Expression profiles of HuR , PP2A and miRNA let - 7a are connected to IL - 6 expression in RAW 264 . 7 cells . Data obtained from experiments described in previous figures are summed up to plot the changes in IL - 6 mRNA levels against time of LPS treatment along with changes in PP2A , miRNA let - 7a and HuR ( D ) .

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Expression profiles of HuR, PP2A and miRNA let-7a are connected to IL-6 expression in RAW 264.7 cells. Data obtained from experiments described in previous figures are summed up to plot the changes in IL-6 mRNA levels against time of LPS treatment along with changes in PP2A, miRNA let-7a and HuR (D).


Change in expression of IL - 6 is connected to changes in let - 7a concentration . The mathematical equation fitting the curve are shown here . Y represents concentration of IL - 6 and X represents miRNA let - 7a concentration . A1 is the initial IL - 6 levels and A2 is the changed level . LogX0 is the mid - point of value 0 . 34 , a concentration of let - 7a where Il - 6 expression is reduced to

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Change in expression of IL-6 is connected to changes in let-7a concentration. The mathematical equation fitting the curve are shown here. Y represents concentration of IL-6 and X represents miRNA let-7a concentration. A1 is the initial IL-6 levels and A2 is the changed level. LogX0 is the mid-point of value 0.34, a concentration of let-7a where Il-6 expression is reduced to half


variations of IL - 6 expression with the altered level of PP2A ( black ) and HuR ( red ) . For both cases , the data were fitted to an equation , which is shown above . The parameter m denotes the expression level of PP2A / HuR which corresponds to the maximum IL - 6 expression ; w denotes the width of the distributions for both cases . For PP2A and HuR , the values of m were found to be 0 . 6 and 0 . 9 respectively and A is a constant ( F ) .

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variations of IL-6 expression with the altered level of PP2A (black) and HuR (red). For both cases, the data were fitted to an equation, which is shown above. The parameter m denotes the expression level of PP2A/HuR which corresponds to the maximum IL-6 expression; w denotes the width of the distributions for both cases. For PP2A and HuR, the values of m were found to be 0.6 and 0.9 respectively and A is a constant (F).


Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on internalized parasite number and cytokine expression in RAW 264 . 7 cells . HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells . HA - HuR was immunostained with anti - HA antibody and detected by secondary antibody tagged with AlexaR564 ( A ) .

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Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number and cytokine expression in RAW 264.7 cells. HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. HA-HuR was immunostained with anti-HA antibody and detected by secondary antibody tagged with AlexaR564 (A).


Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on internalized parasite number HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells . In the zoomed part of the merged picture are used to show the internalized parasite . Internalized parasites present in HA - HuR positive cells were counted and compared against untransfected cells without HA - HuR expression ( B ) .

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Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. In the zoomed part of the merged picture are used to show the internalized parasite. Internalized parasites present in HA-HuR positive cells were counted and compared against untransfected cells without HA-HuR expression (B).


Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on cytokine expression in RAW 264 . 7 cells . HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Levels of expression of different cytokines are measured in control or HA - HuR expressing RAW 264 . 7 cells infected with Ld ( C ) . Scale bar 20 µm . Values are mean + / - s . e . m . and , n = 3 .

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Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on cytokine expression in RAW 264.7 cells. HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Levels of expression of different cytokines are measured in control or HA-HuR expressing RAW 264.7 cells infected with Ld (C). Scale bar 20 µm. Values are mean+/- s.e.m. and, n=3.


Effect of Ld infection on the HuR expression in mouse liver . BALB / c mice of 6 weeks of age were infected with Ld via cardiac puncture . Animals were sacrificed after subsequent days ; protein was extracted from liver tissue followed by western blot analysis for HuR . β - Actin was used as loading control ( D , left panel ) . Each lane have designated individual animal . Band intensities were determined by ImageJ . In each case , HuR level was normalized by its respective β - Actin bands ( D , right panel mean + / - s . e . m . , n = 5 ) .

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Effect of Ld infection on the HuR expression in mouse liver. BALB/c mice of 6 weeks of age were infected with Ld via cardiac puncture. Animals were sacrificed after subsequent days; protein was extracted from liver tissue followed by western blot analysis for HuR. β- Actin was used as loading control (D, left panel). Each lane have designated individual animal. Band intensities were determined by ImageJ. In each case, HuR level was normalized by its respective β- Actin bands (D, right panel mean +/- s.e.m., n=5).


Animals were injected with GP63 containing liposome ( gp1 - 3 ) or empty liposome ( C1 - 2 ) and after 24 hrs of injection , HuR level was checked against β - Actin in liver tissue . β - Actin was used as loading control ( E ) .

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Animals were injected with GP63 containing liposome (gp1-3) or empty liposome (C1-2) and after 24 hrs of injection, HuR level was checked against β-Actin in liver tissue. β-Actin was used as loading control (E).


Effect of expression of HA - HuR on Ld infection in mice liver . Scheme for animal experiment done with HA - HuR expression in liver is shown . Also the HA - HuR level was detected in the liver tissue by western blot analysis using anti - HA antibody ( F ) .

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Effect of expression of HA-HuR on Ld infection in mice liver. Scheme for animal experiment done with HA-HuR expression in liver is shown. Also the HA-HuR level was detected in the liver tissue by western blot analysis using anti-HA antibody (F).


Effect of expression of HA - HuR on Ld infection in mice liver . Parasite load in liver tissues were estimated by measuring Ld DNA in infected tissue using specific primer for Ld minicircle kDNA . Values are mean + / - s . e . m . and , n = 5 ( G ) .

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Effect of expression of HA-HuR on Ld infection in mice liver. Parasite load in liver tissues were estimated by measuring Ld DNA in infected tissue using specific primer for Ld minicircle kDNA. Values are mean+/- s.e.m. and, n=5 (G).


Effect of expression of HA - HuR on Ld infection in mice liver . TNF - α ( Values are mean + / - s . e . m . and , n = 5 ) and IL - 10 ( Values are mean + / - s . e . m . and , n = 4 ) mRNA levels were checked from RNA isolated from liver tissues and 18S rRNA normalized Ct values were plotted ( H and I , left panels ) . Relative fold change was calculated by ΔΔCt method and the values plotted for Ld count , TNF - α and IL - 10 mRNA levels ( G mean + / - s . e . m . , ( n = 5 ) , H mean + / - s . e . m . , ( n = 6 ) and I mean + / - s . e . m . , ( n = 4 ) , right panel ) .

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Effect of expression of HA-HuR on Ld infection in mice liver. TNF-α (Values are mean+/- s.e.m. and, n=5) and IL-10 (Values are mean+/- s.e.m. and, n=4) mRNA levels were checked from RNA isolated from liver tissues and 18S rRNA normalized Ct values were plotted (H and I, left panels). Relative fold change was calculated by ΔΔCt method and the values plotted for Ld count, TNF-α and IL-10 mRNA levels (G mean +/- s.e.m., (n=5), H mean +/- s.e.m., (n=6) and I mean +/- s.e.m., (n=4), right panel).


Effect of HA - HuR plasmid tail - vein injection on mice spleen . Levels of HA - HuR were undetectable in mice spleen after expression plasmid was injected . Sample from one liver tissue of HA - HuR injected group was used as a positive control of HA - HuR expression in moue liver after tail vein injection of HuR encoding plasmid ( J ) .

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Effect of HA-HuR plasmid tail-vein injection on mice spleen. Levels of HA-HuR were undetectable in mice spleen after expression plasmid was injected. Sample from one liver tissue of HA-HuR injected group was used as a positive control of HA-HuR expression in moue liver after tail vein injection of HuR encoding plasmid (J).


Effect of HA - HuR plasmid tail - vein injection on mice spleen . Simultaneously , TNF - α mRNA levels were checked in spleen total RNA ( K ) .

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Effect of HA-HuR plasmid tail-vein injection on mice spleen. Simultaneously, TNF-α mRNA levels were checked in spleen total RNA (K).


Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on cellular cytokine level RAW 264 . 7 cells were infected with Antimony sensitive ( LdS - Ag83 ) and antimony resistant ( LdR - BHU569 ) form of Ld for 24 hours along with uninfected cells kept as control . IL - 10 ( A ) and TNF - α ( B ) were measured at mRNA level by qRT - PCR after infection ( mean + / - s . e . m . and , n = 4 ) .

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Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on cellular cytokine level RAW 264.7 cells were infected with Antimony sensitive (LdS-Ag83) and antimony resistant (LdR-BHU569) form of Ld for 24 hours along with uninfected cells kept as control. IL-10 (A) and TNF-α (B) were measured at mRNA level by qRT-PCR after infection (mean+/- s.e.m. and, n=4).


Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on on phospho - ERK level . ( D ) . Densitometry analysis was done for p - ERK level by ImageJ and relative values were plotted ( C ) . β - Actin was used as loading control Values are mean + / - s . e . m . and , n = 3 .

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Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on on phospho-ERK level. (D). Densitometry analysis was done for p-ERK level by ImageJ and relative values were plotted (C). β-Actin was used as loading control Values are mean+/- s.e.m. and, n=3.


Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on phospho - ERK and MDR1 level . p - ERK , ERK and MDR1 level were also measured by western blot ( D ) .

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Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on phospho-ERK and MDR1 level. p-ERK, ERK and MDR1 level were also measured by western blot (D).


Effect of Antimony sensitive and antimony resistant form of the Ld on HuR and PP2A . RAW 264 . 7 cells were infected with LdS and LdR for 12 hrs and HuR level was measured by western blot ( left panel ) . PP2A level was also measured by western blot after 4 and 12 hrs of LdS and LdR infection ( right panel ) . β - Actin was used as loading control .

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Effect of Antimony sensitive and antimony resistant form of the Ld on HuR and PP2A. RAW 264.7 cells were infected with LdS and LdR for 12 hrs and HuR level was measured by western blot (left panel). PP2A level was also measured by western blot after 4 and 12 hrs of LdS and LdR infection (right panel). β-Actin was used as loading control.


Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell . RAW 264 . 7 cells were transfected with HA - HuR or pCIneo followed by 2 hrs of pretreatment with OA . These cells were then infected with LdR for 24 hrs . Pro - inflammatory cytokines TNF - α ( right panel ; mean + / - s . e . m . , n = 3 ) and IL - 1β ( left panel ; mean + / - s . e . m . and , n = 4 ) were measured at mRNA level and relative values are plotted . Values without OA treatment was set as control .

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Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell. RAW 264.7 cells were transfected with HA-HuR or pCIneo followed by 2 hrs of pretreatment with OA. These cells were then infected with LdR for 24 hrs. Pro-inflammatory cytokines TNF-α (right panel; mean+/- s.e.m., n=3) and IL-1β (left panel; mean+/- s.e.m. and, n=4) were measured at mRNA level and relative values are plotted. Values without OA treatment was set as control.


Effect of HuR over expression on LdR infection in mice liver . Pro - inflammatory cytokines TNF - α ( left panel ) and IL - 1β ( right panel ) level were measured at mRNA level in liver ( H ) .

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Effect of HuR over expression on LdR infection in mice liver. Pro-inflammatory cytokines TNF-α (left panel) and IL-1β (right panel) level were measured at mRNA level in liver (H).


Effect of HuR over expression on LdR infection in mice liver . Parasite load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle DNA of Ld ( I ) . Values are mean + / - s . e . m . and , n = 6 ) .

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Effect of HuR over expression on LdR infection in mice liver. Parasite load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle DNA of Ld (I). Values are mean+/- s.e.m. and, n=6).


A Actin expression levels shown by western blotting for strains expressing S . cerevisiae ' s actin protein from various coding sequences , with tubulin ( Tub1p ) as a loading control . B Quantification of actin expression levels , showing a decrease when more silent mutations are present . Data are presented as mean + / - SD ( n = 4 for Sc , n = 8 for ScNI ; n = 12 for Sc [ Ca ] , Sc [ Sp ] and Sc [ At ] ; 2 biological replicates with n / 2 technical replicates each ) . * P < 0 . 05 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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A Actin expression levels shown by western blotting for strains expressing S. cerevisiae's actin protein from various coding sequences, with tubulin (Tub1p) as a loading control. B Quantification of actin expression levels, showing a decrease when more silent mutations are present. Data are presented as mean +/- SD (n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each). *P<0.05 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


F In vivo actin network deviation indexes , defined to evaluate the patch - cable balance compared to S . cerevisiae haploid cells ( value is 0 in S . cerevisiae ' s cells , 1 when cells contain only actin patches and - 1 when cells contain only cables ) . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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F In vivo actin network deviation indexes, defined to evaluate the patch-cable balance compared to S. cerevisiae haploid cells (value is 0 in S. cerevisiae's cells, 1 when cells contain only actin patches and -1 when cells contain only cables). Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


E Phalloidin staining of F - actin organization . Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed . Micrographs of Sc and Sc _ NI cells are reproduced from Fig 2E . Scale bar : 3 µm .

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E Phalloidin staining of F-actin organization. Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed. Micrographs of Sc and Sc_NI cells are reproduced from Fig 2E. Scale bar: 3 µm.


F In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

F In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


I Effect of CK - 666 ( 75 µM ) on the organization of the actin cytoskeleton . Cells were stained with phalloidin after 30 min incubation with CK - 666 . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm J Quantification of actin patch resistance to CK - 666 treatment . Bar graphs represent the percentage of cells with a given number of visible actin patches after CK - 666 treatment . ( n = 41 for ScNI , 55 for N2 , 31 for Ca , 41 for Op , 55 for Hs ) .

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I Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm J Quantification of actin patch resistance to CK-666 treatment. Bar graphs represent the percentage of cells with a given number of visible actin patches after CK-666 treatment. (n = 41 for ScNI, 55 for N2, 31 for Ca, 41 for Op, 55 for Hs).


A ( Top ) Snapshots of actin networks assembled from three different actins sources : Act _ Sc , Act _ N2 and Act _ Ca . ( Bottom left ) Quantification of actin fluorescence on beads . Data are presented as mean + / - SD ( n = 52 for Act _ Sc branched , n = 70 for Act _ N2 branched , n = 81 for Act _ Ca branched , n = 32 for Act _ Sc linear , n = 34 for Act _ N2 linear and n = 47 for Act _ Ca linear ) . * * P < 0 . 01 , * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) . ( Bottom right ) In vitro actin network deviation indexes .

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A (Top) Snapshots of actin networks assembled from three different actins sources: Act_Sc, Act_N2 and Act_Ca. (Bottom left) Quantification of actin fluorescence on beads. Data are presented as mean +/- SD (n = 52 for Act_Sc branched, n = 70 for Act_N2 branched, n = 81 for Act_Ca branched, n = 32 for Act_Sc linear, n = 34 for Act_N2 linear and n = 47 for Act_Ca linear). **P<0.01, ***P<0.001 (Kruskal-Wallis test, with multiple comparisons). (Bottom right) In vitro actin network deviation indexes.


B ( Left ) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa - 488 - labeled ADF / cofilin ( replacement of unlabeled ADF / cofilin ) . ( Right ) Quantification of ADF / cofilin ' s fluorescence intensity relative to actin . Data are presented as mean + / - SD ( n = 34 for Act _ Sc branched , n = 28 for Act _ N2 branched , n = 43 for Act _ Ca branched , n = 24 for Act _ Sc linear , n = 15 for Act _ N2 linear and n = 27 for Act _ Ca linear ) . * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) .

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B (Left) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa-488-labeled ADF/cofilin (replacement of unlabeled ADF/cofilin). (Right) Quantification of ADF/cofilin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 34 for Act_Sc branched, n = 28 for Act_N2 branched, n = 43 for Act_Ca branched, n = 24 for Act_Sc linear, n = 15 for Act_N2 linear and n = 27 for Act_Ca linear). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).


C ( Left ) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488 - tropomyosin . ( Right ) Quantification of tropomyosin ' s fluorescence intensity relative to actin . Data are presented as mean + / - SD ( n = 45 for Act _ Sc branched , n = 63 for Act _ N2 branched , n = 57 for Act _ Ca branched , n = 22 for Act _ Sc linear , n = 32 for Act _ N2 linear and n = 27 for Act _ Ca linear ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

C (Left) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488-tropomyosin. (Right) Quantification of tropomyosin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 45 for Act_Sc branched, n = 63 for Act_N2 branched, n = 57 for Act_Ca branched, n = 22 for Act_Sc linear, n = 32 for Act_N2 linear and n = 27 for Act_Ca linear). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


C Phalloidin staining depicting F - actin organization . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm . D In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) . E Polarity Indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. D In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). E Polarity Indexes. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).


F Effect of CK - 666 ( 75 µM ) on the organization of the actin cytoskeleton . Cells were stained with phalloidin after 30 min incubation with CK - 666 . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm . G In vivo actin network deviation indexes of cells treated with DMSO or CK - 666 . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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

F Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. G In vivo actin network deviation indexes of cells treated with DMSO or CK-666. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


( B ) Coomassie - stained SDS - PAGE of the SEC fractions highlighted in ( A ) with a horizontal dashed line on top .

O O 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) Coomassie-stained SDS-PAGE of the SEC fractions highlighted in (A) with a horizontal dashed line on top.


( C - D ) ITC measurements of purified CEP19 titrated with CrRabL2 in the presence of GTP ( C ) or GDP ( D ) .

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

(C-D) ITC measurements of purified CEP19 titrated with CrRabL2 in the presence of GTP (C) or GDP (D).


( G ) SEC profile ( top ) and Coomassie stained SDS gel ( bottom ) of the complex between RabL2 and a minimal binding - region of CEP19 ( residues 107 - 195 ) demonstrating a direct interaction .

O O O O O O O O O O O O O O O O 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) SEC profile (top) and Coomassie stained SDS gel (bottom) of the complex between RabL2 and a minimal binding-region of CEP19 (residues 107-195) demonstrating a direct interaction.


( A ) SEC profile of CrRabL2Q83L co - purification with CrIFT81 / 74 / 27 / 25 / 22 ( top ) in the presence of non - hydrolysable GTP homologue , GTPγS . Coomassie stained SDS gel of SEC fraction highlighted by dashed lines ( bottom ) . ( B ) SEC profile for the purification of the HsIFT81 / 74 / 27 / 25 / 22 / RabL2 complex in the presence of GTPγS ( top ) and the corresponding Coomassie stained SDS gel ( bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SEC profile of CrRabL2Q83L co-purification with CrIFT81/74/27/25/22 (top) in the presence of non-hydrolysable GTP homologue, GTPγS. Coomassie stained SDS gel of SEC fraction highlighted by dashed lines (bottom). (B) SEC profile for the purification of the HsIFT81/74/27/25/22/RabL2 complex in the presence of GTPγS (top) and the corresponding Coomassie stained SDS gel (bottom).


( E - H ) GTPases assays with the indicated proteins following the release of inorganic phosphate upon GTP hydrolysis as a function of time . Each experiment was carried out in three technical replicates . Concentrations for experiments in ( E ) were 55µM CrIFT - B1 pentamer / CrRabL2 , 60µM CrIFT - B1 pentamer and 70µM CrIFT - B1 tetramer and 1mM GTP . Concentrations for experiments in ( G ) were 60µM protein and 30µM GTP . The curves represent averages and error bars indicate standard deviation of measurements for every 100s for panel ( E ) and every 200s for panel ( G ) . ( F , H ) Quantification of the reaction rates ( arbitrary units of Absorbance ( Abs ) per second ( s ) ) for each experiment shown in panels ( E ) calculated using linear regression of the first 500s and ( H ) using single exponential fit , and agreement with the fit to the curves in ( E , G ) are indicated by the error bars and R2 - value .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) GTPases assays with the indicated proteins following the release of inorganic phosphate upon GTP hydrolysis as a function of time. Each experiment was carried out in three technical replicates. Concentrations for experiments in (E) were 55µM CrIFT-B1 pentamer/CrRabL2, 60µM CrIFT-B1 pentamer and 70µM CrIFT-B1 tetramer and 1mM GTP. Concentrations for experiments in (G) were 60µM protein and 30µM GTP. The curves represent averages and error bars indicate standard deviation of measurements for every 100s for panel (E) and every 200s for panel (G). (F, H) Quantification of the reaction rates (arbitrary units of Absorbance (Abs) per second(s)) for each experiment shown in panels (E) calculated using linear regression of the first 500s and (H) using single exponential fit, and agreement with the fit to the curves in (E, G) are indicated by the error bars and R2-value.


( A ) SEC profile showing that a minimal CrIFT81460 - 533 / CrIFT74460 - 532 complex co - purifies with CrRabL2Q83L in the presence of GTPγS ( left ) . The right panel displays the Coomassie stained SDS gel of SEC fractions ( horizontal top dashed line ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SEC profile showing that a minimal CrIFT81460-533/CrIFT74460-532 complex co-purifies with CrRabL2Q83L in the presence of GTPγS (left). The right panel displays the Coomassie stained SDS gel of SEC fractions (horizontal top dashed line).


( B ) An N - terminal hexa - histidine tagged CrIFT81460 - 533 / 74460 - 532 complex interacts with untagged CrRabL2Q83L in a GTPγS - dependent manner in pulldown assays .

O 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

(B) An N-terminal hexa-histidine tagged CrIFT81460-533/74460-532 complex interacts with untagged CrRabL2Q83L in a GTPγS-dependent manner in pulldown assays.


( E ) Quantification of GTPase reaction rates ( arbitrary units of Absorbance ( Abs ) per second ( s ) ) using single exponential fit ( D ) , error bars and R2 - value indicate the agreement of the fit to the curves in ( D ) .

(E) Quantification of GTPase reaction rates (arbitrary units of Absorbance (Abs) per second(s)) using single exponential fit (D), error bars and R2-value indicate the agreement of the fit to the curves in (D).


( C ) SEC profile showing that human RabL2B co - purifies with Chlamydomonas IFT - B1 pentamer ( left ) . Coomassie stained SDS gel of SEC fractions highlighted with dashed lines on the SEC profile ( right ) .

O O O O O O O O O O O O O O O O O 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) SEC profile showing that human RabL2B co-purifies with Chlamydomonas IFT-B1 pentamer (left). Coomassie stained SDS gel of SEC fractions highlighted with dashed lines on the SEC profile (right).


( E ) Quantification of the reaction rates of the GTPase reaction based on single exponential fit of curves in ( D ) error bars and R2 indicate the agreement of the fit .

O O O O O O O O O O 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) Quantification of the reaction rates of the GTPase reaction based on single exponential fit of curves in (D) error bars and R2 indicate the agreement of the fit.


( B ) Affinity pull - down of CrRabL2 with His - tagged WT or mutant CrIFT74T484R / 81 complex in the presence or absence of nucleotides .

O O O O 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) Affinity pull-down of CrRabL2 with His-tagged WT or mutant CrIFT74T484R/81 complex in the presence or absence of nucleotides.


( E ) Immunoblot ( IB ) analysis of expression of IFT74 , IFT81 , RabL2B and α - tubulin in control ( sgSafe ) or IFT74 KO RPE cells stably expressing either empty vector , FLAG - IFT74 , or FLAG - IFT74 T438R . Representative images from three experimental replicates are shown . The cells were grown to confluence ( without serum starvation ) , lysed and analyzed by Immunoblot . Molecular weights ( kDa ) estimated from a protein marker are indicated . Red asterisk : endogenous IFT74 ; black asterisks : FLAG tagged IFT74 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot (IB) analysis of expression of IFT74, IFT81, RabL2B and α-tubulin in control (sgSafe) or IFT74 KO RPE cells stably expressing either empty vector, FLAG-IFT74, or FLAG-IFT74 T438R. Representative images from three experimental replicates are shown. The cells were grown to confluence (without serum starvation), lysed and analyzed by Immunoblot. Molecular weights (kDa) estimated from a protein marker are indicated. Red asterisk: endogenous IFT74; black asterisks: FLAG tagged IFT74.


( F ) Quantification of the immunoblots of IFT74 shown in ( E ) . Data are combined from three independent experiments . Error bars represent ± SD . Statistics obtained through one - way analysis of variance ( ANOVA ) with Tukey ' s multiple comparison 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

(F) Quantification of the immunoblots of IFT74 shown in (E). Data are combined from three independent experiments. Error bars represent ± SD. Statistics obtained through one-way analysis of variance (ANOVA) with Tukey's multiple comparison test.


( G - I ) Box plots showing centrosomal signal intensity of FLAG ( G ) , IFT81 ( H ) , or RabL2 ( I ) in the cells described in ( D ) serum starved for 24 hours . The relative fluorescence signal intensity compared with the average of the control ( IFT74 KO + FLAG - IFT74 WT for ( G ) and sgSafe + empty for ( H ) and ( I ) ) are shown . Data are combined from three experimental replicates . Statistics obtained through nested one - way ANOVA with Šídák ' s multiple comparison test . ( G ) The number of mother centrioles analyzed in each sample per replicate are : 78 , 77 , 78 ( sgSafe + empty ) ; 102 , 95 , 101 ( IFT74 KO + empty ) ; 54 , 77 , 85 ( IFT74 KO + FLAG - IFT74 WT ) ; 49 , 83 , 99 ( IFT74 KO + FLAG - IFT74 T438R ) , ( H ) The number of mother centrioles analyzed in each sample per replicate are : 93 , 137 , 138 ( sgSafe + empty ) ; 109 , 133 , 193 ( IFT74 KO + empty ) ; 62 , 146 , 131 ( IFT74 KO + FLAG - IFT74 WT ) ; 41 , 143 , 133 ( IFT74 KO + FLAG - IFT74 T438R ) , ( I ) The number of mother centrioles analyzed in each sample per replicate are : 64 , 101 , 106 ( sgSafe + empty ) ; 84 , 105 , 129 ( IFT74 KO + empty ) ; 52 , 96 , 121 ( IFT74 KO + FLAG - IFT74 WT ) ; 73 , 92 , 102 ( IFT74 KO + FLAG - IFT74 T438R ) .

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

(G-I) Box plots showing centrosomal signal intensity of FLAG (G), IFT81 (H), or RabL2 (I) in the cells described in (D) serum starved for 24 hours. The relative fluorescence signal intensity compared with the average of the control (IFT74 KO + FLAG-IFT74 WT for (G) and sgSafe + empty for (H) and (I)) are shown. Data are combined from three experimental replicates. Statistics obtained through nested one-way ANOVA with Šídák's multiple comparison test. (G) The number of mother centrioles analyzed in each sample per replicate are: 78, 77, 78 (sgSafe + empty); 102, 95, 101 (IFT74 KO + empty); 54, 77, 85 (IFT74 KO + FLAG-IFT74 WT); 49, 83, 99 (IFT74 KO + FLAG-IFT74 T438R), (H) The number of mother centrioles analyzed in each sample per replicate are: 93, 137, 138 (sgSafe + empty); 109, 133, 193 (IFT74 KO + empty); 62, 146, 131 (IFT74 KO + FLAG-IFT74 WT); 41, 143, 133 (IFT74 KO + FLAG-IFT74 T438R), (I) The number of mother centrioles analyzed in each sample per replicate are: 64, 101, 106 (sgSafe + empty); 84, 105, 129 (IFT74 KO + empty); 52, 96, 121 (IFT74 KO + FLAG-IFT74 WT); 73, 92, 102 (IFT74 KO + FLAG-IFT74 T438R).


( K ) Box plots showing the quantification of number of IFT88 particles per cilium in the experiment described in ( J ) . Data are combined from three experimental replicates : The number of cilia analyzed in each sample per replicate are : 20 , 20 , 18 ( sgSafe + empty ) ; 20 , 20 , 18 ( IFT74 KO + WT ) ; 20 , 20 , 19 ( IFT74 KO + FLAG - IFT74 WT ) . The number of IFT particles in individual cells in each experiment are available from Figures S7A , D , and G . Statistics obtained through nested one - way ANOVA with Tukey ' s multiple comparison 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

(K) Box plots showing the quantification of number of IFT88 particles per cilium in the experiment described in (J). Data are combined from three experimental replicates: The number of cilia analyzed in each sample per replicate are: 20, 20, 18 (sgSafe + empty); 20, 20, 18 (IFT74 KO + WT); 20, 20, 19 (IFT74 KO + FLAG-IFT74 WT). The number of IFT particles in individual cells in each experiment are available from Figures S7A, D, and G. Statistics obtained through nested one-way ANOVA with Tukey's multiple comparison test.


( B ) Left , orthogonal view of a 20μm tumor section labelled with antibodies against EpCAM ( in red ) and DNA in blue . Right , dot plot bar representing cell heights in cells from tumor sections , n = 60 cells from 1 tumor .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Left, orthogonal view of a 20μm tumor section labelled with antibodies against EpCAM (in red) and DNA in blue. Right, dot plot bar representing cell heights in cells from tumor sections, n=60 cells from 1 tumor.


( C ) On the left , images of low magnification views of healthy tissues labelled with antibodies against pericentrin ( PCNT ) and CDK5RAP2 , shown in red and green , respectively . DNA is in blue . The white dashed squares represent the regions shown in higher magnifications on the right . One centrosome was considered as such when PCNT and CDK5RAP2 signals co - localized ( Inset 1 ) . Lack of co - localization ( Insets 2 and 3 ) was noticed and discarded during quantification .

(C) On the left, images of low magnification views of healthy tissues labelled with antibodies against pericentrin (PCNT) and CDK5RAP2, shown in red and green, respectively. DNA is in blue. The white dashed squares represent the regions shown in higher magnifications on the right. One centrosome was considered as such when PCNT and CDK5RAP2 signals co-localized (Inset 1). Lack of co-localization (Insets 2 and 3) was noticed and discarded during quantification.


( D ) Super - resolution microscopy of healthy tissues labelled for the centriole marker CEP135 ( in green ) and PCNT ( in red ) .

(D) Super-resolution microscopy of healthy tissues labelled for the centriole marker CEP135 (in green) and PCNT (in red).


( E - F ) Representative images of two different fields of the same tumor ( E ) and of a tumor section to highlight different centrososome phenotypes , labelled as described in ( C ) . The pink dashed line highlights an area of cells without centrosomes . The white dashed squares represent the regions shown in higher magnifications in insets in ( G and I ) and the pink dashed linesurrounds nuclei without centrosomes . ( G ) Insets from ( F ) . ( H ) Examples of tumor sections showing small ( left ) and large ( right ) regions with nuclei without centrosomes .

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

(E-F) Representative images of two different fields of the same tumor (E) and of a tumor section to highlight different centrososome phenotypes, labelled as described in (C). The pink dashed line highlights an area of cells without centrosomes. The white dashed squares represent the regions shown in higher magnifications in insets in (G and I) and the pink dashed linesurrounds nuclei without centrosomes. (G) Insets from (F). (H) Examples of tumor sections showing small (left) and large (right) regions with nuclei without centrosomes.


( I ) Insets from ( F ) showing different types of centrosome amplification events .

(I) Insets from (F) showing different types of centrosome amplification events.


( J ) Super - resolution microscopy of tumor tissues labelled as in ( C ) .

(J) Super-resolution microscopy of tumor tissues labelled as in (C).


( K ) Maximum Z - projection of a tumor section labelled with EpCAM antibodies ( Red ) and DNA in blue . The dashed line shows the absence of EpCAM staining , arrows show a disorganized region .

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

(K) Maximum Z-projection of a tumor section labelled with EpCAM antibodies (Red) and DNA in blue. The dashed line shows the absence of EpCAM staining, arrows show a disorganized region.


( 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 .

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

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

(B-C) 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 O O O O O O O O O O O O O O O O O O O O O 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) 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.


( 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 .

(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 .

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

(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 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

(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.


( a ) Expression of Ulk1 and Atg5 - 12 in the indicated erythroid cells .

(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 ) .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O

(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).


( 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 .

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

(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 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 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 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

(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).


( 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 .

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

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

(b) Representative 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 ) .

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

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

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 O O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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).


( 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 .

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

(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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


( 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 .

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

(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 ) .

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

(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 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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


( 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 ) .

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

(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 ) ) .

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

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

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

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(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 ) .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(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.


Gata6 - 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 .

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Gata6-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 .

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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 .

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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 .

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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 .

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.


Normalised 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 .

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Normalised 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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 .

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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 .

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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 .

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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.


IL - 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 .

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IL-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 .

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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 .

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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 .

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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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.


IL - 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 .

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IL-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 .

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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 .

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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.