Figure 2 ( A ) Images of EOC tumors labelled with antibodies against PCNT ( red ) and CDKRAP2 ( green ) . Nuclei are shown in blue . The colored arrows reflect different centrosome number . The list of centrosome behaviors annotated it is not exhaustive to allow comprehension of the events . # TT72 , shows a cyst - like arrangement of EOC nuclei where it is impossible to correspond centrosomes to individual nuclei . The white bars with arrows of different colors highlight possible interpretations of the same condition . In # TT79 , regions without centrosomes can be identified . In the inset , a single centrosome localized in the lumen can be identified and it is not possible to know to which nucleus ( white dashed lines ) it corresponds to . # TT88 presents only very small clusters of centrosomes , few nuclei without centrosomes and many conditions with 2 centrosomes . In # TT61 , regions without centrosomes can be distinguished , in addition to nuclei associated with single centrosomes . In # TT46 , clusters containing large ( inset 1 ) or small clusters ( inset 3 ) can be identified . In cyst - like arrangements several centrosomes can be identified ( inset 2 ) , suggesting that all cells have at least one centrosome or , in contrast just one centrosome ( inset 4 ) , illustrating lack of centrosomes . ( B - C ) Plots showing the frequency of centrosome amplification events ( B ) and the frequency of nuclei without centrosomes ( C ) in tumor tissues . Note that in B and C , the order of the tumors is conserved between the two plots to allow for comparison . For each tumor , 10 random fields were chosen and analysed , with an average of 5248 nuclei counted per tumor . ( D ) Tumor sections labelled with CEP135 ( green ) and PCNT ( red ) on the left and CEP135 ( green ) and CEP192 ( red ) on the right . The white squares represent the regions shown in higher magnifications on the right .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Images of EOC tumors labelled with antibodies against PCNT (red) and CDKRAP2 (green). Nuclei are shown in blue. The colored arrows reflect different centrosome number. The list of centrosome behaviors annotated it is not exhaustive to allow comprehension of the events. #TT72, shows a cyst-like arrangement of EOC nuclei where it is impossible to correspond centrosomes to individual nuclei. The white bars with arrows of different colors highlight possible interpretations of the same condition. In #TT79, regions without centrosomes can be identified. In the inset, a single centrosome localized in the lumen can be identified and it is not possible to know to which nucleus (white dashed lines) it corresponds to. #TT88 presents only very small clusters of centrosomes, few nuclei without centrosomes and many conditions with 2 centrosomes. In #TT61, regions without centrosomes can be distinguished, in addition to nuclei associated with single centrosomes. In #TT46, clusters containing large (inset 1) or small clusters (inset 3) can be identified. In cyst-like arrangements several centrosomes can be identified (inset 2), suggesting that all cells have at least one centrosome or, in contrast just one centrosome (inset 4), illustrating lack of centrosomes.(B-C) Plots showing the frequency of centrosome amplification events (B) and the frequency of nuclei without centrosomes (C) in tumor tissues. Note that in B and C, the order of the tumors is conserved between the two plots to allow for comparison. For each tumor, 10 random fields were chosen and analysed, with an average of 5248 nuclei counted per tumor.(D) Tumor sections labelled with CEP135 (green) and PCNT (red) on the left and CEP135 (green) and CEP192 (red) on the right. The white squares represent the regions shown in higher magnifications on the right.


Figure 3 ( B ) Stills of a time - lapse movie of Ctrl spheroids . Time is shown in hours ( hrs ) . ( C ) z - view of Ctrl cells as shown in ( B ) . Note the red - colored cancer cells at the beginning of the movie on top of the mesothelial layer ( green ) while at later time points , they have cleared through the mesothelial cells . ( D ) Graph bars of the normalized clearance in A . U . of iOVCAR8 ( left ) and iSKOV3 ( right ) spheroids after the indicated treatments . The two cell lines show different clearance capacity . For each cell line , three independent experiments were performed . For iOVCAR8 , we analyzed 47 spheroids in DMSO and 56 spheroids in centrinone . For iSKOV3 we analyzed 56 spheroids in DMSO and 48 spheroids in centrinone . Dots represent the mean and the shadow ± SEM . Statistical significance was assessed with an ANCOVA test . Dots represent the mean and the shadow ± SEM . Statistical significance was assessed with an ANOVA test . ( F ) Representative images of DMSO ( left ) and centrinone ( right ) spheroids . Mean ± SD . Red nuclei represent false colored invading nuclei . ( G ) Dot plot showing the quantification of the number of nuclei detected on the bottom side of the BM . Three or four positions from three BM inserts were analyzed in each condition . Mean ± SD . Statistical significance was assessed with the Mann - Whitney test . For all the experiments , the centrosome number was verified in parallel to confirm the decreased centrosome conditions compared to DMSO .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Stills of a time-lapse movie of Ctrl spheroids. Time is shown in hours (hrs). (C) z- view of Ctrl cells as shown in (B). Note the red-colored cancer cells at the beginning of the movie on top of the mesothelial layer (green) while at later time points, they have cleared through the mesothelial cells.(D) Graph bars of the normalized clearance in A.U. of iOVCAR8 (left) and iSKOV3 (right) spheroids after the indicated treatments. The two cell lines show different clearance capacity. For each cell line, three independent experiments were performed. For iOVCAR8, we analyzed 47 spheroids in DMSO and 56 spheroids in centrinone. For iSKOV3 we analyzed 56 spheroids in DMSO and 48 spheroids in centrinone. Dots represent the mean and the shadow±SEM. Statistical significance was assessed with an ANCOVA test. Dots represent the mean and the shadow±SEM. Statistical significance was assessed with an ANOVA test.(F) Representative images of DMSO (left) and centrinone (right) spheroids. Mean±SD. Red nuclei represent false colored invading nuclei. (G) Dot plot showing the quantification of the number of nuclei detected on the bottom side of the BM. Three or four positions from three BM inserts were analyzed in each condition. Mean±SD. Statistical significance was assessed with the Mann-Whitney test. For all the experiments, the centrosome number was verified in parallel to confirm the decreased centrosome conditions compared to DMSO.


f1 ( a ) Expression of Ulk1 and Atg5 - 12 in the indicated erythroid cells . ( b - e ) Induction of conventional macroautophagy in WT and Ulk1gt / gt , but not in Atg5 − / − , embryonic erythroid cells . Ter119 + erythroid cells from WT , Atg5 − / − , and Ulk1gt / gt embryonic mice ( E18 . 5 ) were treated with 1 μM rapamycin ( b , c ) and 1 μM STS ( d , e ) , and then harvested at the indicated times . ( b , d ) Representative protein expression of LC3 and p62 measured by western blot . Actin was a loading control . Uncropped images are shown in Supplementary Fig . 10 . ( c , e ) Semi - quantitative analysis of LC3 - II / LC3 - I and p62 protein expression ( n = 3 , mean ± s . d . ) . Asterisks indicate a significant difference at P0 . 05 ( analysis of variance ( ANOVA ) ) . # P0 . 05 versus value of WT 24 h ( ANOVA ) . ' NS ' indicates not significant ( ANOVA ) . ( b - e ) Induction of conventional macroautophagy in WT and Ulk1gt / gt , but not in Atg5 − / − , embryonic erythroid cells . Ter119 + erythroid cells from WT , Atg5 − / − , and Ulk1gt / gt embryonic mice ( E18 . 5 ) were treated with 1 μM rapamycin ( b , c ) and 1 μM STS ( d , e ) , and then harvested at the indicated times . ( b , d ) Representative protein expression of LC3 and p62 measured by western blot . Actin was a loading control . Uncropped images are shown in Supplementary Fig . 10 . ( c , e ) Semi - quantitative analysis of LC3 - II / LC3 - I and p62 protein expression ( n = 3 , mean ± s . d . ) . Asterisks indicate a significant difference at P0 . 05 ( analysis of variance ( ANOVA ) ) . # P0 . 05 versus value of WT 24 h ( ANOVA ) . ' NS ' indicates not significant ( ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1(a) Expression of Ulk1 and Atg5-12 in the indicated erythroid cells.(b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).(b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).


f2 ( a ) Induction of alternative macroautophagy in WT and Atg5 − / − , but not Ulk1gt / gt and DKO , erythroid cells . Erythroblasts and reticulocytes from the liver of embryonic mice ( E18 . 5 ) were incubated with or without STS ( 1 μM ) for 24 h , followed by staining with anti - Lamp2 ( red ) , anti - Ter119 ( green ) and DAPI ( blue ) . DAPI - positive erythroblasts and DAPI - negative reticulocytes are shown . Lamp2 image and merged image ( DAPI , Ter119 and Lamp2 ) are shown . Scale bar , 1 μm . Large dots for Lamp2 are observed in STS - treated WT and Atg5 − / − cells , but not STS - treated Ulk1gt / gt or DKO cells . ( b ) Representative electron micrographs of EC incubated with or without STS . Erythroid cells were harvested from the liver of embryonic mice ( E18 . 5 ) , incubated with or without STS ( 1 μM ) for 24 h , and analysed by electron microscopy ( EM ) . Scale bar , 1 μm . Insets of WT and Atg5 − / − cells show mitophagy ( Scale bar , 0 . 5 μm ) . Inset of a Ulk1gt / gt cell showing mitochondria that have not been engulfed ( Scale bar , 0 . 5 μm ) . Arrows point to non - engulfed mitochondria and the arrowheads indicate engulfed mitochondria . ( c - e ) Quantitative analysis of mitophagy after STS treatment , calculated from EM photos . Population of reticulocytes with mitophagy ( c ) , number of mitochondria per reticulocytes ( d ) and population of reticulocytes showing macroautophagy ( e ) were calculated ( n > 35 cells per mouse ) . The data are shown as mean ± s . d . ( n = 3 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2(a) Induction of alternative macroautophagy in WT and Atg5−/−, but not Ulk1gt/gt and DKO, erythroid cells. Erythroblasts and reticulocytes from the liver of embryonic mice (E18.5) were incubated with or without STS (1 μM) for 24 h, followed by staining with anti-Lamp2 (red), anti-Ter119 (green) and DAPI (blue). DAPI-positive erythroblasts and DAPI-negative reticulocytes are shown. Lamp2 image and merged image (DAPI, Ter119 and Lamp2) are shown. Scale bar, 1 μm. Large dots for Lamp2 are observed in STS-treated WT and Atg5−/− cells, but not STS-treated Ulk1gt/gt or DKO cells.(b) Representative electron micrographs of EC incubated with or without STS. Erythroid cells were harvested from the liver of embryonic mice (E18.5), incubated with or without STS (1 μM) for 24 h, and analysed by electron microscopy (EM). Scale bar, 1 μm. Insets of WT and Atg5−/− cells show mitophagy (Scale bar, 0.5 μm). Inset of a Ulk1gt/gt cell showing mitochondria that have not been engulfed (Scale bar, 0.5 μm). Arrows point to non-engulfed mitochondria and the arrowheads indicate engulfed mitochondria. (c-e) Quantitative analysis of mitophagy after STS treatment, calculated from EM photos. Population of reticulocytes with mitophagy (c), number of mitochondria per reticulocytes (d) and population of reticulocytes showing macroautophagy (e) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).


f3 ( a ) Three - colour flow cytometry of erythroid cells from WT embryonic liver ( E14 . 5 ) stained with anti - Ter119 , anti - CD71 and Mitotracker Deep Red . ( Left upper panel ) Ter119 versus CD71 fluorescence . ( Other panels ) FSC versus Mitotracker fluorescence of the R1 ( right upper panel ) , R2 ( left lower panel ) and R3 ( right lower panel ) fractions . ( b , c ) Percentage of cells with a low number of mitochondria ( mitochondrialow cells ) among CD71 − Ter119 + EC . Erythroid cells from the liver ( E14 . 5 and E18 . 5 ) and peripheral blood ( E18 . 5 ) of indicated mice were stained with anti - Ter119 , anti - CD71 and Mitotracker Deep Red . ( b ) Representative histograms of mitochondrial content in CD71 − Ter119 + cells . Numbers indicate the population of mitochondrialow cells . ( c ) The percentage of CD71 − Ter119 + cells without mitochondria was determined by gating the mitochondrialow fraction , as shown in ( b ) ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) . ( d ) Percent mitochondrialow cells in the CD71 − Ter119 + EC of Atg7F / FCre embryo . Erythroid cells from the liver and peripheral blood of WT and Atg7F / FCre embryo ( E18 . 5 ) were stained with anti - Ter119 , anti - CD71 , and Mitotracker Deep Red ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) . ( e ) Percentage of mitochondrialow cells in Syto16low cells . Liver erythroid cells ( E18 . 5 ) were stained with Syto16 ( DNA ) and Mitotracker Deep Red . Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig . 5 , the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( ANOVA ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) . ( f ) Immunofluorescent analysis of mitochondrial and lysosomal proteins . Liver Ter119 + cells ( E18 . 5 ) of the indicated mice were stained with anti - Tom20 ( mitochondrial marker ) and anti - Lamp2 ( lysosomal marker ) antibodies , and observed by fluorescent microscopy . Scale bar , 2 μm . Green , red , white , and blue indicate Tom20 , Lamp2 , Ter119 and DAPI ( DNA ) , respectively . Mitochondrial and lysosomal markers have separate distributions in Ulk1gt / gt cells and DKO cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a) Three-colour flow cytometry of erythroid cells from WT embryonic liver (E14.5) stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red. (Left upper panel) Ter119 versus CD71 fluorescence. (Other panels) FSC versus Mitotracker fluorescence of the R1 (right upper panel), R2 (left lower panel) and R3 (right lower panel) fractions. (b,c) Percentage of cells with a low number of mitochondria (mitochondrialow cells) among CD71−Ter119+ EC. Erythroid cells from the liver (E14.5 and E18.5) and peripheral blood (E18.5) of indicated mice were stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red.(b) Representative histograms of mitochondrial content in CD71−Ter119+ cells. Numbers indicate the population of mitochondrialow cells.(c) The percentage of CD71−Ter119+ cells without mitochondria was determined by gating the mitochondrialow fraction, as shown in (b) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(d) Percent mitochondrialow cells in the CD71−Ter119+ EC of Atg7F/FCre embryo. Erythroid cells from the liver and peripheral blood of WT and Atg7F/FCre embryo (E18.5) were stained with anti-Ter119, anti-CD71, and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test).(e) Percentage of mitochondrialow cells in Syto16low cells. Liver erythroid cells (E18.5) were stained with Syto16 (DNA) and Mitotracker Deep Red. Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig. 5, the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction (mean±s.d., n=6). *P0.05 versus value of WT (ANOVA); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(f) Immunofluorescent analysis of mitochondrial and lysosomal proteins. Liver Ter119+ cells (E18.5) of the indicated mice were stained with anti-Tom20 (mitochondrial marker) and anti-Lamp2 (lysosomal marker) antibodies, and observed by fluorescent microscopy. Scale bar, 2 μm. Green, red, white, and blue indicate Tom20, Lamp2, Ter119 and DAPI (DNA), respectively. Mitochondrial and lysosomal markers have separate distributions in Ulk1gt/gt cells and DKO cells.


f4 ( a - f ) Representative EM of WT ( a ) , ATG5 − / − ( b ) , Ulk1gt / gt ( c , d ) and DKO ( e , f ) reticulocytes from embryonic liver ( E14 . 5 ) . ( a , b ) Mitophagy was detected in WT and ATG5 − / − cells ( scale bar , 1 μm ) . Insets indicate mitophagy ( scale bar , 0 . 2 μm ) . ( c , e ) In Ulk1gt / gt and DKO cells , numerous mitochondria were in contact with membrane structures ( scale bar , 1 μm ) . Enlarged images are shown on the right side ( scale bar , 0 . 2 μm ) . ( d , f ) Some Ulk1gt / gt and DKO cells showed plasma membrane blebs containing mitochondria ( scale bar , 1 μm ) . ( g , h ) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18 . 5 . Population of reticulocytes with mitophagy ( g ) , and number of mitochondria per reticulocytes ( h ) were calculated ( n > 35 cells per mouse ) . The data are shown as mean ± s . d . ( n = 3 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4(a-f) Representative EM of WT (a), ATG5−/− (b), Ulk1gt/gt (c,d) and DKO (e,f) reticulocytes from embryonic liver (E14.5). (a,b) Mitophagy was detected in WT and ATG5−/− cells (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm). (c,e) In Ulk1gt/gt and DKO cells, numerous mitochondria were in contact with membrane structures (scale bar, 1 μm). Enlarged images are shown on the right side (scale bar, 0.2 μm). (d,f) Some Ulk1gt/gt and DKO cells showed plasma membrane blebs containing mitochondria (scale bar, 1 μm). (g,h) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18.5. Population of reticulocytes with mitophagy (g), and number of mitochondria per reticulocytes (h) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).


f5 ( a ) Erythroblasts ( EB ; mitochondriahigh Syto16high ) , reticulocytes ( RC ; mitochondriahigh Syto16low ) and EC ( EC ; Ter119 + PBC ) were collected from the indicated embryos ( E18 . 5 ) , and the expression of each protein was examined by western blot . The asterisk indicates a nonspecific band . Uncropped images are shown in Supplementary Figs 11 and 12 . ( b ) Semi - quantitative analysis of the expression level of each protein and the LC3 - II / LC3 - I ratio ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT EC ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − EC ( ANOVA ) ; ' NS ' indicates not significant versus value of WT EC ( ANOVA ) . ( c ) Modification of Ulk1 and Atg13 during erythrocyte maturation . Cell lysates were treated with λ - phosphatase ( 400 U ) for 45 min at 30 ° C , and band shift was examined by anti - Ulk1 and anti - Atg13 antibodies . Asterisk indicates non - specific band . Uncropped images are shown in Supplementary Fig . 12 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(a) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated embryos (E18.5), and the expression of each protein was examined by western blot. The asterisk indicates a nonspecific band. Uncropped images are shown in Supplementary Figs 11 and 12.(b) Semi-quantitative analysis of the expression level of each protein and the LC3-II/LC3-I ratio (mean±s.d., n=3). *P0.05 versus value of WT EC (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− EC (ANOVA); 'NS' indicates not significant versus value of WT EC (ANOVA).(c) Modification of Ulk1 and Atg13 during erythrocyte maturation. Cell lysates were treated with λ-phosphatase (400 U) for 45 min at 30 °C, and band shift was examined by anti-Ulk1 and anti-Atg13 antibodies. Asterisk indicates non-specific band. Uncropped images are shown in Supplementary Fig. 12.


f6 ( a ) Morphology of cultured erythroid cells on day 0 , 1 and 3 of differentiation . Erythroblasts were purified from embryonic liver ( E14 . 5 ) and incubated for the indicated times in erythrocyte differentiation medium . The cells were stained with a Diff - quick kit . ( b ) Percentage of cells without nuclei ( Syto16low cells ) on the indicated days of differentiation ( mean ± s . d . , n = 6 ) . Loss of nuclei occurred normally in all mice . ( c , d ) Percentage of Syto16low cells without mitochondria . Differentiated cells were stained with Syto16 and Mitotracker Deep Red . Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in ( c ) . Numbers indicate the population of mitochondrialow cells . Representative dot plots are also available in Supplementary Fig . 13 . ( d ) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in ( c ) ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) . ( e - h ) Impact of various drugs on mitochondrial clearance during in vitro differentiation . Differentiated cells from WT embryos were incubated with or without wortmannin ( e ) , 3 - methyladenine ( 3 - MA ) ( f ) , rapamycin ( g ) or compound C ( h ) at the indicated concentrations and time periods . Percentage of mitochondrialow cells / Syto16low cells was measured by flow cytometry ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of no drug ( ANOVA ) . ( i ) Impact of various drugs on mitochondrial clearance during in vitro differentiation . with cells from Atg5 − / − embryos exposed to wortmannin or 3 - MA ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of no drug ( ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f6(a) Morphology of cultured erythroid cells on day 0, 1 and 3 of differentiation. Erythroblasts were purified from embryonic liver (E14.5) and incubated for the indicated times in erythrocyte differentiation medium. The cells were stained with a Diff-quick kit. (b) Percentage of cells without nuclei (Syto16low cells) on the indicated days of differentiation (mean±s.d., n=6). Loss of nuclei occurred normally in all mice.(c,d) Percentage of Syto16low cells without mitochondria. Differentiated cells were stained with Syto16 and Mitotracker Deep Red. Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in (c). Numbers indicate the population of mitochondrialow cells. Representative dot plots are also available in Supplementary Fig. 13. (d) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in (c) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(e-h) Impact of various drugs on mitochondrial clearance during in vitro differentiation. Differentiated cells from WT embryos were incubated with or without wortmannin (e), 3-methyladenine (3-MA) (f), rapamycin (g) or compound C (h) at the indicated concentrations and time periods. Percentage of mitochondrialow cells/Syto16low cells was measured by flow cytometry (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).(i)Impact of various drugs on mitochondrial clearance during in vitro differentiation.with cells from Atg5−/− embryos exposed to wortmannin or 3-MA (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).


f7 ( a ) Number of EC and haemoglobin content in the blood of the indicated mice ( 10 - 20 weeks old ; mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) . ( b ) Analysis of early erythrocyte development . Bone marrow cells were harvested from the indicated mice ( 10 - 20 weeks old ) and stained with anti - cKit and anti - Sca1 antibodies . The population of each cell type was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) . ( c ) Percentage of mitochondria - containing Ter119 + blood cells . Cells in peripheral blood from the indicated mice ( 10 - 20 weeks old ) were stained with anti - Ter119 antibody together with Mitotracker Deep Red . The population of mitochondriahigh cells among Ter119 - positive cells was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( ANOVA ) . ( d ) Percentage of mitochondria - containing Syto16low blood cells . Cells in peripheral blood from the indicated mice ( 10 - 20 weeks old ) were stained with Syto16 and Mitotracker Deep Red . The population of mitochondriahigh cells among Syto16 - negative cells was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( ANOVA ) . ( e ) Representative electron micrographs of bone marrow reticulocytes in 10 week - old WT and Ulk1gt / gt mice ( scale bar , 1 μm ) . Insets indicate mitophagy ( scale bar , 0 . 2 μm ) . ( f , g ) Erythroblasts ( EB ; mitochondriahigh Syto16high ) , reticulocytes ( RC ; mitochondriahigh Syto16low ) and EC ( EC ; Ter119 + PBC ) were collected from the indicated mice ( 10 - 20 weeks old ) , and the expression of each protein was examined by western blot . Uncropped images are shown in Supplementary Fig . 14 . ( g ) Semi - quantitative analysis of the expression level of each protein ( mean ± s . d . , n = 3 ) . ' NS ' indicates not significant versus value of WT EC ( ANOVA ) . ( h , i ) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis . ( h ) Representative images of erythrocyte in PHZ - treated WT and Ulk1gt / gt mice ( 14 weeks old ) . Blood cells were stained with anti - Ter119 ( erythroid cell marker ) and anti - Tom20 ( mitochondrial marker ) antibodies and observed by fluorescence microscopy . Green and red indicate Ter119 and Tom20 , respectively . Scale bar , 10 μm . ( i ) Percentage of mitochondrialow cells among Syto16low cells . Erythroid cells were stained with Syto16 ( DNA ) and Mitotracker Deep Red ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) . Mitochondrial retention was observed in Ulk1gt / gt mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f7(a) Number of EC and haemoglobin content in the blood of the indicated mice (10-20 weeks old; mean±s.d., n=6). *P0.05 versus value of WT (Student's t-test).(b) Analysis of early erythrocyte development. Bone marrow cells were harvested from the indicated mice (10-20 weeks old) and stained with anti-cKit and anti-Sca1 antibodies. The population of each cell type was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)).(c) Percentage of mitochondria-containing Ter119+ blood cells. Cells in peripheral blood from the indicated mice (10-20 weeks old) were stained with anti-Ter119 antibody together with Mitotracker Deep Red. The population of mitochondriahigh cells among Ter119-positive cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA).(d) Percentage of mitochondria-containing Syto16low blood cells. Cells in peripheral blood from the indicated mice (10-20 weeks old) were stained with Syto16 and Mitotracker Deep Red. The population of mitochondriahigh cells among Syto16-negative cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA).(e) Representative electron micrographs of bone marrow reticulocytes in 10 week-old WT and Ulk1gt/gt mice (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm).(f,g) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated mice (10-20 weeks old), and the expression of each protein was examined by western blot. Uncropped images are shown in Supplementary Fig. 14. (g) Semi-quantitative analysis of the expression level of each protein (mean±s.d., n=3). 'NS' indicates not significant versus value of WT EC (ANOVA).(h,i) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis. (h) Representative images of erythrocyte in PHZ-treated WT and Ulk1gt/gt mice (14 weeks old). Blood cells were stained with anti-Ter119 (erythroid cell marker) and anti-Tom20 (mitochondrial marker) antibodies and observed by fluorescence microscopy. Green and red indicate Ter119 and Tom20, respectively. Scale bar, 10 μm. (i) Percentage of mitochondrialow cells among Syto16low cells. Erythroid cells were stained with Syto16 (DNA) and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test). Mitochondrial retention was observed in Ulk1gt/gt mice.


Figure 1Gata6 - WT and Gata6 - KOmye peritoneal macrophages ( pMɸ ) were unstimulated ( - ) or stimulated with TLR2L Pam3CSK4 ( 500 ng . ml - 1 ) , TLR3L Poly I : C ( 1 µg . ml - 1 ) , TLR4L purified LPS ( 100 ng . ml - 1 ) , TLR5L Flagellin ( 100 ng . ml - 1 ) , TLR7 and 8L R848 ( 1 µg . ml - 1 ) or TLR9L CpG ODN1826 ( 5 µM ) . Culture supernatants were collected 24 h after the start of stimulation and IL - 1β and TNF ELISA were performed . n = 5 , two - way ANOVA analysis with Tukeys multiple comparison post - test . IL - 1β ( C ) and TNF ( D ) ELISA of Gata6 - WT and Gata6 - KOmye pMɸ stimulated for 24 h with the indicated LPS concentrations . n = 3 , two - way ANOVA analysis with Sidak ' s multiple comparison post - test . IL - 1β ( E ) and TNF ( F ) ELISA from Gata6 - WT and Gata6 - KOmye pMɸ stimulated with LPS ( 100 ng . ml - 1 ) for the indicated times . n = 3 , two - way ANOVA analysis with Sidak ' s multiple comparison post - test . IL - 1β ELISA of Gata6 - WT and Gata6 - KOmye pMɸ stimulated for 18 h with 100 ng . ml - 1 LPS or recombinant IL - 1 receptor antagonist ( rIL - 1ra ) , n = 4 to 5 , two - way ANOVA analysis with Tukey ' s multiple comparison post - test . IL - 1β ELISA of Gata6 - WT and Gata6 - KOmye pMɸ stimulated for 18 h with 100 ng . ml - 1 LPS or 100 ng . ml - 1 recombinant TNF ( recTNF ) , n = 4 to 5 , two - way ANOVA analysis with Tukey ' s multiple comparison post - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Gata6-WT and Gata6-KOmye peritoneal macrophages (pMɸ) were unstimulated (-) or stimulated with TLR2L Pam3CSK4 (500 ng.ml-1), TLR3L Poly I:C (1 µg.ml-1), TLR4L purified LPS (100 ng.ml-1), TLR5L Flagellin (100 ng.ml-1), TLR7 and 8L R848 (1 µg.ml-1) or TLR9L CpG ODN1826 (5 µM). Culture supernatants were collected 24 h after the start of stimulation and IL-1β and TNF ELISA were performed. n=5, two-way ANOVA analysis with Tukeys multiple comparison post-test.IL-1β (C) and TNF (D) ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 24 h with the indicated LPS concentrations. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.IL-1β (E) and TNF (F) ELISA from Gata6-WT and Gata6-KOmye pMɸ stimulated with LPS (100 ng.ml-1) for the indicated times. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or recombinant IL-1 receptor antagonist (rIL-1ra), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or 100 ng.ml-1 recombinant TNF (recTNF), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.


Figure 2Normalised expression of Tlr and Cd14 genes following microarray analysis performed on unstimulated cell - sorted Gata6 - WT and Gata6 - KOmye pMɸ . Data are shown as mean + / - SEM from three biological replicates . Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin - Hochberg procedure . n . d = non detectable . Mean fluorescence intensity ( MFI ) of extracellular TLR2 , TLR4 and CD14 expression on naïve Gata6 - WT and Gata6 - KOmye pMɸ . n = 4 to 7 individual mice per group . Il1b and Tnf mRNA relative expression of Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h and 6 h respectively . Data shown are representative of at least 3 independent experiments . IL - 1β Western blot protein analysis ( D ) of Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h and 6 h respectively . Data shown are representative of at least 3 independent experiments . Western blot was performed on whole cell lysates . Representative dot plot , percentage and mean fluorescence intensity ( MFI ) analysis of pro - IL - 1β + Gata6 - WT and Gata6 - KOmye pMФ flow cytometry analysis 3 h after stimulation with 100 ng . ml - 1 LPS . n = at least 3 independent experiments . Nlrp3 mRNA relative expression of Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h . Data shown are pooled from 3 independent experiments . Western blot protein analysis of Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 6 h . Data shown are representative of at least 3 independent experiments . Western blot was performed on whole cell lysates . IL - 1β ELISA of supernatants collected from Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS and either vehicle control ( Vh , DMSO ) or 10 µM MCC950 for 24 h ( n = 5 ) . Data shown in ( H ) are pooled from 5 independent replicates . Western blot protein analysis ( I ) of supernatants collected from Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS and either vehicle control ( Vh , DMSO ) or 10 µM MCC950 for 24 h ( n = 5 ) . Caspase1 ( Casp1 ) mRNA relative expression of Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h and 6 h respectively . Data shown are pooled from 3 independent experiments . Western blot protein analysis ( K ) of Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h and 6 h respectively . Data shown are pooled from 3 independent experiments . IL - 1β ELISA of Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS and either vehicle control ( Vh , DMSO ) or Ac - YVAD - cmk for 24 h . Data shown are pooled of 5 independent replicates . IL - 1β ELISA of Gata6 - WT and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h , followed by a 30 min pulse with either vehicle control ( Vh ) , 5 mM ATP or 20 µM Nigericin . Data shown are pooled of 5 independent replicates . Representative picture of confocal immunofluorescence analysis of Gata6 - WT and Gata6 - KOmye pMФ stimulated with 100 ng . ml - 1 LPS for 3 h , followed by a 30 min pulse with 5 mM ATP . The white arrows show ASC specks . Scale bar = 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Normalised expression of Tlr and Cd14 genes following microarray analysis performed on unstimulated cell-sorted Gata6-WT and Gata6-KOmye pMɸ. Data are shown as mean +/- SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin-Hochberg procedure. n.d = non detectable.Mean fluorescence intensity (MFI) of extracellular TLR2, TLR4 and CD14 expression on naïve Gata6-WT and Gata6-KOmye pMɸ. n = 4 to 7 individual mice per group.Il1b and Tnf mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments.IL-1β Western blot protein analysis (D) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.Representative dot plot, percentage and mean fluorescence intensity (MFI) analysis of pro-IL-1β+ Gata6-WT and Gata6-KOmye pMФ flow cytometry analysis 3 h after stimulation with 100 ng.ml-1 LPS. n = at least 3 independent experiments.Nlrp3 mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h. Data shown are pooled from 3 independent experiments.Western blot protein analysis of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 6 h. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.IL-1β ELISA of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5). Data shown in (H) are pooled from 5 independent replicates.Western blot protein analysis (I) of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5).Caspase1 (Casp1) mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.Western blot protein analysis (K) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or Ac-YVAD-cmk for 24 h. Data shown are pooled of 5 independent replicates.IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 µM Nigericin. Data shown are pooled of 5 independent replicates.Representative picture of confocal immunofluorescence analysis of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with 5 mM ATP. The white arrows show ASC specks. Scale bar = 10 µm.


Figure 3IL - 1β ELISA analysis of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ in monoculture or co - cultured ( ratio 1 : 1 ) and stimulated for 24 h with 100 ng . ml - 1 LPS . Data shown are pooled from 3 independent experiments . One - way ANOVA statistical analysis with Tukey ' s multiple comparison test was performed . IL - 1β ELISA analysis of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ co - cultured in the same well ( co - culture ) or using Transwell system ( ratio 1 : 1 ) and stimulated for 24 h with 100 ng . ml - 1 LPS . Data shown are pooled from 3 independent experiments . One - way ANOVA statistical analysis with Tukey ' s multiple comparison test was performed . Microarray analysis of Ptgis , Tbxas1 , Ptges2 and Ptgs1 expression from Gata6 - WT and Gata6 - KOmye pMɸ isolated from naïve mice . Data is shown as mean + / - SEM from three biological replicates . Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin - Hochberg procedure . Western blot analysis of Ptgis protein level of unstimulated pMФ from Gata6 - WT and Gata6 - KOmye mice . Mass spectrometry analysis of 6 - keto - PGF1α , TXB2 and PGE2 content of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated for 3 h with 100 ng . ml - 1 LPS . n = 6 . Two - way ANOVA statistical analysis with Tukey ' s multiple comparison post - test was performed .

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Figure 3IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ in monoculture or co-cultured (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ co-cultured in the same well (co-culture) or using Transwell system (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.Microarray analysis of Ptgis, Tbxas1, Ptges2 and Ptgs1 expression from Gata6-WT and Gata6-KOmye pMɸ isolated from naïve mice. Data is shown as mean +/- SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin-Hochberg procedure.Western blot analysis of Ptgis protein level of unstimulated pMФ from Gata6-WT and Gata6-KOmye mice.Mass spectrometry analysis of 6-keto-PGF1α, TXB2 and PGE2 content of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated for 3 h with 100 ng.ml-1 LPS. n = 6. Two-way ANOVA statistical analysis with Tukey's multiple comparison post-test was performed.


Figure 4IL - 10 ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS . Data shown are representative of at least 5 independent experiments . IL - 10 ( B ) and IL - 1β ( C ) ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , beraprost ( 1 and 10 µM ) , cicaprost ( 1 and 10 µM ) , Iloprost ( 1 or 10 nM ) or vehicle control ( Vh , DMSO ) . Data shown are representative of at least 3 independent experiments . IL - 10 ( D ) and IL - 1β ( E ) ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 µM PGE2 , 10 µM U46619 , 100 µM picotamide or vehicle control ( Vh , methyl acetate ) . Data shown are representative of at least 3 independent experiments . IL - 1β ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 ng . ml - 1 IL - 10 , 10 µM beraprost or vehicle control ( Vh ) . Data shown are representative of at least 3 independent experiments . IL - 1β ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM beraprost , 5 µg . ml - 1 αIL - 10R or isotype antibody . Data shown are representative of at least 3 independent experiments . IL - 1β ( H ) and IL - 10 ( I ) ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM MCC950 , 5 µg . ml - 1 αIL - 10R or isotype antibody All stimulations were performed for 16 h . Data shown are representative of at least 3 independent experiments . IL - 1β ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 ng . ml - 1 IL - 10 and 5 mM ATP . Data shown are a pool of 3 independent experiments .

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Figure 4IL-10 ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS. Data shown are representative of at least 5 independent experiments.IL-10 (B) and IL-1β (C) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, beraprost (1 and 10 µM), cicaprost (1 and 10 µM), Iloprost (1 or 10 nM) or vehicle control (Vh, DMSO). Data shown are representative of at least 3 independent experiments.IL-10 (D) and IL-1β (E) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 10 µM PGE2, 10 µM U46619, 100 µM picotamide or vehicle control (Vh, methyl acetate). Data shown are representative of at least 3 independent experiments.IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10, 10 µM beraprost or vehicle control (Vh). Data shown are representative of at least 3 independent experiments.IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 5 µg.ml-1 αIL-10R or isotype antibody. Data shown are representative of at least 3 independent experiments.IL-1β (H) and IL-10 (I) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM MCC950, 5 µg.ml-1 αIL-10R or isotype antibody All stimulations were performed for 16 h. Data shown are representative of at least 3 independent experiments.IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10 and 5 mM ATP. Data shown are a pool of 3 independent experiments.


Figure 5Flow cytometry analysis of Mitotracker ( MT ) Green and Red integration in Gata6 - WT and Gata6 - KOmye naïve pMɸ ( gated on F4 / 80hi / + Tim4 + ) in vivo , 30 min after intraperitoneal injection ( i . p . ) of 1 µM of Mitotracker Green and Red . Data shown are representative of 3 to 5 independent mice of each genotype and are expressed as mean + / - SEM . Student ' s t test analysis was performed . Flow cytometry analysis of Mitotracker green and red staining in Gata6 - WT and Gata6 - KOmye pMɸ ( gated on F4 / 80hi / + Tim4 + ) unstimulated ( - ) or after LPS ( 100 ng . ml - 1 ) , IL - 10 ( 10 ng . ml - 1 ) , αIL - 10R ( 5 µg . ml - 1 ) or isotype ( 5 µg . ml - 1 ) stimulation for 16 h in vitro . Data shown are pooled from 3 independent experiments and normalized to WT unstimulated . Flow cytometry analysis of MitoSOX staining in Gata6 - WT and Gata6 - KOmye pMɸ ( gated on F4 / 80hi / + Tim4 + ) unstimulated ( - ) or after LPS ( 100 ng . ml - 1 ) , IL - 10 ( 10 ng . ml - 1 ) and αIL - 10R ( 5 µg . ml - 1 ) stimulation for 16 h in vitro . Data shown are pooled from 2 independent experiments and normalized to WT unstimulated . IL - 1β ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 500 µM MitoTempo , 10 mM NAC , 0 . 5 µM MitoQ for 16 h . Data are representative of at least 3 experiments .

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Figure 5Flow cytometry analysis of Mitotracker (MT) Green and Red integration in Gata6-WT and Gata6-KOmye naïve pMɸ (gated on F4/80hi/+ Tim4+) in vivo, 30 min after intraperitoneal injection (i.p.) of 1 µM of Mitotracker Green and Red. Data shown are representative of 3 to 5 independent mice of each genotype and are expressed as mean +/- SEM. Student's t test analysis was performed.Flow cytometry analysis of Mitotracker green and red staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1), αIL-10R (5 µg.ml-1) or isotype (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 3 independent experiments and normalized to WT unstimulated.Flow cytometry analysis of MitoSOX staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1) and αIL-10R (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 2 independent experiments and normalized to WT unstimulated.IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 500 µM MitoTempo, 10 mM NAC, 0.5 µM MitoQ for 16 h. Data are representative of at least 3 experiments.


Figure 6mRNA expression analysis of Il1b , Nlrp3 and Caspase1 ( Casp1 ) of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype . Data are representative of at least 3 independent experiments . Western blot analysis ( left ) and quantification ( right ) of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 6 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 or 5 µg . ml - 1 αIL - 10R . Data are representative of 3 independent experiments . Caspase1 activity analysis of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 16 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype . n = 5 to 8 individual mice . Mean fluorescence intensity ( MFI ) of pro - IL - 1β of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype for 16 h or freshly isolated . n = 6 to 13 individual mice . Data were log transformed before performing statistical analysis . mRNA expression analysis of Il10 and Tnf of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 or 5 µg . ml - 1 αIL - 10R . Data are representative of at least 3 independent experiments .

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Figure 6mRNA expression analysis of Il1b, Nlrp3 and Caspase1 (Casp1) of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype. Data are representative of at least 3 independent experiments.Western blot analysis (left) and quantification (right) of Gata6-WT and Gata6-KOmye pMФ stimulated for 6 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10 or 5 µg.ml-1 αIL-10R. Data are representative of 3 independent experiments.Caspase1 activity analysis of Gata6-WT and Gata6-KOmye pMФ stimulated for 16 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype. n = 5 to 8 individual mice.Mean fluorescence intensity (MFI) of pro-IL-1β of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype for 16 h or freshly isolated. n = 6 to 13 individual mice. Data were log transformed before performing statistical analysis.mRNA expression analysis of Il10 and Tnf of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10 or 5 µg.ml-1 αIL-10R. Data are representative of at least 3 independent experiments.


Figure 2B , C Colocalization of N1 - 54 : mVenus : OsCRT3 with OFP - HDEL . OFP - HDEL is an ER lumen marker harboring a C - terminal HDEL ER - retention sequence . ( C ) shows a magnification of the box represented in ( B ) . Bar : 20 μm . D Colocalization analyses along the white arrow in ( C ) . E , F Colocalization of N1 - 54 : mVenus : OsCRT3 with the ER membrane marker AtCNX1 - mCherry . ( F ) shows a magnification of the box represented in ( E ) . Bar : 20 μm . G Colocalization analyses along the white arrow in ( F ) .

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Figure 2B, C Colocalization of N1-54:mVenus:OsCRT3 with OFP-HDEL. OFP-HDEL is an ER lumen marker harboring a C-terminal HDEL ER-retention sequence. (C) shows a magnification of the box represented in (B). Bar: 20 μm. D Colocalization analyses along the white arrow in (C).E, F Colocalization of N1-54:mVenus:OsCRT3 with the ER membrane marker AtCNX1-mCherry. (F) shows a magnification of the box represented in (E). Bar: 20 μm. G Colocalization analyses along the white arrow in (F).


Figure 3C Cold response of cytosolic free Ca2 + concentration in root cells expressing NES - YC3 . 6 . Superimposition of representative normalized ratio variations for wild - type , oscrt3 - 1 , and the complemented transgenic pOsCRT3 root cells . The rectangle represents the regions of interest ( ROIs ) for the ratio measurements . The blue background indicates duration of cold treatment . Bar : 50 μm .

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Figure 3C Cold response of cytosolic free Ca2+ concentration in root cells expressing NES-YC3.6. Superimposition of representative normalized ratio variations for wild-type, oscrt3-1, and the complemented transgenic pOsCRT3 root cells. The rectangle represents the regions of interest (ROIs) for the ratio measurements. The blue background indicates duration of cold treatment. Bar: 50 μm.


Figure 4B Yeast two - hybrid analysis to test the OsCRT3 - OsCIPK7 interaction . The interactions between OsCRT3 , OsCRT3N , OsCRT3P or OsCRT3C with OsCIPK7 , OsCIPK7N , OsCIPK7C , OsCIPK7NAF , or OsCIPK7PPI were verified . C Pull - down assay of the interaction between OsCRT3 and OsCIPK7 . E Localization of OsCIPK7 - GFP in N . benthamiana leaf epidermal cells . OFP - HDEL was used as an ER marker . Bar : 20 μm . F Magnification of the box represented in ( E ) . Colocalization analyses along the white arrow are shown . Bar : 20 μm .

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Figure 4B Yeast two-hybrid analysis to test the OsCRT3-OsCIPK7 interaction. The interactions between OsCRT3, OsCRT3N, OsCRT3P or OsCRT3C with OsCIPK7, OsCIPK7N, OsCIPK7C, OsCIPK7NAF, or OsCIPK7PPI were verified.C Pull-down assay of the interaction between OsCRT3 and OsCIPK7.E Localization of OsCIPK7-GFP in N. benthamiana leaf epidermal cells. OFP-HDEL was used as an ER marker. Bar: 20 μm. F Magnification of the box represented in (E). Colocalization analyses along the white arrow are shown. Bar: 20 μm.


Figure 5A OsCIPK7 interacts with OsCBLs in a yeast two - hybrid system . Interactions were verified by growing the yeast on selective medium ( SD / - Leu - Trp , SD / - Leu - Trp - His - Ade ) and conducting β - Galactosidase assays . B Subcellular localization of OsCBL7 and OsCBL8 in N . benthamiana leaf epidermal cells . Pictures were taken after 2 and 3 days of expression . Bar : 50 μm . C Bimolecular fluorescence complementation ( BiFC ) assay confirming the interaction between OsCBL7 / 8 and OsCIPK7 in N . benthamiana leaf epidermal cells . 19K was used as a negative control . Bar : 50 μm . D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three - hybrid assay . E Constructs and OsCRT3 protein expression level in ( D ) . F Luciferase Complementation Imaging assay ( LCI ) to detect the interaction between OsCIPK7 - nluc and OsCBL7 - cluc or OsCBL8 - cluc , with co - expression of N1 - 54 : mVenus : OsCRT3 ( mVenus - OsCRT3 ) or GFP , respectively . Quantification of luciferase activity was measured and shown . Values are mean ± SD . At least 6 leaves were used for each sample . * P < 0 . 05 , Student ' s t - test . Experiments were repeated at least twice with similar results . G Immunoblot confirming the expression of OsCIPK7 , OsCBL7 , OsCBL8 , and OsCRT3 in ( F ) respectively . H Pull - down assay showing that the interaction between OsCIPK7 and OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3 . MBP was used as a negative control . Relative expression analysis was conducted using Image J software .

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Figure 5A OsCIPK7 interacts with OsCBLs in a yeast two-hybrid system. Interactions were verified by growing the yeast on selective medium (SD/-Leu-Trp, SD/-Leu-Trp-His-Ade) and conducting β-Galactosidase assays.B Subcellular localization of OsCBL7 and OsCBL8 in N. benthamiana leaf epidermal cells. Pictures were taken after 2 and 3 days of expression. Bar: 50 μm.C Bimolecular fluorescence complementation (BiFC) assay confirming the interaction between OsCBL7/8 and OsCIPK7 in N. benthamiana leaf epidermal cells. 19K was used as a negative control. Bar: 50 μm.D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three-hybrid assay.E Constructs and OsCRT3 protein expression level in (D).F Luciferase Complementation Imaging assay (LCI) to detect the interaction between OsCIPK7-nluc and OsCBL7-cluc or OsCBL8-cluc, with co-expression of N1-54:mVenus:OsCRT3 (mVenus-OsCRT3) or GFP, respectively. Quantification of luciferase activity was measured and shown. Values are mean ± SD. At least 6 leaves were used for each sample. *P < 0.05, Student's t-test. Experiments were repeated at least twice with similar results.G Immunoblot confirming the expression of OsCIPK7, OsCBL7, OsCBL8, and OsCRT3 in (F) respectively.H Pull-down assay showing that the interaction between OsCIPK7 and OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3. MBP was used as a negative control. Relative expression analysis was conducted using Image J software.


Figure 7A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro . The loaded protein is indicated by red arrows . Autoradiograph of γ - 32P - labeled OsCIPK7 indicates autophosphorylation of OsCIPK7 . B OsCIPK7 didn ' t phosphorylate OsCBL7 or OsCBL8 in vitro . Loaded proteins stained with Coomassie brilliant blue ( CBB ) are shown in the top panel and indicated by red arrows . Autoradiograph of γ - 32P - labeled myelin basic protein and OsCIPK7 are shown in the bottom panel . C The kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 protein . SDS - PAGE gel showing CBB - stained OsCRT3 , myelin basic protein , and OsCIPK7 ( top panel ) . γ - 32P - labeled myelin basic protein and OsCIPK7 substrate phosphorylation ( bottom panel ) . The amount of OsCRT3 added was 0 μg , 0 . 2 μg , 0 . 4 μg , 0 . 6 μg , 0 . 8 μg in the SDS - PAGE gel lanes , respectively .

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Figure 7A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro. The loaded protein is indicated by red arrows. Autoradiograph of γ-32P-labeled OsCIPK7 indicates autophosphorylation of OsCIPK7.B OsCIPK7 didn't phosphorylate OsCBL7 or OsCBL8 in vitro. Loaded proteins stained with Coomassie brilliant blue (CBB) are shown in the top panel and indicated by red arrows. Autoradiograph of γ-32P-labeled myelin basic protein and OsCIPK7 are shown in the bottom panel.C The kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 protein. SDS-PAGE gel showing CBB-stained OsCRT3, myelin basic protein, and OsCIPK7 (top panel). γ-32P-labeled myelin basic protein and OsCIPK7 substrate phosphorylation (bottom panel). The amount of OsCRT3 added was 0 μg, 0.2 μg, 0.4 μg, 0.6 μg, 0.8 μg in the SDS-PAGE gel lanes, respectively.


Figure 1A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000 . Primary spheres were re - plated to evaluate secondary sphere formation . Bars indicate mean ± s . d . of 10 wells . B Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16 . ( n = 3 ) . C mRNA levels of the indicated genes in GB2 , GB4 and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT - PCR . The results were normalized with the values for GAPDH . Bars indicate mean ± s . d . ( n = 3 - 4 ) . The sphere formation capacity of CD133 - positive and CD133 - negative cells sorted by FACS directly from a tumor sample . GB17 was infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Bars indicate mean ± s . d . of 8 wells . E The sphere formation capacity of CD133 - positive and CD133 - negative cells sorted by FACS directly from a tumor sample . ( Left panel ) GB18 was treated with AGK2 ( 10 μM ) or DMSO . ( Right panel ) Secondary sphere formation of GB18 was examined in the absence of AGK2 . Bars indicate mean ± s . d . of 8 wells .

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Figure 1A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000. Primary spheres were re-plated to evaluate secondary sphere formation. Bars indicate mean ± s.d. of 10 wells.B Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16. (n = 3).C mRNA levels of the indicated genes in GB2, GB4 and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT-PCR. The results were normalized with the values for GAPDH. Bars indicate mean ± s.d. (n = 3-4).The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. GB17 was infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Bars indicate mean ± s.d. of 8 wells.E The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. (Left panel) GB18 was treated with AGK2 (10 μM) or DMSO. (Right panel) Secondary sphere formation of GB18 was examined in the absence of AGK2. Bars indicate mean ± s.d. of 8 wells.


Figure 2A Kaplan - Meier overall survival curves of mice transplanted with GB2 cells ( Left panel ) , GB13 cells ( Middle panel ) or GB16 cells ( Right panel ) infected with the indicated lentivirus ( 1 × 104 cells , 7 mice per group ) . The y - axis indicates the percent survival . B Mice were transplanted with the indicated number of GB2 cells infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Six weeks after transplantation , mice ( 3 or 4 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA were quantified by qRT - PCR . C H & E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Scale bar , 1 mm . An image with higher magnification is shown on the right ( scale bar , 50 μm ) . D The effect of AK7 on the deacetylation of acetyl - tubulin ( Left panel ) and the proliferation of GB2 cells ( Right panel ) . ( Left panel ) Immunoblotting analysis of the effect of AK7 ( 20 μM ) on deacetylation of acetyl - tubulin / tubulin were performed on day 3 in Right panel . Bars indicate mean ± s . d . ( n = 3 ) . E Ten days after intracranial transplantation of GB2 cells ( 1 . 0 × 104 cells ) , AK7 was intraperitoneally administrated for 4 weeks ( 15mg / kg , twice / week ) . After eight weeks , mice ( 4 or 5 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA was quantified by qRT - PCR .

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Figure 2A Kaplan-Meier overall survival curves of mice transplanted with GB2 cells (Left panel), GB13 cells (Middle panel) or GB16 cells (Right panel) infected with the indicated lentivirus (1 × 104 cells, 7 mice per group). The y-axis indicates the percent survival.B Mice were transplanted with the indicated number of GB2 cells infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Six weeks after transplantation, mice (3 or 4 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.C H&E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control (Empty) or shSIRT2-expressing (shS2#1) lentivirus. Scale bar, 1 mm. An image with higher magnification is shown on the right (scale bar, 50 μm).D The effect of AK7 on the deacetylation of acetyl-tubulin (Left panel) and the proliferation of GB2 cells (Right panel). (Left panel) Immunoblotting analysis of the effect of AK7 (20 μM) on deacetylation of acetyl-tubulin/tubulin were performed on day 3 in Right panel. Bars indicate mean ± s.d. (n = 3).E Ten days after intracranial transplantation of GB2 cells (1.0 × 104 cells), AK7 was intraperitoneally administrated for 4 weeks (15mg/kg, twice/week). After eight weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA was quantified by qRT-PCR.


Figure 3B Immunoblotting analysis of p73 in GB2 cells infected with a control ( Empty ) or shTP73 - expressing lentivirus for 96 h . C Growth curve of GB16 cells infected with the indicated lentiviruses . Bars indicate mean ± s . d . ( n = 3 ) . Ectopic expression of the ∆ N isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells . Four days after lentiviral infection , the expression levels of PUMA and GADD45 mRNA were measured by qRT - PCR . Bars indicate mean ± s . d . ( n = 3 ) . E GB2 and GB4 cells [ 5 × 104 and 2 × 104 cells , respectively ( indicated by the dashed lines ) ] were infected with the indicated lentiviruses . After 96 h , the number of viable cells was counted . Bars indicate mean ± s . d . ( n = 4 ) . F HEK293 cells were transfected with TAp73α along with ΔNp73β . PUMA mRNA was measured by qRT - PCR analyses . Bars indicate mean ± s . d . ( n = 3 ) . GB2 cells ( 5 × 104 cells , dashed line ) were infected with the indicated lentiviruses , respectively . After 96 h , the number of viable cells was counted . shTP73 corresponds to both α and β isoforms of p73 . Bars indicate mean ± s . d . ( n = 4 ) . H GB2 cells were transfected with TAp73α along with NLS - SIRT2 ( nuclear - localizing mutant of SIRT2 ) or 3mut ( deacetylase - inactive , nuclear - localizing mutant of SIRT2 ) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene ( Left panel ) . Reporter activities were determined by Dual luciferase assays ( Right panel ) . Bars indicate mean ± s . d . ( n = 3 ) . I GB2 cells were electroporated with TAp73α along with NLS - SIRT2 ( nuclear - localizing mutant of SIRT2 ) or 3mut ( deacetylase - inactive , nuclear - localizing mutant of SIRT2 ) . The expression of PUMA and GADD45 was measured by qRT - PCR analysis . Bars indicate mean ± s . d . ( n = 3 )

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Figure 3B Immunoblotting analysis of p73 in GB2 cells infected with a control (Empty) or shTP73-expressing lentivirus for 96 h.C Growth curve of GB16 cells infected with the indicated lentiviruses. Bars indicate mean ± s.d. (n = 3).Ectopic expression of the ∆N isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells. Four days after lentiviral infection, the expression levels of PUMA and GADD45 mRNA were measured by qRT-PCR. Bars indicate mean ± s.d. (n = 3).E GB2 and GB4 cells [5 × 104 and 2 × 104 cells, respectively (indicated by the dashed lines)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).F HEK293 cells were transfected with TAp73α along with ΔNp73β. PUMA mRNA was measured by qRT-PCR analyses. Bars indicate mean ± s.d. (n = 3).GB2 cells (5 × 104 cells, dashed line) were infected with the indicated lentiviruses, respectively. After 96 h, the number of viable cells was counted. shTP73 corresponds to both α and β isoforms of p73. Bars indicate mean ± s.d. (n = 4).H GB2 cells were transfected with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene (Left panel). Reporter activities were determined by Dual luciferase assays (Right panel). Bars indicate mean ± s.d. (n = 3).I GB2 cells were electroporated with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2). The expression of PUMA and GADD45 was measured by qRT-PCR analysis. Bars indicate mean ± s.d. (n = 3)


Figure 4A Cells transfected with FLAG - tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti - FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG . Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO , respectively , for 6 h and subjected to immunoprecipitation with anti - FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG . E HEK293T cells transfected with FLAG - tagged TAp73α were treated with 20 μM AGK2 ( pre - AGK2 + ) or DMSO ( pre - AGK2 - ) for 6 h . p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 ( 10 U ) , NAD ( 1 mM ) and / or AGK2 ( 3 . 5 μM ) as indicated . F qRT - PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses . Bars indicate mean ± s . e . m . ( n = 4 - 5 ) . G Expression of p73 in ( F ) was determined by immunoblotting with anti - p73 antibody . RFP was used as a control . H GB glioblastima neurospheres [ 5 × 104 cells ( indicated by the dashed line ) ] were infected with the indicated lentiviruses . After 96 h , the number of viable cells was counted . Bars indicate mean ± s . d . ( n = 4 ) . GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice . After 8 weeks , mice ( 3 to 5 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA were quantified by qRT - PCR .

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Figure 4A Cells transfected with FLAG-tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.E HEK293T cells transfected with FLAG-tagged TAp73α were treated with 20 μM AGK2 (pre-AGK2 +) or DMSO (pre-AGK2 -) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM) and/or AGK2 (3.5 μM) as indicated.F qRT-PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses. Bars indicate mean ± s.e.m. (n = 4-5).G Expression of p73 in (F) was determined by immunoblotting with anti-p73 antibody. RFP was used as a control.H GB glioblastima neurospheres [5 × 104 cells (indicated by the dashed line)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3 to 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.


Figure 1 ( A ) Ribbon representation of the Atg12 - Atg5 conjugate bound to Atg16N . Atg12 , Atg5 and Atg6 are coloured salmon pink , green and orange , respectively . Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model . The disordered C ‐ terminal region of Atg12 ( residues 182 - 184 ) is indicated with a broken line . The left figure was obtained by a 90 ° rotation of the right figure along the vertical axis . The amino ‐ and carboxy ‐ termini are denoted as N and C , respectively . All of the figures representing the molecular structures were generated with PyMOL [ 26 ] . ( B ) Stereo view of the detailed interactions between Atg12 and Atg5 . The side chains of the residues involved in the Atg12 - Atg5 interaction are shown with a stick model , in which nitrogen and oxygen atoms are coloured red and blue , respectively . ( C ) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149 . The simulated annealing Fo - Fc difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 , and is shown with black meshes at 5 . 0σ . ( D ) Superimposition of the Atg5 ‐ Atg16N complex structure ( PDB ID 2DYM ) on that of the Atg12 - Atg5 ‐ Atg16N complex . The Atg12 - Atg5 ‐ Atg16N complex is coloured red , while the Atg5 ‐ Atg16N complex is coloured grey . Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model . ( E ) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non ‐ covalent Atg12 ‐ Atg5 interactions . Yeast cells with or without starvation were lysed and subjected to urea SDS - polyacrylamide gel electrophoresis ( PAGE ) followed by western blotting . Ape1 , aminopeptidase I ; mApe1 , mature form of Ape1 ; PE , phosphatidylethanolamine ; prApe1 , preform of Ape1 ; WT , wild ‐ type .

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Figure 1(A) Ribbon representation of the Atg12-Atg5 conjugate bound to Atg16N. Atg12, Atg5 and Atg6 are coloured salmon pink, green and orange, respectively. Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model. The disordered C‐terminal region of Atg12 (residues 182-184) is indicated with a broken line. The left figure was obtained by a 90° rotation of the right figure along the vertical axis. The amino‐ and carboxy‐termini are denoted as N and C, respectively. All of the figures representing the molecular structures were generated with PyMOL [ 26].(B) Stereo view of the detailed interactions between Atg12 and Atg5. The side chains of the residues involved in the Atg12-Atg5 interaction are shown with a stick model, in which nitrogen and oxygen atoms are coloured red and blue, respectively.(C) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149. The simulated annealing Fo-Fc difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149, and is shown with black meshes at 5.0σ.(D) Superimposition of the Atg5‐Atg16N complex structure (PDB ID 2DYM) on that of the Atg12-Atg5‐Atg16N complex. The Atg12-Atg5‐Atg16N complex is coloured red, while the Atg5‐Atg16N complex is coloured grey. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model.(E) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non‐covalent Atg12‐Atg5 interactions. Yeast cells with or without starvation were lysed and subjected to urea SDS-polyacrylamide gel electrophoresis (PAGE) followed by western blotting. Ape1, aminopeptidase I; mApe1, mature form of Ape1; PE, phosphatidylethanolamine; prApe1, preform of Ape1; WT, wild‐type.


Figure 2 ( A ) In vitro pull ‐ down assay using recombinant plant Atg homologues . CBB , Coomassie Brilliant Blue staining ; GST , S ; HA , haemagglutinin ; PE , phosphatidylethanolamine ; WT , wild ‐ type . ( B ) Mapping of the mutated residues on the Atg12 - Atg5 ‐ Atg16N complex structure . Atg5 and Atg16N are shown with a ribbon model , whereas Atg12 is shown with both surface and ribbon models . The side chains of Tyr 147 , Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey ( Tyr 147 ) or blue ( Tyr 149 and Phe 154 ) . Corresponding AtAtg12b residues are indicated in parentheses . Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model .

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Figure 2(A) In vitro pull‐down assay using recombinant plant Atg homologues.CBB, Coomassie Brilliant Blue staining; GST, S; HA, haemagglutinin; PE, phosphatidylethanolamine; WT, wild‐type.(B) Mapping of the mutated residues on the Atg12-Atg5‐Atg16N complex structure. Atg5 and Atg16N are shown with a ribbon model, whereas Atg12 is shown with both surface and ribbon models. The side chains of Tyr 147, Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey (Tyr 147) or blue (Tyr 149 and Phe 154). Corresponding AtAtg12b residues are indicated in parentheses. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model.


Figure 1 ( B , C ) Exercise capacity of MCK - KO and WT mice from the low intensity regiment was conducted . ( n = 4 WT , n = 5 KO mice , mean ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( E , F ) Exercise capacity of MCK - KO and WT mice from the high intensity regiment ( n = 5 mice , means ± SEM , two - tailed student ' s t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B, C) Exercise capacity of MCK-KO and WT mice from the low intensity regiment was conducted. (n = 4 WT, n = 5 KO mice, mean ±SEM, * p < 0.05, two-tailed student's t-test).(E, F) Exercise capacity of MCK-KO and WT mice from the high intensity regiment (n = 5 mice, means ± SEM, two-tailed student's t-test).


Figure 2 ( A ) The H & E staining of MCK - KO and WT soleus muscle after a bout of low intensity treadmill running ( top 40X , bottom 100X magnifications ) . Black arrow indicates centralized nuclei . ( B ) The percentage of myofibers with centralized nuclei was determined by manual counting 300 myofibers in 20X magnification . ( n = 3 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( C - D ) The analogous set of data is shown with WT and MCK - KO GA muscles . ( n = 3 , means ± SEM , two - tailed student ' s t - test . GA : Gastrocnemius ) Hydrogen peroxide ( H2O2 ) levels were measured in WT and MCK - KO soleus ( E ) at rest and after a bout of exercise ( n = 6 per group , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) The H&E staining of MCK-KO and WT soleus muscle after a bout of low intensity treadmill running (top 40X, bottom 100X magnifications). Black arrow indicates centralized nuclei. (B) The percentage of myofibers with centralized nuclei was determined by manual counting 300 myofibers in 20X magnification. (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test). (C-D) The analogous set of data is shown with WT and MCK-KO GA muscles. (n = 3, means ± SEM, two-tailed student's t-test. GA: Gastrocnemius)Hydrogen peroxide (H2O2) levels were measured in WT and MCK-KO soleus (E) at rest and after a bout of exercise (n = 6 per group, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).