFG3L2 , were identified using a two - way ANOVA . An interaction p - value of 0 . 01 was used as a cutoff . Log2 - transformed LFQ intensities of MitoCarta 3 . 0 proteins were Z - Score normalized and visualized in a hierarchical cluster analysis ( Euclidean distance , complete method ) . The dendrogram is omitted . Proteins accumulating upon depletion of AFG3L2 ( cluster 1 , C1 ) and proteins whose steady state levels are decreased upon depletion of AFG3L2 ( cluster 2 , C2 ) are shown .

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

Alterations in mitochondrial proteome in WT and TMBIM5-/- HeLa cells upon depletion of AFG3L2. WT and TMBIM5-/- HeLa cells were transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Proteins, which were downregulated in the absence of TMBIM5 and whose steady state levels was altered upon depletion of AFG3L2, were identified using a two-way ANOVA. An interaction p-value of 0.01 was used as a cutoff. Log2-transformed LFQ intensities of MitoCarta 3.0 proteins were Z-Score normalized and visualized in a hierarchical cluster analysis (Euclidean distance, complete method). The dendrogram is omitted. Proteins accumulating upon depletion of AFG3L2 (cluster 1, C1) and proteins whose steady state levels are decreased upon depletion of AFG3L2 (cluster 2, C2) are shown.


Representative immunoblot of TMBIM5 - / - HeLa cells transfected with scrambled siRNA ( control ) or siRNA targeting AFG3L2 for 48 h . Samples were treated with the protein synthesis inhibitor cycloheximide ( CHX ; 10 μg / ml ) and collected at the indicated time points ( n = 3 independent 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 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

Representative immunoblot of TMBIM5-/- HeLa cells transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Samples were treated with the protein synthesis inhibitor cycloheximide (CHX; 10 μg/ml) and collected at the indicated time points (n=3 independent experiments).


Volcano plot of mitochondrial protein ( MitoCarta 3 . 0 ) changes in WT HeLa cells treated with oligomycin ( 10 μM for 16 h ) when compared to untreated cells . Before MitoCarta 3 . 0 - filtering , significantly altered proteins were identified using a two - sided t - test followed by permutation - based FDR estimation to 0 . 05 and are colored in blue ( n = 5 independent experiments ) .

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

Volcano plot of mitochondrial protein (MitoCarta 3.0) changes in WT HeLa cells treated with oligomycin (10 μM for 16 h) when compared to untreated cells. Before MitoCarta 3.0-filtering, significantly altered proteins were identified using a two-sided t-test followed by permutation-based FDR estimation to 0.05 and are colored in blue (n=5 independent experiments).


Representative immunoblot of WT HeLa cells treated with the indicated drugs for 16 h . Antimycin A ( AA ; 10 μM ) , piericidin A ( 10 μM ) and oligomycin ( 10 μM ) .

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

Representative immunoblot of WT HeLa cells treated with the indicated drugs for 16 h. Antimycin A (AA; 10 μM), piericidin A (10 μM) and oligomycin (10 μM).


Representative immunoblot of WT HeLa cells transfected with scrambled siRNA ( control ) and siRNA targeting AFG3L2 for 48 h , treated when indicated with oligomycin ( 10 μM ) for 16 h . Quantification of TMBIM5 levels is shown on the right panel . TMBIM5 levels in untreated WT cells was set to 1 ( n = 3 independent experiments ; mean ± SD ) .

O 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

Representative immunoblot of WT HeLa cells transfected with scrambled siRNA (control) and siRNA targeting AFG3L2 for 48 h, treated when indicated with oligomycin (10 μM) for 16 h. Quantification of TMBIM5 levels is shown on the right panel. TMBIM5 levels in untreated WT cells was set to 1 (n=3 independent experiments; mean ±SD).


TMBIM5 abundance determined by mass spectrometry in WT HeLa cells treated with oligomycin ( 10 µM ; 16 h ) and depleted of AFG3L2 when indicated . n = 5 independent experiments . P - value was calculated using a two - sided unpaired t - test . The central band of each box is the median value , and the box defines the 25th ( lower ) and 75th ( higher ) quantile . The whiskers represent the minimum and maximum value in the data excluding outliers ( greater than 1 . 5 * inter quantile range distance to median - no outliers found here ) .

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

TMBIM5 abundance determined by mass spectrometry in WT HeLa cells treated with oligomycin (10 µM; 16 h) and depleted of AFG3L2 when indicated. n=5 independent experiments. P-value was calculated using a two-sided unpaired t-test. The central band of each box is the median value, and the box defines the 25th (lower) and 75th (higher) quantile. The whiskers represent the minimum and maximum value in the data excluding outliers (greater than 1.5 * inter quantile range distance to median - no outliers found here).


Zoom in to selected clusters of hierarchical clustering of Z - Score normalized log2 LFQ intensities . Proteins that were significantly ( FDR < 0 . 05 ) accumulated between HeLa WT and TMBIM5 - / - cells after oligomycin treatment ( 10 µM ) for 16 h were clustered using Euclidean distance and the complete method on protein features . The figure shows identified cluster C4 and C5 with gene names and MitoCarta 3 . 0 localization annotations . The row dendrogram is omitted .

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

Zoom in to selected clusters of hierarchical clustering of Z-Score normalized log2 LFQ intensities. Proteins that were significantly (FDR<0.05) accumulated between HeLa WT and TMBIM5-/- cells after oligomycin treatment (10 µM) for 16 h were clustered using Euclidean distance and the complete method on protein features. The figure shows identified cluster C4 and C5 with gene names and MitoCarta 3.0 localization annotations. The row dendrogram is omitted.


A . AICD was induced in hPBT cells as described in Materials and methods . Apoptotic cells were detected at indicated times after AICD induction by flow cytometry as AnnexinV / PI double positive cells and the ratio between AICD and Ctrl values obtained are shown . Data represent mean ± SE of six independent 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

A. AICD was induced in hPBT cells as described in Materials and methods. Apoptotic cells were detected at indicated times after AICD induction by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown. Data represent mean ± SE of six independent experiments.


B . AICD was induced in Jurkat cells as described in Materials and methods . Apoptotic cells were detected as in ( A ) . Data represent mean ± SE of five independent 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

B. AICD was induced in Jurkat cells as described in Materials and methods. Apoptotic cells were detected as in (A). Data represent mean ± SE of five independent experiments.


C . hPBT cells were transfected with mtYFP and after 24h AICD was induced . Representative reconstructions of confocal z - stacks of the mtYFP fluorescence 30 ' after AICD induction is shown . Scale bar , 5μm .

O O O O O O O O O O O O O O O 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. hPBT cells were transfected with mtYFP and after 24h AICD was induced. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 30' after AICD induction is shown. Scale bar, 5μm.


D . Morphometric analysis of mitochondrial shape of cells treated as in ( B ) . Data represent mean ± SE of five independent experiments ( n = 30 cells per condition in each experiment ) .

O O O O O O O O O O O O O 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. Morphometric analysis of mitochondrial shape of cells treated as in (B). Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


E . Jurkat cells were transfected with mtYFP , 24h after AICD induction cells were fixed . Representative reconstructions of confocal z - stacks of the mtYFP fluorescence 24h after AICD induction are shown . Scale bar , 5μm . F . Morphometric analysis of mitochondrial shape in cells treated as in ( E ) is shown . Data represent mean ± SE of five independent experiments ( n = 30 cells per condition in each experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Jurkat cells were transfected with mtYFP, 24h after AICD induction cells were fixed. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.F. Morphometric analysis of mitochondrial shape in cells treated as in (E) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


G . Representative electron micrographs of hPBT cells treated as indicated . 30min after AICD induction cells were fixed and processed for electron microscopy . Scale bar , 2μm . H . 24h after AICD induction Jurkat cells were fixed and processed for electron microscopy . Representative electron micrographs of cells treated as indicated are shown . In EM images white arrowheads point to autophagic vesicles ( late autophagosomes or most likely autophagolysosomes , Aφ ) ; black arrows point to mitochondria ( mito ) with remodelled cristae , specifically touching a representative disorganized crista . Scale bar , 2μm .

G. Representative electron micrographs of hPBT cells treated as indicated. 30min after AICD induction cells were fixed and processed for electron microscopy. Scale bar, 2μm.H. 24h after AICD induction Jurkat cells were fixed and processed for electron microscopy. Representative electron micrographs of cells treated as indicated are shown. In EM images white arrowheads point to autophagic vesicles (late autophagosomes or most likely autophagolysosomes, Aφ); black arrows point to mitochondria (mito) with remodelled cristae, specifically touching a representative disorganized crista. Scale bar, 2μm.


I . Representative confocal microscopy images of hPBT cells transfected with mtYFP , fixed 30 ' after AICD induction and immunostained with an anti - cytochrome c antibody ( red ) . J . Cytochrome c localization index was calculated from 30 randomly selected cells treated as in ( I ) . Data represent mean ± SE of three independent experiments . K . Representative confocal images of Jurkat cells transfected with mtYFP , fixed at 32h after AICD induction and immunostained with an anti - cytochrome c antibody . L . Cytochrome c localization index was calculated from 30 randomly selected images per condition . Data represent mean ± SE of five independent experiments .

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I. Representative confocal microscopy images of hPBT cells transfected with mtYFP, fixed 30' after AICD induction and immunostained with an anti-cytochrome c antibody (red).J. Cytochrome c localization index was calculated from 30 randomly selected cells treated as in (I). Data represent mean ± SE of three independent experiments.K. Representative confocal images of Jurkat cells transfected with mtYFP, fixed at 32h after AICD induction and immunostained with an anti-cytochrome c antibody.L. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of five independent experiments.


A . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGEand immunoblotted with the indicated antibodies . WB are representative of at least three independent 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

A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGEand immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.


B . Jurkat cells were transfected with pE - GFP and the indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as the percentage of GFP - positive , annexin - V - PE - negative cells . Data represent mean ± SE of five independent 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

B. Jurkat cells were transfected with pE-GFP and the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of five independent experiments.


C . Jurkat cells were transfected as indicated . Representative reconstructions of confocal z - stacks of the mtYFP fluorescence 24h after AICD induction are shown . Scale bar , 5μm . D . Morphometric analysis of mitochondrial shape of cells transfected as in ( C ) is shown . Data represent mean ± SE of five independent experiments ( n = 30 cells per condition in each experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Jurkat cells were transfected as indicated. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.D. Morphometric analysis of mitochondrial shape of cells transfected as in (C) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


E . AICD was induced in Jurkat cells over expressing pE - GFP and the indicated plasmids and sorted for GFP positive fluorescence . 24h after AICD induction cells were fixed and processed for electron microscopy . Representative electron micrographs of randomly selected mitochondria from cells treated as indicated are shown . White arrow points to tight cristae structure , black arrow points to remodelled cristae and to a swollen cristae junction . Scale bar , 0 . 2μm . F . Morphometric analysis of cristae width in cells transfected as in ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. AICD was induced in Jurkat cells over expressing pE-GFP and the indicated plasmids and sorted for GFP positive fluorescence. 24h after AICD induction cells were fixed and processed for electron microscopy. Representative electron micrographs of randomly selected mitochondria from cells treated as indicated are shown. White arrow points to tight cristae structure, black arrow points to remodelled cristae and to a swollen cristae junction. Scale bar, 0.2μm.F. Morphometric analysis of cristae width in cells transfected as in (E).


G . Representative confocal microscopy images of cells transfected as indicated , fixed 32h after AICD induction and immunostained with an anti - cytochrome c antibody ( red ) . H . Cytochrome c localization index was calculated from 30 randomly selected cells ( per condition ) transfected as in ( G ) . Data represent mean ± SE of five independent 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

G. Representative confocal microscopy images of cells transfected as indicated, fixed 32h after AICD induction and immunostained with an anti-cytochrome c antibody (red).H. Cytochrome c localization index was calculated from 30 randomly selected cells (per condition) transfected as in (G). Data represent mean ± SE of five independent experiments.


A . Representative confocal microscopy images of cells transfected GFP - LC3 and Parkin - Cherry , treated as indicated , fixed 24h after AICD induction and immunostained with anti - TOM20 antibody are shown . Scale bar , 5μm .

O O O O O O O O O O O O O O O O O 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 confocal microscopy images of cells transfected GFP-LC3 and Parkin-Cherry, treated as indicated, fixed 24h after AICD induction and immunostained with anti-TOM20 antibody are shown. Scale bar, 5μm.


B . Statistical analysis of Parkin localization in cells subjected to AICD or to 10μM CCCP for the indicated times is shown . Data represent mean ± SE of three independent experiments ( n = 30 cells per condition in each experiment ) .

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

B. Statistical analysis of Parkin localization in cells subjected to AICD or to 10μM CCCP for the indicated times is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).


C . Cells were treated as in ( A ) . Where indicated , lysosomal proteases inhibitors E64D and PepA were added . LC3 - Parkin - mitochondria co - localization was calculated from 30 randomly selected cells per condition . Data represent mean ± SE of three independent experiments . Scale bar ,

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

C. Cells were treated as in (A). Where indicated, lysosomal proteases inhibitors E64D and PepA were added. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected cells per condition. Data represent mean ± SE of three independent experiments. Scale bar, 5μm


D . hPBT cells were transfected with LC3 - cherry and treated as indicated . 30min after AICD induction cells were fixed and stained with TUNEL and DAPI . Representative reconstructions of confocal z - stacks of the LC3 - Cherry fluorescence and single stacks for DAPI are shown . Scale bar , 5μm . E . Statistical analysis of the autophagy in cells treated as in ( D ) is shown . Data represent mean ± SE of three independent experiments ( n = 20 cells per condition in each experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. hPBT cells were transfected with LC3-cherry and treated as indicated. 30min after AICD induction cells were fixed and stained with TUNEL and DAPI. Representative reconstructions of confocal z-stacks of the LC3-Cherry fluorescence and single stacks for DAPI are shown. Scale bar, 5μm.E. Statistical analysis of the autophagy in cells treated as in (D) is shown. Data represent mean ± SE of three independent experiments (n = 20 cells per condition in each experiment).


F . Jurkat cells were transfected with GFP - LC3 and treated as indicated . Representative reconstructions of confocal z - stacks of the GFP - LC3 fluorescence 16h after AICD induction are shown . Scale bar , 5μm . G . Statistical analysis of the autophagic flux in cells treated as in ( F ) is shown . Data represent mean ± SE of three independent experiments ( n = 30 cells per condition in each experiment ) .

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

F. Jurkat cells were transfected with GFP-LC3 and treated as indicated. Representative reconstructions of confocal z-stacks of the GFP-LC3 fluorescence 16h after AICD induction are shown. Scale bar, 5μm.G. Statistical analysis of the autophagic flux in cells treated as in (F) is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).


H . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Where indicated , lysosomal proteases inhibitors E64D and PepA were added . WB are representative of at least three independent 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O

H. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated, lysosomal proteases inhibitors E64D and PepA were added. WB are representative of at least three independent experiments.


A - B . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Wb are representative of at least four independent experiments . Levels of proteins and phosphorylation status of AMPK were quantified and analyzed as shown . In these panels , Time states for " time , hours , after AICD treatment " and indicates at which time upon AICD induction samples have been collected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Wb are representative of at least four independent experiments. Levels of proteins and phosphorylation status of AMPK were quantified and analyzed as shown. In these panels, Time states for "time, hours, after AICD treatment" and indicates at which time upon AICD induction samples have been collected.


C . Jurkat cells were transfected with the indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as the percentage of GFP - positive , annexin - V - PE - negative cells . Data represent mean ± SE of three independent 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

C. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.


D . Protein samples from Jurkat cells over expressing empty vector or AMPKS173A - S485A were immunoblotted for LC3 and LC3 - II / Actin ratio was quantified and analyzed as shown . Data represent mean ± SE of three independent 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

D. Protein samples from Jurkat cells over expressing empty vector or AMPKS173A-S485A were immunoblotted for LC3 and LC3-II/Actin ratio was quantified and analyzed as shown. Data represent mean ± SE of three independent experiments.


E - F . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . WB are representative of at least three independent experiments . Levels of proteins and phosphorylation status of LC3 were quantified and analyzed as shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments. Levels of proteins and phosphorylation status of LC3 were quantified and analyzed as shown.


G . Jurkat cells were transfected with the indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as described above .

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. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as described above.


H - I . Cells where treated as in ( A ) and where indicated 10μM H89 was added . Quantitative analysis of P - AMPK Ser485 - 491 phosphorylation ( H ) and of LC3 - II / LC3 - I ratio ( I ) during AICD are shown .

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

H-I. Cells where treated as in (A) and where indicated 10μM H89 was added. Quantitative analysis of P-AMPK Ser485-491 phosphorylation (H) and of LC3-II/LC3-I ratio (I) during AICD are shown.


J - K . AICD was induced in Jurkat cells ( J ) and in hPBT cells ( K ) in presence of 10μM H89 . At indicated time points ( hours ) after AICD induction , apoptotic cells were detected by flow cytometry as AnnexinV / PI double positive cells and the ratio between AICD and Ctrl values obtained are shown .

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

J-K. AICD was induced in Jurkat cells (J) and in hPBT cells (K) in presence of 10μM H89. At indicated time points (hours) after AICD induction, apoptotic cells were detected by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown.


A . Apoptotic cells were detected at indicated time points after AICD induction in hPBT cells by flow cytometry as AnnexinV / PI double positive cells . Where indicated hPBT cells were pre - incubated with 100nM rapamycin for 24h before AICD induction . Data represent mean ± SE of six independent experiments .

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A. Apoptotic cells were detected at indicated time points after AICD induction in hPBT cells by flow cytometry as AnnexinV/PI double positive cells. Where indicated hPBT cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Data represent mean ± SE of six independent experiments.


B . AICD was induced in Jurkat cells and where indicated 100nM rapamycin was added at time 0h of AICD induction . Apoptotic cell death analysis was carried out as in ( A ) , except that cells were analysed 32h after AICD induction . Data represent mean ± SE of six independent experiments .

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B. AICD was induced in Jurkat cells and where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of six independent experiments.


C . Morphometric analysis of mitochondrial shape 24h after AICD induction . Jurkat cells were transfected with mtYFP 24h before AICD induction and , where indicated , 100nM rapamycin was added . Data represent mean ± SE of four independent experiments ( n = 30 cells per condition in each experiment ) .

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C. Morphometric analysis of mitochondrial shape 24h after AICD induction. Jurkat cells were transfected with mtYFP 24h before AICD induction and, where indicated, 100nM rapamycin was added. Data represent mean ± SE of four independent experiments (n = 30 cells per condition in each experiment).


D . Representative electron micrographs of cells subjected to AICD untreated ( upper panel ) or treated with 100nM rapamycin ( lower panel ) . 24h after AICD induction cells were fixed and processed for electron microscopy , Scale bar , 2μm .

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D. Representative electron micrographs of cells subjected to AICD untreated (upper panel) or treated with 100nM rapamycin (lower panel). 24h after AICD induction cells were fixed and processed for electron microscopy, Scale bar, 2μm.


E . Jurkat cells were transfected with mtYFP and treated as indicated . 32h after AICD induction cells were fixed and immunostained with an anti - cytochrome c antibody . Cytochrome c localization index was calculated from 30 randomly selected images per condition . Data represent mean ± SE of four independent experiments .

E. Jurkat cells were transfected with mtYFP and treated as indicated. 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of four independent experiments.


F . Jurkat cells were transfected with DRP1S637A - GFP , 24h after transfection AICD was induced . 32h after AICD induction viability was determined cytofluorimetrically as the percentage of GFP - positive , annexin - V - PE - negative cells . Data represent mean ± SE of three independent experiments .

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F. Jurkat cells were transfected with DRP1S637A-GFP, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.


G - H . Jurkat cells were co - transfected with mt - RFP and the indicated plasmids , 32h after AICD induction cells were fixed and immunostained with an anti - cytochrome c antibody . Cytochrome c localization index ( G ) and mitochondrial morphology ( H ) were calculated from 30 randomly selected images . Data represent mean ± SE of three independent experiments .

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G-H. Jurkat cells were co-transfected with mt-RFP and the indicated plasmids, 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index (G) and mitochondrial morphology (H) were calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments.


I . Jurkat cells were transfected with DRP1K38A , 24h after transfection AICD was induced . 32h after AICD induction viability was determined cytofluorimetrically as the percentage AnnexinV / PI double negative cells . Data represent mean ± SE of three independent experiments .

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I. Jurkat cells were transfected with DRP1K38A, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage AnnexinV/PI double negative cells. Data represent mean ± SE of three independent experiments.


A . Protein samples ( 20 μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . WB are representative of at least three independent experiments . B - C . Quantitative analysis of the ratio between the protein levels of mitochondrial markers and Actin during AICD is shown . Data represent mean ± SE of three independent experiments .

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A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.B-C. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and Actin during AICD is shown. Data represent mean ± SE of three independent experiments.


D . Jurkat cells were co - transfected with GFP - LC3 and Parkin - Cherry , 32h after AICD induction cells were fixed and immunostained with an anti - TOM20 antibody . Representative images for each condition are presented . Scale bar , 5μm . E . LC3 - Parkin - mitochondria co - localization was calculated from 30 randomly selected images . Data represent mean ± SE of three independent experiments . Data referring to Ctrl and AICD - stimulated cells without rapamycin correspond to the ones presented in Fig . 3C .

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D. Jurkat cells were co-transfected with GFP-LC3 and Parkin-Cherry, 32h after AICD induction cells were fixed and immunostained with an anti-TOM20 antibody. Representative images for each condition are presented. Scale bar, 5μm.E. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments. Data referring to Ctrl and AICD-stimulated cells without rapamycin correspond to the ones presented in Fig.3C.


A . Splenocytes were isolated from Ambragt / + and wt C57 / BL6mice and activated as indicated in materials and methods . At day 7 after activation , AICD was induced with 0 . 1μg / ml of plate - bound anti - mCD3 ( Clone 17A2 ) . Apoptotic cells were detected 12h after AICD induction by flow cytometry as AnnexinV / PI double positive cells .

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A. Splenocytes were isolated from Ambragt/+ and wt C57/BL6mice and activated as indicated in materials and methods. At day 7 after activation, AICD was induced with 0.1μg/ml of plate-bound anti-mCD3 (Clone 17A2). Apoptotic cells were detected 12h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.


B . Splenocytes were isolated from ATG7 fl / fl and wt C57 / BL6mice . Activation of splenocytes and AICD were performed as previously described . At day 5 after activation , cells were infected with retroviral constructs expressing CRE recombinase . Where indicated , cells were pre - incubated with 100nM rapamycin for 24h before AICD induction . Apoptotic cells were detected 2h after AICD induction by flow cytometry as AnnexinV / PI double positive cells .

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B. Splenocytes were isolated from ATG7 fl/fl and wt C57/BL6mice. Activation of splenocytes and AICD were performed as previously described. At day 5 after activation, cells were infected with retroviral constructs expressing CRE recombinase. Where indicated, cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Apoptotic cells were detected 2h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.


C . Jurkat cells were transfected with scramble or ATG7 siRNA 24h before AICD induction . AICD was induced as described in materials and methods . Where indicated 100nM rapamycin was added at time 0h of AICD induction . Apoptotic cell death analysis was carried out as in ( A ) , except that cells were analysed 32h after AICD induction . Data represent mean ± SE of five independent experiments .

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C. Jurkat cells were transfected with scramble or ATG7 siRNA 24h before AICD induction. AICD was induced as described in materials and methods. Where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of five independent experiments.


D . Mitochondrial depolarization upon AICD induction was analysed in cells treated as indicated . Data represent mean ± SE of five independent experiments .

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D. Mitochondrial depolarization upon AICD induction was analysed in cells treated as indicated. Data represent mean ± SE of five independent experiments.


E . Protein samples ( 20μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . WB are representative of three independent experiments . F . Quantitative analysis of the ratio between the protein levels of mitochondrial markers and GRP75 during AICD is shown . Data represent mean ± SE of three independent experiments .

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E. Protein samples (20μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of three independent experiments.F. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and GRP75 during AICD is shown. Data represent mean ± SE of three independent experiments.


A Concentrations of amylase , lipase , CRP , and ANGPTL4 in the sera of patients ( n = 90 ) diagnosed with pancreatitis . The box plots show a typical display consisting of a median value depicted by the line in the center of the box ; an interquartile range ( IQR ; 25th to the 75th percentile ) depicted by the box ; and the maximum ( Q3 + 1 . 5 * IQR ) and minimum ( Q1 - 1 . 5 * IQR ) values depicted by the whisker . Statistical significance of Mann - Whitney U - tests is indicated ( * * P < 0 . 01 and * * * P < 0 . 001 ) . Exact P values are shown in Appendix Table S2 . ANGPTL4 and CRP concentration from pancreatitis patients were determined by sandwich ELISA . The correlation between ANGPTL4 and CRP is depicted with Pearson correlation coefficient ( R ) . Each dot represents the levels of ANGPTL4 and CRP from an individual patient ( n = 90 ) .

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A Concentrations of amylase, lipase, CRP, and ANGPTL4 in the sera of patients (n = 90) diagnosed with pancreatitis. The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (** P < 0.01 and *** P < 0.001). Exact P values are shown in Appendix Table S2. ANGPTL4 and CRP concentration from pancreatitis patients were determined by sandwich ELISA. The correlation between ANGPTL4 and CRP is depicted with Pearson correlation coefficient (R). Each dot represents the levels of ANGPTL4 and CRP from an individual patient (n = 90).


B Pancreatitis and adjacent normal regions ( n = 6 ) were stained by immunofluorescence for amylase and ANGPTL4 . Scale bar represents 30

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B Pancreatitis and adjacent normal regions (n = 6) were stained by immunofluorescence for amylase and ANGPTL4. Scale bar represents 30 µm


C H & E staining , ANGPTL4 and amylase expression by immunofluorescence analysis after the induction of mild ( AP ) and severe acute pancreatitis ( SAP ) for 2 h ( n = 15 , each group ) . Scale bar represents 50 µm .

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C H&E staining, ANGPTL4 and amylase expression by immunofluorescence analysis after the induction of mild (AP) and severe acute pancreatitis (SAP) for 2 h (n = 15, each group). Scale bar represents 50 µm.


D Amylase , lipase , and ANGPTL4 were assessed in the serum from AP and SAP animal models and ANGPTL4 protein expression were determined in the blood and pancreas ( n = 15 , each group ) .

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D Amylase, lipase, and ANGPTL4 were assessed in the serum from AP and SAP animal models and ANGPTL4 protein expression were determined in the blood and pancreas (n = 15, each group).


A AP was induced by 5 injections ( IP ) of cerulein at a dosage of 50 μg / kg . SAP was induced by AP and one injection of lipopolysaccharide ( LPS , IP ) at a dosage of 20 mg / kg . AP + ANGPTL4 ( 2 mg / kg ) was induced by AP and one injection of ANGPTL4 ( IP ) at a dosage of 2 mg / kg . ANGPTL4 ( 4 mg / kg ) was induced by two injections of ANGPTL4 ( IP ) at a dosage of 2 mg / kg . Mice were sacrificed 6 h after pancreatitis induction . ( H & E and immunofluorescent staining for IL - 1β ( red ) and amylase ( green ) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues ( n = 10 , each group ) . Scale bar represents 50

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A AP was induced by 5 injections (IP) of cerulein at a dosage of 50 μg/kg. SAP was induced by AP and one injection of lipopolysaccharide (LPS, IP) at a dosage of 20 mg/kg. AP+ANGPTL4 (2 mg/kg) was induced by AP and one injection of ANGPTL4 (IP) at a dosage of 2 mg/kg. ANGPTL4 (4 mg/kg) was induced by two injections of ANGPTL4 (IP) at a dosage of 2 mg/kg. Mice were sacrificed 6 h after pancreatitis induction. (H&E and immunofluorescent staining for IL-1β (red) and amylase (green) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues (n = 10, each group). Scale bar represents 50 µm


B Concentrations of the inflammatory cytokines TGF - β , IL - 1β , and IFN - γ as well as ANGPTL4 in the sera ( n = 10 , each group ) .

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B Concentrations of the inflammatory cytokines TGF-β, IL-1β, and IFN-γ as well as ANGPTL4 in the sera (n = 10, each group).


C TUNEL staining for apoptosis and vacuoles formation ( TEM ) . Red arrow , apoptotic cells ; white dotted line , vacuoles region .

C TUNEL staining for apoptosis and vacuoles formation (TEM). Red arrow, apoptotic cells; white dotted line, vacuoles region.


D T . E . M . ANGPTL4 immunogold labelling were performed in pancreas tissues from the AP , SAP and ANGPTL4 - treated models ( n = 5 , each group ) .

D T.E.M. ANGPTL4 immunogold labelling were performed in pancreas tissues from the AP, SAP and ANGPTL4-treated models (n = 5, each group).


A AP and SAP were induced in wild type ( WT ) and ANGPTL4 - / - mice ( n = 15 , each group ) , as described in the material and methods . H & E staining , TUNEL staining for apoptosis , and immunofluorescence staining for ANGPTL4 ( red ) and amylase ( green ) were performed in the pancreatic tissues . ANGPTL4 and IL - 6 protein expressions were also determined in pancreatic tissues from ANGPTL4 WT and - / - mice by Western blotting . Scale bar represents 100 µm .

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A AP and SAP were induced in wild type (WT) and ANGPTL4 -/- mice (n = 15, each group), as described in the material and methods. H&E staining, TUNEL staining for apoptosis, and immunofluorescence staining for ANGPTL4 (red) and amylase (green) were performed in the pancreatic tissues. ANGPTL4 and IL-6 protein expressions were also determined in pancreatic tissues from ANGPTL4 WT and -/- mice by Western blotting. Scale bar represents 100 µm.


B Levels of ANGPTL4 , amylase , lipase , and MPO in serum ( n = 15 , each group ) .

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B Levels of ANGPTL4, amylase, lipase, and MPO in serum (n = 15, each group).


C Immunofluorescence staining of TNF - α and IL - 6 , and the levels of the inflammatory cytokines TGF - β , IFN - γ , and IL - 1β in the sera from ANGPTL4 WT and - / - mice ( n = 10 , each group ) . Scale bar represents 150

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C Immunofluorescence staining of TNF-α and IL-6, and the levels of the inflammatory cytokines TGF-β, IFN-γ, and IL-1β in the sera from ANGPTL4 WT and -/- mice (n = 10, each group). Scale bar represents 150 µm


A For macrophage depletion models , mice ( six weeks ) were injected with GdCl3 ( 15 mg / kg ) . After the induction of macrophage depletion in the mice , pancreatitis was induced by cerulein , LPS , and ANGPTL4 for 24 h ( n = 10 , each group ) . H & E staining of pancreatic tissue , and levels of amylase and lipase in the serum . Scale bar represents 100

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A For macrophage depletion models, mice (six weeks) were injected with GdCl3 (15 mg/kg). After the induction of macrophage depletion in the mice, pancreatitis was induced by cerulein, LPS, and ANGPTL4 for 24 h (n = 10, each group). H&E staining of pancreatic tissue, and levels of amylase and lipase in the serum. Scale bar represents 100 µm


B Immunohistochemistry for the macrophage marker CD11b and ANGPTL4 in pancreas tissues . Levels of ANGPTL4 in the serum were measured in the AP , SAP , and ANGPTL4 ( 4 mg / kg ) groups of macrophage WT or depletion mice h ( n = 10 , each group ) . Scale bar represents 100

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B Immunohistochemistry for the macrophage marker CD11b and ANGPTL4 in pancreas tissues. Levels of ANGPTL4 in the serum were measured in the AP, SAP, and ANGPTL4 (4 mg/kg) groups of macrophage WT or depletion mice h (n = 10, each group). Scale bar represents 100 µm


C Immunofluorescent staining of the F4 / 80 macrophage marker and quantification of macrophage - positive cells in the AP and SAP groups from the ANGPTL4 - / - and WT mice ( n = 10 ) . Scale bar represents 150

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C Immunofluorescent staining of the F4/80 macrophage marker and quantification of macrophage-positive cells in the AP and SAP groups from the ANGPTL4 -/-and WT mice (n = 10). Scale bar represents 150 µm


A Cell morphology and the mRNA levels of inflammatory cytokines , including and TNF - α , IL - 6 , and IL - 1β in mouse primary macrophages treated with LPS and ANGPTL4 ( 100 ng / ml ) using qPCR and RT - PCR , and levels of TGF - β and NO in the LPS - and ANGPTL4 - treated primary macrophages ( n = 5 ) . Scale bar represents 100

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A Cell morphology and the mRNA levels of inflammatory cytokines, including and TNF-α, IL-6, and IL-1β in mouse primary macrophages treated with LPS and ANGPTL4 (100 ng/ml) using qPCR and RT-PCR, and levels of TGF-β and NO in the LPS- and ANGPTL4-treated primary macrophages (n = 5). Scale bar represents 100 µm


B Migration assays were performed with mouse primary acinar cells and macrophages using IBIDI removable 2 - well silicone culture inserts that were placed in a cell culture μ - Dish and transwell at 24 h ( n = 5 ) . Scale bar represents 50

B Migration assays were performed with mouse primary acinar cells and macrophages using IBIDI removable 2-well silicone culture inserts that were placed in a cell culture μ-Dish and transwell at 24 h (n = 5). Scale bar represents 50 µm


C Mouse primary acinar cells and macrophages were labelled with fluorescence dyes ( macrophages , red and acinar cells , green ) . Co - cultured cells treated with 100 ng / ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h . Scale bars represent 80 µm ( red ) and 40 µm ( white ) .

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C Mouse primary acinar cells and macrophages were labelled with fluorescence dyes (macrophages, red and acinar cells, green). Co-cultured cells treated with 100 ng/ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h. Scale bars represent 80 µm (red) and 40 µm (white).


A Macrophage cells were treated with ANGPTL4 ( 100 ng / ml ) for 24 h and then cytokine levels were analyzed by a mouse cytokine profiler assay ( n = 3 ) . Mean pixel density was quantified using ImageJ software analysis .

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A Macrophage cells were treated with ANGPTL4 (100 ng/ml) for 24 h and then cytokine levels were analyzed by a mouse cytokine profiler assay (n=3). Mean pixel density was quantified using ImageJ software analysis.


B ANGPTL4 and C5a expression levels were measured in bone marrow - derived macrophages from pancreatitis - induced ANGPTL4 - / - and WT mice .

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B ANGPTL4 and C5a expression levels were measured in bone marrow-derived macrophages from pancreatitis-induced ANGPTL4-/- and WT mice.


C Phospho - kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1 - derived macrophage cells ( n = 3 ) . Cells were treated with ANGPTL4 ( 100 ng / ml ) for 6 h . After phospho - kinase array analysis , the expression of AMPKα , JNK , AKT , P70S6K , and EGFR was confirmed by western blotting .

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C Phospho-kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1-derived macrophage cells (n=3). Cells were treated with ANGPTL4 (100 ng/ml) for 6 h. After phospho-kinase array analysis, the expression of AMPKα, JNK, AKT, P70S6K, and EGFR was confirmed by western blotting.


D To explore whether ANGPTL4 affects JNK , AMPK , and PI3K / AKT signaling , each inhibitor ( SP600125 , compound C , and HS - 173 , 1 µM ) was treated to in THP1 - derived macrophages for 2 h after ANGPTL4 treatment ( 100 ng / ml ) for 6 h .

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D To explore whether ANGPTL4 affects JNK, AMPK, and PI3K/AKT signaling, each inhibitor (SP600125, compound C, and HS-173, 1 µM) was treated to in THP1-derived macrophages for 2 h after ANGPTL4 treatment (100 ng/ml) for 6 h.


E C5a , C5aR , TNF - α , IL - 6 , and IL - 1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS ( 100 ng / ml ) . In the transwell experiment , mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 ( 100 ng / ml ) . Expression of ANGPTL4 , C5aR , and C5a was determined in siANGPTL4 - treated THP1 - derived macrophage after LPS activation .

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E C5a, C5aR, TNF-α, IL-6, and IL-1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS (100 ng/ml). In the transwell experiment, mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 (100 ng/ml). Expression of ANGPTL4, C5aR, and C5a was determined in siANGPTL4-treated THP1-derived macrophage after LPS activation.


F In the transwell experiment , TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist ( W - 54011 , 10 nM ) pretreatment for 12 h prior to treatment with ANGPTL4 , LPS , and C5a ( 100 ng / ml ) for 24 h to macrophage in transwell . Scale bar represents 20

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F In the transwell experiment, TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist (W-54011, 10 nM) pretreatment for 12 h prior to treatment with ANGPTL4, LPS, and C5a (100 ng/ml) for 24 h to macrophage in transwell. Scale bar represents 20 µm


A Concentrations of C5a in the sera from pancreatitis patients , and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients ( n = 90 ) . The box plots show a typical display consisting of a median value depicted by the line in the center of the box ; an interquartile range ( IQR ; 25th to the 75th percentile ) depicted by the box ; and the maximum ( Q3 + 1 . 5 * IQR ) and minimum ( Q1 - 1 . 5 * IQR ) values depicted by the whisker . Statistical significance of Mann - Whitney U - tests is indicated ( * * * P < 0 . 001 ) . Exact P values are shown in Appendix Table S2 . The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient ( R ) . Each dot represents the levels of C5a and ANGPTL4 from an individual patient ( n = 90 ) . The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues . Scale bar represents 50

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A Concentrations of C5a in the sera from pancreatitis patients, and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients (n = 90). The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (*** P < 0.001). Exact P values are shown in Appendix Table S2. The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient (R). Each dot represents the levels of C5a and ANGPTL4 from an individual patient (n = 90). The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues. Scale bar represents 50 µm


B Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 - / - and WT animals and macrophage depletion / WT models with pancreatitis . Scale bar represents 150 µm . Levels of C5a were measured in serum ( n = 7 ) .

B Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 -/- and WT animals and macrophage depletion/WT models with pancreatitis. Scale bar represents 150 µm. Levels of C5a were measured in serum (n = 7).


C After macrophages were activated with LPS ( 100 ng / ml ) , the cells were treated with either neutralizing ANGPTL4 or C5a antibody ( 100 ng / ml ) or an isotype control ( IgG ) . ANGPTL4 , C5a , TNF - α , and IL - 6 levels were measured in culture media ( n = 5 ) .

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C After macrophages were activated with LPS (100 ng/ml), the cells were treated with either neutralizing ANGPTL4 or C5a antibody (100 ng/ml) or an isotype control (IgG). ANGPTL4, C5a, TNF-α, and IL-6 levels were measured in culture media (n = 5).


D The neutralizing ANGPTL4 antibody ( 10 mg / kg ) was injected twice ( IP ) into the AP and SAP models . H & E staining of pancreatic tissues and the concentrations of ANGPTL4 , C5a , TNF - α , and amylase in the serum ( n = 10 , each group ) . Scale bar represents 50

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D The neutralizing ANGPTL4 antibody (10 mg/kg) was injected twice (IP) into the AP and SAP models. H&E staining of pancreatic tissues and the concentrations of ANGPTL4, C5a, TNF-α, and amylase in the serum (n = 10, each group). Scale bar represents 50 µm


( A ) . Human neutrophils stained for CD66b after incubation with RBCs infected with GFP + P . falciparum parasites ( white arrows ) . Nuclei were stained with DAPI ( blue ) , neutrophils were stained with CD66b ( red ) , GFP expressing parasites ( green ) . Scale bar , 5 µm . The upper and lower panel show two different cells .

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(A). Human neutrophils stained for CD66b after incubation with RBCs infected with GFP+ P. falciparum parasites (white arrows). Nuclei were stained with DAPI (blue), neutrophils were stained with CD66b (red), GFP expressing parasites (green). Scale bar, 5 µm. The upper and lower panel show two different cells.


( B ) . Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P . falciparum parasites ( black arrows ) .

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(B). Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P. falciparum parasites (black arrows).


( C ) . Flow cytometry analysis of human neutrophils incubated with opsonized or non - opsonized GFP + - late - stage iRBCs . Free neutrophils were used as control .

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(C). Flow cytometry analysis of human neutrophils incubated with opsonized or non-opsonized GFP+-late-stage iRBCs. Free neutrophils were used as control.


( D ) . Quantification of GFP + iRBCs phagocytosed by neutrophils . The phagocytosis was analysed by flow cytometry ( FACS ) . Data information :

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(D). Quantification of GFP+ iRBCs phagocytosed by neutrophils. The phagocytosis was analysed by flow cytometry (FACS). Data information:


( A ) . Interaction between neutrophils and opsonized ( red ) or non - opsonized ( blue ) GFP + iRBCs by time in a " pulse - chase " experiment . The percentage of GFP + - neutrophils following 5 min of co - culture was set as 100 % .

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(A). Interaction between neutrophils and opsonized (red) or non-opsonized (blue) GFP+ iRBCs by time in a "pulse-chase" experiment. The percentage of GFP+-neutrophils following 5 min of co-culture was set as 100%.


( C ) . Parasitemia ( percentage % ) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils .

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(C). Parasitemia (percentage %) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils.


( D ) . Parasitemia ( percentage % ) of cultures of RBC infected by different P . falciparum lines ( NF54 , Dd2 , SL , PfEMP1 - KO ) . The parasites were kept in culture for six days in the presence ( Neut . ) or absence ( Cont . ) of neutrophils .

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(D). Parasitemia (percentage %) of cultures of RBC infected by different P. falciparum lines (NF54, Dd2, SL, PfEMP1-KO). The parasites were kept in culture for six days in the presence (Neut.) or absence (Cont.) of neutrophils.


( E ) . Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines ( NF54 , Dd2 , SL , PfEMP1 - KO ) .

(E). Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines (NF54, Dd2, SL, PfEMP1-KO).


( F ) . Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression . Neutrophils were stained with an anti - CD11b antibody , while iRBCs were labeled with SYBR green . Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population , excluding the neutrophils that were SYBR green negative , but CD11b positive .

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(F). Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression. Neutrophils were stained with an anti-CD11b antibody, while iRBCs were labeled with SYBR green. Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population, excluding the neutrophils that were SYBR green negative, but CD11b positive.


( G ) . Short term neutrophil killing of late - stage NF54 - luciferase positive RBCs in the presence ( red ) or absence ( blue ) of catalase .

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(G). Short term neutrophil killing of late-stage NF54-luciferase positive RBCs in the presence (red) or absence (blue) of catalase.


( H ) . Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase .

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(H). Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase.


( I ) . Short - term luciferase - based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D .

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(I). Short-term luciferase-based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D.


( A ) . Representative flow cytometric analysis of human neutrophils cultured alone ( Control ) , incubated with late - stage NF54 parasites ( wild type ) or PfEMP1 Knock out ( PfEMP1 KO ) GFP + iRBC .

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(A). Representative flow cytometric analysis of human neutrophils cultured alone (Control), incubated with late-stage NF54 parasites (wild type) or PfEMP1 Knock out (PfEMP1 KO) GFP+ iRBC.


( B ) . Flow cytometric quantification of the effect of trypsin treatment on neutrophil interaction with wild type and PfEMP1 KO GFP + iRBC .

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(B). Flow cytometric quantification of the effect of trypsin treatment on neutrophil interaction with wild type and PfEMP1 KO GFP+ iRBC.


( C ) . Luciferase - based killing assay and neutrophil elimination of late - stage wild type ( wt ) and PfEMP1ko iRBC .

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(C). Luciferase-based killing assay and neutrophil elimination of late-stage wild type (wt) and PfEMP1ko iRBC.


( B ) . Short - term ( 6h ) neutrophil killing of unselected NF54 and antibody - selected PFD1235w expressing parasite lines .

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(B). Short-term (6h) neutrophil killing of unselected NF54 and antibody-selected PFD1235w expressing parasite lines.


( C ) . Flow cytometric quantification of neutrophil interaction with MitoTracker ( APC + ) stained NF54 iRBCs and antibody - selected PFD1235w expressing iRBC .

(C). Flow cytometric quantification of neutrophil interaction with MitoTracker (APC+) stained NF54 iRBCs and antibody-selected PFD1235w expressing iRBC.


( D ) . Percent reduction in parasitemia of NF54 and antibody - selected PFD1235w expressing parasites after 5 days of co - culture with neutrophils , compared to unchallenged

(D). Percent reduction in parasitemia of NF54 and antibody-selected PFD1235w expressing parasites after 5 days of co-culture with neutrophils, compared to unchallenged parasites


( E ) . var gene transcription profiles measured by qRT - PCR of antibody - selected PFD1235w expressing parasite line cultured in the absence ( upper panel ) or presence ( lower panel ) of neutrophils . Steady state mRNA levels of each individual var gene was calculated as in A . I .

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(E). var gene transcription profiles measured by qRT-PCR of antibody-selected PFD1235w expressing parasite line cultured in the absence (upper panel) or presence (lower panel) of neutrophils. Steady state mRNA levels of each individual var gene was calculated as in A. I.


( A ) QRT - PCR assay of PAX6 , SOX1 , NKX2 - 1 , NKX2 - 2 , TBR1 and TBR2 during dorsal ( up ) and ventral ( down ) neural differentiation . ( B ) QRT - PCR assay of SOX1 - OT V1 during dorsal ( up ) and ventral ( down ) neural differentiation Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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(A) QRT-PCR assay of PAX6, SOX1, NKX2-1, NKX2-2, TBR1 and TBR2 during dorsal (up) and ventral (down) neural differentiation. (B) QRT-PCR assay of SOX1-OT V1 during dorsal (up) and ventral (down) neural differentiation Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( F and G ) Immunostaining assay of PAX6 ( green ) and SOX1 ( red ) in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( F ) , and quantiﬁcation of SOX1 + cells ( G ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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(F and G) Immunostaining assay of PAX6 (green) and SOX1 (red) in control and V1-PAKI cells on day 16 of dorsal neural differentiation (F), and quantiﬁcation of SOX1+ cells (G), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( H and I ) Immunostaining assay of MAP2 ( green ) in control and V1 - PAKI cells on day 35 of dorsal neural differentiation ( H ) , and quantiﬁcation of MAP2 + cells ( I ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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(H and I) Immunostaining assay of MAP2 (green) in control and V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( J and K ) Immunostaining assay of PAX6 ( green ) and SOX1 ( red ) in control and V1 - PAKI cells on day 16 of ventral neural differentiation ( J ) , and quantiﬁcation of SOX1 + cells ( K ) , p = 0 . 0003 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

(J and K) Immunostaining assay of PAX6 (green) and SOX1 (red) in control and V1-PAKI cells on day 16 of ventral neural differentiation (J), and quantiﬁcation of SOX1+ cells (K), p=0.0003. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( L and M ) Immunostaining assay of GABA ( green ) in control and V1 - PAKI cells on day 35 of ventral neural differentiation ( L ) , and quantiﬁcation of GABA + cells ( M ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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(L and M) Immunostaining assay of GABA (green) in control and V1-PAKI cells on day 35 of ventral neural differentiation (L), and quantiﬁcation of GABA+ cells (M), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( C and D ) Immunostaining assay of MAP2 ( green ) in control and SOX1KO cells on day 35 of dorsal neural differentiation ( C ) , and quantiﬁcation of MAP2 + cells ( D ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

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(C and D) Immunostaining assay of MAP2 (green) in control and SOX1KO cells on day 35 of dorsal neural differentiation (C), and quantiﬁcation of MAP2+ cells (D), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( F and G ) Immunostaining assay of GABA ( green ) in control and SOX1KO cells on day 35 of ventral neural differentiation ( F ) , and quantiﬁcation of GABA + cells ( G ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

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(F and G) Immunostaining assay of GABA (green) in control and SOX1KO cells on day 35 of ventral neural differentiation (F), and quantiﬁcation of GABA+ cells (G), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( H and I ) Immunostaining assay of MAP2 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 35 of dorsal neural differentiation ( H ) , and quantiﬁcation of MAP2 + cells ( I ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( V1 - PAKI + ptSOX1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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(H and I) Immunostaining assay of MAP2 (green) after SOX1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001 (V1-PAKI), p=0.0001 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( J and K ) Immunostaining assay of GABA ( green ) after SOX1 overexpression in V1 - PAKI cells on day 35 of ventral neural differentiation ( J ) , and quantiﬁcation of GABA + cells ( K ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0002 ( V1 - PAKI + ptSOX1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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(J and K) Immunostaining assay of GABA (green) after SOX1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (J), and quantiﬁcation of GABA+ cells (K), p=0.0001 (V1-PAKI), p=0.0002 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Enrichment of H3K14Ac ( left ) and H3K27Ac ( right ) on SOX1 promoter region in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( A ) , p = 0 . 0046 ( H3K14Ac ) , p = 0 . 0223 ( H3K27Ac ) and ventral neural differentiation ( B ) , p = 0 . 0499 ( H3K14Ac ) , p = 0 . 0022 ( H3K27Ac ) . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

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(A and B) Enrichment of H3K14Ac (left) and H3K27Ac (right) on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (A), p=0.0046 (H3K14Ac), p=0.0223 (H3K27Ac) and ventral neural differentiation (B), p=0.0499 (H3K14Ac), p=0.0022 (H3K27Ac). Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01 versus Ctrl group (unpaired two-tailed Student's t-test).


( C Immunostaining assay of SOX1 ( red ) after 4 HDAC inhibitors ( 4i ) , including RG2833 ( 2833 , 2μM ) , CAY10683 ( CAY , 2μM ) , PCI - 34051 ( 34051 , 5μM ) and Bufexamac ( Buf , 12 . 5μM ) , treatment or remove one inhibitor from these 4 HDAC inhibitors ( 4i ) in V1 - PAKI cells on day 16 of dorsal neural differentiation Scale bar , 100 μm .

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(C Immunostaining assay of SOX1 (red) after 4 HDAC inhibitors (4i), including RG2833 (2833, 2μM), CAY10683 (CAY, 2μM), PCI-34051 (34051, 5μM) and Bufexamac (Buf, 12.5μM), treatment or remove one inhibitor from these 4 HDAC inhibitors (4i) in V1-PAKI cells on day 16 of dorsal neural differentiation Scale bar, 100 μm.


( G and H ) Immunostaining assay of SOX1 ( red ) after Bufexamac treatment in V1 - PAKI cells on day 16 of dorsal neural differentiation ( G ) , and quantiﬁcation of SOX1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( + Buf ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ; ( one - way ANOVA ) .

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 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

(G and H) Immunostaining assay of SOX1 (red) after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).


( J and K ) Immunostaining assay of MAP2 ( green ) after Bufexamac treatment in V1 - PAKI cells on day 35 of dorsal neural differentiation ( J ) , and quantiﬁcation of MAP2 + cells ( K ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( + Buf ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ( one - way ANOVA ) .

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 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

(J and K) Immunostaining assay of MAP2 (green) after Bufexamac treatment in V1-PAKI cells on day 35 of dorsal neural differentiation (J), and quantiﬁcation of MAP2+ cells (K), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group (one-way ANOVA).


( L ) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1 - PAKI cells on day 16 of dorsal ( left , p = 0 . 0007 ( V1 - PAKI ) , p = 0 . 0002 ( + Buf ) ) and ventral ( right , p = 0 . 0083 ( V1 - PAKI ) , p = 0 . 0276 ( + Buf ) ) neural differentiation . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ; ( one - way ANOVA ) .

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 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-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

(L) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal (left, p=0.0007 (V1-PAKI), p=0.0002 (+Buf)) and ventral (right, p=0.0083 (V1-PAKI), p=0.0276 (+Buf)) neural differentiation. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).


( B and C ) RIP analysis for endogenous interaction between HDAC10 and SOX1 - OT V1 on day 16 of dorsal neural differentiation ( B ) , p = 0 . 0085 and ventral neural differentiation ( C ) , p = 0 . 0152 . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus IgG group ( unpaired two - tailed Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C) RIP analysis for endogenous interaction between HDAC10 and SOX1-OT V1 on day 16 of dorsal neural differentiation (B), p=0.0085 and ventral neural differentiation (C), p=0.0152. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus IgG group (unpaired two-tailed Student's t-test).


( D and E ) Enrichment of HDAC10 on SOX1 promoter region in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( D ) , p = 0 . 0052 and ventral neural differentiation ( E ) , p = 0 . 0001 . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

(D and E) Enrichment of HDAC10 on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (D), p=0.0052 and ventral neural differentiation (E), p=0.0001. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( G and H ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 , SOX1 - OT V1 - 5P or SOX1 - OT V1 - 3P overexpression in V1 - PAKI cells on day 16 of dorsal neural differentiation ( G ) , and quantiﬁcation of SOX1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptV1 + dox ) , p = 0 . 0001 ( pt5P + dox ) . Scale bar , 100 μm . ( I and J ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 , SOX1 - OT V1 - 5P or SOX1 - OT V1 - 3P overexpression in V1 - PAKI cells on day 16 of ventral neural differentiation ( I ) , and quantiﬁcation of SOX1 + cells ( J ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptV1 + dox ) , p = 0 . 0001 ( pt5P + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and H) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of ventral neural differentiation (I), and quantiﬁcation of SOX1+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Immunostaining assay of ASCL1 ( green ) in control , V1 - PAKI and SOX1KO cells on day 25 of dorsal neural differentiation ( A ) , and quantiﬁcation of ASCL1 + cells ( B ) , p = 0 . 0021 ( V1 - PAKI ) , p = 0 . 0024 ( SOX1KO ) . Scale bar , 100 μm . ( C and D ) Immunostaining assay of ASCL1 ( green ) in control , V1 - PAKI and SOX1KO cells on day 25 of ventral neural differentiation ( C ) , and quantiﬁcation of ASCL1 + cells ( D ) , p = 0 . 0003 ( V1 - PAKI ) , p = 0 . 0004 ( SOX1KO ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of dorsal neural differentiation (A), and quantiﬁcation of ASCL1+ cells (B), p=0.0021 (V1-PAKI), p=0.0024 (SOX1KO). Scale bar, 100 μm. (C and D) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of ventral neural differentiation (C), and quantiﬁcation of ASCL1+ cells (D), p=0.0003 (V1-PAKI), p=0.0004 (SOX1KO). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( E and F ) Immunostaining assay of ASCL1 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 25 of dorsal neural differentiation ( E ) , and quantiﬁcation of ASCL1 + cells ( F ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptSOX1 + dox ) . Scale bar , 100 μm . ( G and H ) Immunostaining assay of ASCL1 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 25 of ventral neural differentiation ( G ) , and quantiﬁcation of ASCL1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptSOX1 + dox ) . Scale bar , 100 μm . ( I and J ) Immunostaining assay of MAP2 ( green ) after ASCL1 overexpression in V1 - PAKI cells on day 35 of dorsal neural differentiation ( I ) , and quantiﬁcation of MAP2 + cells ( J ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptASCL1 + dox ) . Scale bar , 100 μm . ( K and L ) Immunostaining assay of GABA ( green ) after ASCL1 overexpression in V1 - PAKI cells on day 35 of ventral neural differentiation ( K ) , and quantiﬁcation of GABA + cells ( L ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptASCL1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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

(E and F) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of dorsal neural differentiation (E), and quantiﬁcation of ASCL1+ cells (F), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (G and H) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of ventral neural differentiation (G), and quantiﬁcation of ASCL1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of MAP2 (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (I), and quantiﬁcation of MAP2+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. (K and L) Immunostaining assay of GABA (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (K), and quantiﬁcation of GABA+ cells (L), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Immunostaining assay of SOX1 ( red ) in control , V1 - PAKI and SOX1KO cells on day 40 of dorsal ( A ) and ventral ( B ) organoids differentiation . Scale bar , 100 μm . ( C ) Immunostaining assay of MAP2 ( green ) in control , V1 - PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation . Scale bar , 100 μm . ( D ) Immunostaining assay of GABA ( green ) in control , V1 - PAKI and SOX1KO cells on day 40 of ventral organoids differentiation . Scale bar , 100 μm .

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

(A and B) Immunostaining assay of SOX1 (red) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal (A) and ventral (B) organoids differentiation. Scale bar, 100 μm. (C) Immunostaining assay of MAP2 (green) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (D) Immunostaining assay of GABA (green) in control, V1-PAKI and SOX1KO cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.


( E and F ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of dorsal ( E ) and ventral ( F ) organoids differentiation . Scale bar , 100 μm . ( G ) Immunostaining assay of MAP2 ( green ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of dorsal organoids differentiation . Scale bar , 100 μm . ( H ) Immunostaining assay of GABA ( green ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of ventral organoids differentiation . Scale bar , 100 μm .

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

(E and F) Immunostaining assay of SOX1 (red) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal (E) and ventral (F) organoids differentiation. Scale bar, 100 μm. (G) Immunostaining assay of MAP2 (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (H) Immunostaining assay of GABA (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.


B ) Total T cells were enriched from spleens of Ptpn6fl / fl / Ptpn11fl / flWbm , CD4cre Ptpn6fl / fl / Ptpn11fl / flWbm , CD4cre Ptpn6fl / fl mice . Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates , Gapdh used as 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 O O O O O O O O O O O O O O O O O O O O

B) Total T cells were enriched from spleens of Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl mice. Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates, Gapdh used as loading control.


CD4cre Ptpn6fl / fl and control mice were subcutaneously injected with MC38 cells . Tumour growth in individual mice is shown for the indicated genotypes and treatments ; number of mice eradicating the tumour is shown within the graphs ( C ) .

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

CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Tumour growth in individual mice is shown for the indicated genotypes and treatments; number of mice eradicating the tumour is shown within the graphs (C).


CD4cre Ptpn6fl / fl and control mice were subcutaneously injected with MC38 cells . Survival curves are shown ( D ) .

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

CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Survival curves are shown (D).


Tumour growth in individual mice challenged with MC38 and subsequently treated with anti - PD - 1 antibody or isotype control is shown for each indicated genotype ; number of mice eradicating the tumour is shown within the graphs ( G ) .

Tumour growth in individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype; number of mice eradicating the tumour is shown within the graphs (G).


individual mice challenged with MC38 and subsequently treated with anti - PD - 1 antibody or isotype control is shown for each indicated genotype Survival curves of CD4cre Ptpn6fl / fl / Ptpn11fl / flWbm and control mice is shown ( 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

individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype Survival curves of CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm and control mice is shown (H).