B . Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90 ( 269 - 722 ) to activate Arp2 / 3 complex . Reactions contained 3 μM 15 % pyrene labeled actin , 50 nM Bos taurus Arp2 / 3 complex , and 20 μM wild type or mutant SPIN90 ( 269 - 722 ) . C . Plot of maximum polymerization rate versus concentration of SPIN90 ( 269 - 722 ) for reactions as shown in panel ( B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90(269-722) to activate Arp2/3 complex. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex, and 20 μM wild type or mutant SPIN90(269-722). C. Plot of maximum polymerization rate versus concentration of SPIN90(269-722) for reactions as shown in panel (B)


A . Plot of maximum polymerization rate versus concentration of the small molecule Arp2 / 3 complex inhibitor CK - 666 . Reactions contained 3 μM 15 % pyrene labeled actin , 50 nM Bos taurus Arp2 / 3 complex and the indicated concentrations of CK - 666

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR

A. Plot of maximum polymerization rate versus concentration of the small molecule Arp2/3 complex inhibitor CK-666. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex and the indicated concentrations of CK-666


B . SDS - PAGE gel showing results of actin filament copelleting assay . Actin filaments at 3 . 5 μM were used to co - pellet 0 . 6 μM Bos taurus Arp2 / 3 complex in the presence or absence of the indicated concentrations of SPIN90 ( 269 - 722 ) ( S : Soluble fraction , P : Pellet ) C . Plot of co - pelleting data as shown in ( B ) , run in quadruplicate with error bars showing the standard

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SDS-PAGE gel showing results of actin filament copelleting assay. Actin filaments at 3.5 μM were used to co-pellet 0.6 μM Bos taurus Arp2/3 complex in the presence or absence of the indicated concentrations of SPIN90(269-722) (S: Soluble fraction, P: Pellet) C. Plot of co-pelleting data as shown in (B), run in quadruplicate with error bars showing the standard deviation


Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA - mediated p97 depletion under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( A ) ( * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM ) .

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

Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA-mediated p97 depletion under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (A) (***P<0.001; unpaired t-test, n=3, mean +SEM).


Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition ( CB5083 , 10µM for 6h ) under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( C ) ( * P < 0 . 05 , * * * * P < 0 . 0001 ; unpaired t - test , n = 3 , mean + SEM ) .

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

Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10µM for 6h) under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (C) (*P<0.05, ****P<0.0001; unpaired t-test, n=3, mean +SEM).


Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline - induced mild expression of the p97EQ variant under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( E ) ( * * P < 0 . 01 , * * * * P < 0 . 0001 ; unpaired t - test , n = 4 , mean + SEM ) .

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

Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline-induced mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (E) (**P<0.01, ****P<0.0001; unpaired t-test, n=4, mean +SEM).


Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA - mediated p97 depletion . Graph represents the quantifications of ( H ) ( * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) and Western blot for efficacy of siRNA depletion of p97 ( right ) .

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

Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA-mediated p97 depletion.   Graph represents the quantifications of (H) (***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM) and Western blot for efficacy of siRNA depletion of p97 (right).


Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing K48 - linked hyper - ubiquitination of RNF8 after siRNA - mediated p97 depletion .

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

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing K48-linked hyper-ubiquitination of RNF8 after siRNA-mediated p97 depletion.


Western blot analysis of Strep - p97 Co - IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( K ) ( * P < 0 . 05 ; unpaired t - test , n = 2 , mean + SEM ) .

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

Western blot analysis of Strep-p97 Co-IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (K) (*P<0.05; unpaired t-test, n=2, mean +SEM).


Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing the ubiquitination pattern of RNF8 - WT and RNF8 - RING * variant , under siRNA - mediated luciferase ( siLuc ) or p97 - depleted conditions ( sip97 ) .

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

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing the ubiquitination pattern of RNF8-WT and RNF8-RING* variant, under siRNA-mediated luciferase (siLuc) or p97-depleted conditions (sip97).


Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition ( MG132 , 10µM ) . Graph represents the quantifications of ( B ) ( ns ; not significant , P > 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) .

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

Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10µM).   Graph represents the quantifications of (B) (ns; not significant, P>0.05, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).


Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing hyper - ubiquitination of RNF8 after proteasome inhibition ( MG132 , 10µM for 6h ) .

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

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing hyper-ubiquitination of RNF8 after proteasome inhibition (MG132, 10µM for 6h).


Western blot analysis of CHX chase kinetics in U2OS cells , comparing the degradation rate of Flag - RNF8 - WT and Flag - RNF8 - RING * . Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8 . Graph represents the quantifications of ( E ) ( ns P > 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) .

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

Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag-RNF8-WT and Flag-RNF8-RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8.   Graph represents the quantifications of (E) (ns P>0.05, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).


Western blot analysis of Strep - p97 Co - IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR ( 10 Gy ) .

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

Western blot analysis of Strep-p97 Co-IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR (10 Gy).


Western blot analysis of Flag - ATX3 Co - IP in HEK293 cells showing interaction of ATX3 with p97 , RNF8 and PSα6 under physiological conditions and after IR ( 10 Gy ) .

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

Western blot analysis of Flag-ATX3 Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and PSα6 under physiological conditions and after IR (10 Gy).


Western blot analysis of Flag - ATX3 WT or Flag - ATX3 VBM * Co - IP in HEK293 cells showing interaction of ATX3 with p97 , RNF8 and p97 core co - factors Npl4 - Ufd1 . ( * represents unspecific bands ) .

Western blot analysis of Flag-ATX3 WT or Flag-ATX3 VBM* Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and p97 core co-factors Npl4-Ufd1. (* represents unspecific bands).


Western blot analysis of Strep - p97EQ Co - IP in HEK293 cells , in presence or absence ( CRISPR knockout ; ∆ ATX3 ) of ATX3 , showing increased interaction of RNF8 with p97 in absence of ATX3 , under physiological conditions and after IR ( 10 Gy ) . Graph represents quantifications of ( F ) ( * P < 0 . 05 , * * P < 0 . 01 ; unpaired t - test , n = 3 , mean + SEM ) .

Western blot analysis of Strep-p97EQ Co-IP in HEK293 cells, in presence or absence (CRISPR knockout; ∆ATX3) of ATX3, showing increased interaction of RNF8 with p97 in absence of ATX3, under physiological conditions and after IR (10 Gy).   Graph represents quantifications of (F) (*P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).


Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction ( Cytosol and Nucleosol ) of ∆ ATX3 cell extract . Arrow represents the main RNF8 band and asterisks represents unspecific bands . Graphs represent the quantifications of ( A ) . RNF8 level at starting point ( 0h ) was shown without equalisation ( left ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate ( right ) . ( * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) .

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 O O O O O

Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ∆ATX3 cell extract. Arrow represents the main RNF8 band and asterisks represents unspecific bands.   Graphs represent the quantifications of (A). RNF8 level at starting point (0h) was shown without equalisation (left). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate (right). (**P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).


Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction ( Cytosol and Nucleosol ) of ATX3 knockdown HeLa cells . The degradation was completely blocked after simultaneous inhibition of proteasome ( MG132 , 10µM ) .

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 B-CONTROLLED_VAR O O O O

Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ATX3 knockdown HeLa cells. The degradation was completely blocked after simultaneous inhibition of proteasome (MG132, 10µM).


Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing RNF8 hyper - ubiquitination in siRNA - mediated p97 or ATX3 - depleted conditions .

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing RNF8 hyper-ubiquitination in siRNA-mediated p97 or ATX3-depleted conditions.


Western blot analysis of Flag - RNF8 denaturing - IP in HeLa cells showing RNF8 hyper - ubiquitination in ∆ ATX3 condition . RNF8 hyper - ubiquitination was suppressed by reintroduction of GFP - ATX3 - WT but not with GFP - ATX3 - C14A or GFP - ATX3 - UIM * .

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

Western blot analysis of Flag-RNF8 denaturing-IP in HeLa cells showing RNF8 hyper-ubiquitination in ∆ATX3 condition. RNF8 hyper-ubiquitination was suppressed by reintroduction of GFP-ATX3-WT but not with GFP-ATX3-C14A or GFP-ATX3-UIM*.


Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction ( cytoplasm + nucleoplasm ) of HeLa cells , expressing DOX - inducible GFP - ATX3 - C14A variant as compared to GFP - ATX3 - WT . Endogenous ATX3 was depleted with siRNA targeting 3 ' UTR region of ATXN3 . Graph represents the quantifications of ( A ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate . RNF8 level at starting point ( 0h ) was also shown without equalisation . ( * P < 0 . 05 , * * P < 0 . 01 ; two - way ANOVA , n = 2 , mean + SEM ) .

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 O O

Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm+nucleoplasm) of HeLa cells, expressing DOX-inducible GFP-ATX3-C14A variant as compared to GFP-ATX3-WT. Endogenous ATX3 was depleted with siRNA targeting 3'UTR region of ATXN3.   Graph represents the quantifications of (A). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (*P<0.05, **P<0.01; two-way ANOVA, n=2, mean +SEM).


Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction ( cytoplasm + nucleoplasm ) of HeLa cells , expressing DOX - inducible ATX3 - UIM * variant as compared to ATX3 - WT . Endogenous ATX3 was depleted with siRNA ATX3 _ 3 ' UTR . Graph represents the quantifications of ( C ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate . RNF8 level at starting point ( 0h ) was also shown without equalisation . ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) .

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

Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm + nucleoplasm) of HeLa cells, expressing DOX-inducible ATX3-UIM* variant as compared to ATX3-WT. Endogenous ATX3 was depleted with siRNA ATX3_3'UTR.   Graph represents the quantifications of (C). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (ns P>0.05, *P<0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).


Western blot analysis showing endogenous RNF8 protein level in soluble fraction ( Cytosol and Nucleosol ) of HeLa cells under indicated conditions . RNF8 level was significantly reduced after siRNA mediated ATX3 depletion and then was significantly rescued by DOX - inducible expression of GFP - ATX3 - WT but not with GFP - ATX3 - VBM . Graphs represent the quantifications of ( E ) . ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 ; unpaired t - test , n = 3 , mean + SEM ) .

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

Western blot analysis showing endogenous RNF8 protein level in soluble fraction (Cytosol and Nucleosol) of HeLa cells under indicated conditions. RNF8 level was significantly reduced after siRNA mediated ATX3 depletion and then was significantly rescued by DOX-inducible expression of GFP-ATX3-WT but not with GFP-ATX3-VBM.   Graphs represent the quantifications of (E). (ns P>0.05, * P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).


Representative IF images showing γH2AX - positive micronuclei ( white arrows ) under physiological conditions with indicated siRNA mediated depletion . ( Scale bar represents 10 µm ) Graph represents quantification of ( G ) , showing percentage of cells with γH2AX - positive micronuclei in U2OS cells . ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM , at least 200 randomly selected nuclei were counted per condition per experiment ) .

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

Representative IF images showing γH2AX-positive micronuclei (white arrows) under physiological conditions with indicated siRNA mediated depletion. (Scale bar represents 10 µm)   Graph represents quantification of (G), showing percentage of cells with γH2AX-positive micronuclei in U2OS cells. (* P<0.05, **P<0.01, ***P<0.001; unpaired t-test, n=3, mean +SEM, at least 200 randomly selected nuclei were counted per condition per experiment).


Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA - depleted conditions , before and after IR ( 4 Gy ) . ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) .

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

Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA-depleted conditions, before and after IR (4 Gy). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).


Colony forming assay ( CFA ) showing the IR sensitivity of HeLa cells under siRNA - mediated p97 or ATX3 - depleted conditions . ( n = 3 , mean ± SD ) . CFA showing the IR sensitivity of HeLa - WT cells compared to HeLa ∆ ATX3 cells . ( n = 3 , mean ± SD ) .

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

Colony forming assay (CFA) showing the IR sensitivity of HeLa cells under siRNA-mediated p97 or ATX3-depleted conditions. (n=3, mean ±SD).   CFA showing the IR sensitivity of HeLa-WT cells compared to HeLa∆ATX3 cells. (n=3, mean ±SD).


CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after DOX - inducible expression of ATX3 - WT but not with ATX3 - C14A or ATX3 - UIM * variant . ( ns P > 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SD ) . CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after DOX - inducible expression of ATX3 - WT but not with ATX3 - VBM variant . ( ns P > 0 . 05 , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SD ) .

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

CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-C14A or ATX3-UIM* variant. (ns P>0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-VBM variant. (ns P>0.05, *P<0.05, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).


Representative IF micrographs in U2OS cells showing IRIF formation of GFP - RNF8 - WT , 53BP1 and γH2AX ( first panel ) and GFP - RNF8 - ∆ RING , 53BP1 and γH2AX ( second panel ) under indicated siRNA - depleted or p97 - inhibited ( CB5083 ) conditions . ( Scale bar ; 10µm ) . Quantification of ( F ) for GFP - RNF8 - WT . Graph represents the percentage of cells with increased GFP - RNF8 - WT foci ( white bars ) or percentage of cells with > 5 53BP1 foci ( grey bars ) . ( * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM , at least 100 nuclei per condition and experiment ) . Quantification of ( F ) for GFP - RNF8 - ∆ RING . Graph represents the percentage of cells with increased GFP - RNF8 - ∆ RING foci ( white bars ) or percentage of cells with > 5 53BP1 foci ( grey bars ) . ( ns P > 0 . 05 , * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM , at least 100 nuclei per condition and experiment ) .

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

Representative IF micrographs in U2OS cells showing IRIF formation of GFP-RNF8-WT, 53BP1 and γH2AX (first panel) and GFP-RNF8-∆RING, 53BP1 and γH2AX (second panel) under indicated siRNA-depleted or p97-inhibited (CB5083) conditions. (Scale bar; 10µm).   Quantification of (F) for GFP-RNF8-WT. Graph represents the percentage of cells with increased GFP-RNF8-WT foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).   Quantification of (F) for GFP-RNF8-∆RING. Graph represents the percentage of cells with increased GFP-RNF8-∆RING foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (ns P>0.05, *P<0.01, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).


Representative IF images and quantification showing endogenous K63 - Ub and γ - H2AX at UV - A micro - laser - induced DNA damage tracks in U2OS cells under indicated siRNA - depleted conditions . ( Scale bar ; 10µm ) . Quantification of ( I ) . Graph represents the average intensity of the K63 - Ub ( ns P > 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , more than 50 nuclei were analysed ) .

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Representative IF images and quantification showing endogenous K63-Ub and γ-H2AX at UV-A micro-laser-induced DNA damage tracks in U2OS cells under indicated siRNA-depleted conditions. (Scale bar; 10µm).   Quantification of (I). Graph represents the average intensity of the K63-Ub (ns P>0.05, **P<0.01, ***P<0.001; unpaired t-test, n=2, mean +SEM, more than 50 nuclei were analysed).


Representative IF images showing the average intensity of endogenous RNF168 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( A ) . ( * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM , more than 50 nuclei were analysed ) .

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Representative IF images showing the average intensity of endogenous RNF168 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (A). (***P<0.001; unpaired t-test, n=3, mean +SEM, more than 50 nuclei were analysed).


Representative IF images showing the average intensity of endogenous 53BP1 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( C ) . ( ns P > 0 . 05 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , at least 80 nuclei per condition and experiment ) .

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Representative IF images showing the average intensity of endogenous 53BP1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (C). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 80 nuclei per condition and experiment).


Western blot analysis of Flag - histone H1 denaturing - IP , showing ubiquitination pattern of histone H1 after 30 min of IR ( 10 Gy ) treatment under indicated siRNA - depleted conditions .

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Western blot analysis of Flag-histone H1 denaturing-IP, showing ubiquitination pattern of histone H1 after 30 min of IR (10 Gy) treatment under indicated siRNA-depleted conditions.


Representative IF images showing the average intensity of endogenous BRCA1 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( F ) . ( ns P > 0 . 05 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , at least 100 nuclei per condition and experiment ) .

Representative IF images showing the average intensity of endogenous BRCA1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (F). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 100 nuclei per condition and experiment).


GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA - depleted conditions in HEK293 cells . Graph represents the quantifications from three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; one - way ANOVA , n = 3 , mean + SEM ) .

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GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (*P<0.05, **P<0.01, ***P<0.001; one-way ANOVA, n=3, mean +SEM).


GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA - depleted conditions in HEK293 cells . Graph represents the quantifications from three independent experiments ( ns P > 0 . 05 , * * P < 0 . 01 ; one - way ANOVA , n = 3 , mean + SEM ) .

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GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (ns P>0.05, **P<0.01; one-way ANOVA, n=3, mean +SEM).


CFA showing the IR sensitivity of 53BP1 - proficient and deficient MCF7 cells under indicated siRNA - depleted conditions . Data was collected from two individual experiments with triplicates . ( ns P > 0 . 05 ; two - way ANOVA , n = 2 , mean ± SEM ) .

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CFA showing the IR sensitivity of 53BP1-proficient and deficient MCF7 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (ns P>0.05; two-way ANOVA, n=2, mean ±SEM).


CFA showing the IR sensitivity of BRCA2 - proficient and deficient DLD1 cells under indicated siRNA - depleted conditions . Data was collected from two individual experiments with triplicates . ( * * P < 0 . 01 , * * * P < 0 . 001 ; two - way ANOVA , n = 2 , mean ± SEM ) .

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CFA showing the IR sensitivity of BRCA2-proficient and deficient DLD1 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (**P<0.01, ***P<0.001; two-way ANOVA, n=2, mean ±SEM).


Representative IF - images showing Rad51 foci in S - phase ( Edu positive ) U2OS nucleus after 5h of IR ( 2Gy ) exposure . ( Scale bar ; 10µm ) . Graph represents the quantification of ( E ) showing average number of Rad51 foci per S - phase ( Edu positive ) nucleus . ( ns P > 0 . 05 , * * * * P < 0 . 0001 ; unpaired t - test , n = 2 , mean + SEM , at least 50 nuclei per condition and experiment ) .

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Representative IF-images showing Rad51 foci in S-phase (Edu positive) U2OS nucleus after 5h of IR (2Gy) exposure. (Scale bar; 10µm).   Graph represents the quantification of (E) showing average number of Rad51 foci per S-phase (Edu positive) nucleus. (ns P>0.05, ****P<0.0001; unpaired t-test, n=2, mean +SEM, at least 50 nuclei per condition and experiment).


CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after co - depletion ( siRNA ) of RNF8 . ( ns P > 0 . 05 , * P < 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SEM ) .

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CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after co-depletion (siRNA) of RNF8. (ns P>0.05, *P<0.05, ****P<0.0001; two-way ANOVA, n=3, mean ±SEM).


( A ) RT - PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages . NC indicates the no template control .

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(A) RT-PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages. NC indicates the no template control.


( B ) Time - course expression of sinhcaf revealed by whole - mount in situ hybridization . Scale bars , 100

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(B) Time-course expression of sinhcaf revealed by whole-mount in situ hybridization. Scale bars, 100 μ


( C ) RT - PCR analysis for spatial expression of sinhcaf mRNA in adult tissues .

(C) RT-PCR analysis for spatial expression of sinhcaf mRNA in adult tissues.


( E ) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf - / - mutant embryos as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

(E) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf -/- mutant embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test.


( F ) Fluorescence microscopy of 10hpf Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos . Scale bar , 400 μm .

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(F) Fluorescence microscopy of 10hpf Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Scale bar, 400 μm.


( G ) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg ( CMV : EGFP ; CMV : sinhcaf ) embryos as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using 2 - way ANOVA followed by Holm - Sidak post - hoc test .

(G) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg(CMV:EGFP;CMV:sinhcaf) embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test.


( H ) Percentage of male zebrafish in WT ( n = 274 ) , MZsinhcaf - / - ( n = 166 ) , Tg ( CMV : EGFP ; CMV : sinhcaf ) ( n = 158 ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) ( n = 135 ) adult zebrafish . Data shown are mean ± SEM ( n = the number of fish analyzed ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 .

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(H) Percentage of male zebrafish in WT (n = 274), MZsinhcaf -/- (n = 166), Tg(CMV:EGFP;CMV:sinhcaf) (n = 158) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) (n = 135) adult zebrafish. Data shown are mean ± SEM (n = the number of fish analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.


( A and B ) Whole - mount in situ hybridization was performed in WT , MZsinhcaf - / - , Tg ( CMV : EGFP ; CMV : sinhcaf ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos with PGCs markers nanos3 ( A ) and vasa ( B ) . Scale bars , 100 μm . ( C ) Quantitative analysis of PGCs in bud stage WT , MZsinhcaf - / - , Tg ( CMV : EGFP ; CMV : sinhcaf ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos . Data shown are mean ± SEM ( n = 10 , the number of embryos analyzed ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 .

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(A and B) Whole-mount in situ hybridization was performed in WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos with PGCs markers nanos3 (A) and vasa (B). Scale bars, 100 μm. (C) Quantitative analysis of PGCs in bud stage WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Data shown are mean ± SEM (n = 10, the number of embryos analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.


( D ) Maximal intensity projection of a confocal z - stack of 24hpf Tg ( piwil1 : EGFP ) and MZsinhcaf - / - ; Tg ( piwil1 : EGFP ) zebrafish embryos , dorsal views . Arrowheads point to PGCs . Scale bars , 100 μm . ( E ) Quantitative analysis of PGCs in 24hpf Tg ( piwil1 : EGFP ) and MZsinhcaf - / - ; Tg ( piwil1 : EGFP ) zebrafish embryos . Data shown are mean ± SEM ( n = 13 , the number of embryos analyzed ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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(D) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP) zebrafish embryos, dorsal views. Arrowheads point to PGCs. Scale bars, 100 μm. (E) Quantitative analysis of PGCs in 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP)zebrafish embryos. Data shown are mean ± SEM (n = 13, the number of embryos analyzed). Statistical analysis was performed using unpaired two-tailed Student's t-test.


( A ) Whole - mount in situ hybridization was performed in 4 - cell stage WT , MZsinhcaf - / - zebrafish embryos with germ plasm marker vasa . Red arrowheads point to cleavage furrows in the 4 - cell embryos . Scale bars , 100

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(A) Whole-mount in situ hybridization was performed in 4-cell stage WT, MZsinhcaf -/- zebrafish embryos with germ plasm marker vasa. Red arrowheads point to cleavage furrows in the 4-cell embryos. Scale bars, 100 μm


( B ) Relative mRNA levels of germ plasm genes vasa , dnd , nanos3 , dazl and buc in WT and MZsinhcaf - / - mutant eggs , respectively , as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using 2 - way ANOVA followed by the Holm - Sidak post - hoc test ; NS , no significant difference .

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(B) Relative mRNA levels of germ plasm genes vasa, dnd, nanos3, dazl and buc in WT and MZsinhcaf -/- mutant eggs, respectively, as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by the Holm-Sidak post-hoc test; NS, no significant difference.


( C ) Maximal intensity projection of a confocal z - stack of 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP mRNA . White arrowheads point to cleavage furrows of 4 - cell embryos . Scale bars , 100 μm .

(C) Maximal intensity projection of a confocal z-stack of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. White arrowheads point to cleavage furrows of 4-cell embryos. Scale bars, 100 μm.


( D ) Relative GFP - Buc density at the cleavage furrow of 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP mRNA . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data shown are mean ± SEM ( n = 12 , the number of embryos analyzed ) .

(D) Relative GFP-Buc density at the cleavage furrow of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. Statistical analysis was performed using unpaired two-tailed Student's t-test. Data shown are mean ± SEM (n = 12, the number of embryos analyzed).


( E ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test .

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(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test.


( F ) Western blots for GFP - Buc expression in 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP

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(F) Western blots for GFP-Buc expression in 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA


( A , B and C ) Co - immunoprecipitation of zebrafish Sinhcaf - GFP with HA - Hdac1 ( A ) , HA - Sin3a ( B ) or HA - Sap18 ( C ) in HEK293Tcells transfected with both Sinhcaf - GFP and HA - Hdac1 ( A ) , HA - Sin3a ( B ) or HA - Sap18 ( C ) plasmids .

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(A, B and C) Co-immunoprecipitation of zebrafish Sinhcaf-GFP with HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) in HEK293Tcells transfected with both Sinhcaf-GFP and HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) plasmids.


( D ) Relative mRNA level of sinhcaf in zebrafish PG , PV , EV , MV and FG follicles was measured using RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

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(D) Relative mRNA level of sinhcaf in zebrafish PG, PV, EV, MV and FG follicles was measured using RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( E and F ) Western blots for acetyl - histone H3 ( K9 ) ( Ace - H3 - K9 ) , histone H3 in WT ( E ) or MZsinhcaf - / - ( F ) PG , PV , EV , MV and FG follicles . α - tubulin was used as a loading control . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

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(E and F) Western blots for acetyl-histone H3 (K9) (Ace-H3-K9), histone H3 in WT (E) or MZsinhcaf -/- (F) PG, PV, EV, MV and FG follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( G and H ) Statistical analysis of the relative levels of Ace - H3 - K9 in WT ( G ) or MZsinhcaf - / - ( H ) PG , PV , EV , MV and FG follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

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(G and H) Statistical analysis of the relative levels of Ace-H3-K9 in WT (G) or MZsinhcaf -/- (H) PG, PV, EV, MV and FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( I , J and K ) Western blots for Ace - H3 - K9 and H3 in WT and MZsinhcaf - / - EV ( I ) , MV ( J ) and FG ( K ) follicles . α - tubulin was used as a loading control . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III )

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(I, J and K) Western blots for Ace-H3-K9 and H3 in WT and MZsinhcaf -/- EV (I), MV (J) and FG (K) follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)


( L ) Western blots for Ace - H4 - K5 , Ace - H3 - K18 and H3 in WT and MZsinhcaf - / - FG follicles . α - tubulin was used as a loading control . Data information : FG , full - grown stage ( late stage III )

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(L) Western blots for Ace-H4-K5, Ace-H3-K18 and H3 in WT and MZsinhcaf -/- FG follicles. α-tubulin was used as a loading control. Data information: FG, full-grown stage (late stage III)


( M , N and O ) Statistical analysis of the relative levels of Ace - H3 - K9I n WT and MZsinhcaf - / - EV ( M ) , MV ( N ) and FG ( O ) follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III )

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(M, N and O) Statistical analysis of the relative levels of Ace-H3-K9I n WT and MZsinhcaf -/- EV (M), MV (N) and FG (O) follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)


( P and Q ) Statistical analysis of the relative levels of Ace - H3 - K18 ( P ) and Ace - H4 - K5 ( Q ) in WT and MZsinhcaf - / - FG follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data information : ; FG , full - grown stage ( late stage III )

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(P and Q) Statistical analysis of the relative levels of Ace-H3-K18 (P) and Ace-H4-K5 (Q) in WT and MZsinhcaf -/- FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: ; FG, full-grown stage (late stage III)


( A ) Maximal intensity projection of a confocal z - stack of 24hpf Tg ( piwil : egfp ) , Tg ( piwil : egfp ) injected with kif26ab MO and Tg ( piwil : egfp ) injected with control MO embryos . ( B ) Quantitative analysis of PGCs in 24hpf Tg ( piwil : egfp ) ( n = 11 ) , Tg ( piwil : egfp ) injected with kif26ab MO ( n = 12 ) and Tg ( piwil : egfp ) injected with control MO ( n = 13 ) embryos . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil:egfp), Tg(piwil:egfp) injected with kif26ab MO and Tg(piwil:egfp) injected with control MO embryos. (B) Quantitative analysis of PGCs in 24hpf Tg(piwil:egfp) (n = 11), Tg(piwil:egfp) injected with kif26ab MO (n = 12) and Tg(piwil:egfp) injected with control MO (n = 13) embryos. Data shown are mean ± SEM (n = the number of embryos analyzed).


( C ) Maximal intensity projection of a confocal z - stack of 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . White arrowheads point to cleavage furrows of 4 - cell embryos .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximal intensity projection of a confocal z-stack of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. White arrowheads point to cleavage furrows of 4-cell embryos.


( D ) Relative GFP - Buc density at the cleavage furrow of 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA ( n = 11 ) , GFP - Buc mRNA + RFP mRNA + Control MO ( n = 11 ) and GFP - Buc mRNA + RFP mRNA + kif26ab MO ( n = 10 ) . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

(D) Relative GFP-Buc density at the cleavage furrow of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA (n = 11), GFP-Buc mRNA + RFP mRNA + Control MO (n = 11) and GFP-Buc mRNA + RFP mRNA + kif26ab MO (n = 10). Data shown are mean ± SEM (n = the number of embryos analyzed).


( E ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . ( F ) Western blots for GFP - Buc expression in 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . Scale bars , 100 μm .

(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (F) Western blots for GFP-Buc expression in 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.


( G ) Maximal intensity projection of a confocal z - stack of MZsinhcaf - / - or WT 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . White arrowheads point to cleavage furrows of 4 - cell embryos .

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(G) Maximal intensity projection of a confocal z-stack of MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. White arrowheads point to cleavage furrows of 4-cell embryos.


( H ) Relative GFP - Buc density at the cleavage furrow of MZsinhcaf - / - ( n = 12 ) or WT ( n = 13 ) 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative GFP-Buc density at the cleavage furrow of MZsinhcaf -/- (n = 12) or WT (n = 13) 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. Data shown are mean ± SEM (n = the number of embryos analyzed).


( I ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . ( J ) Western blots for GFP - Buc expression in MZsinhcaf - / - or WT 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . Scale bars , 100 μm .

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

(I) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (J) Western blots for GFP-Buc expression in MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.


( a ) Confocal analysis of the subcellular localization of HA - PLD1 ( green ) and endogenous LC3 ( red ) in HeLa cells in normal medium and under nutrient deprivation conditions . Left panels : normal medium condition ( Nm ) ; middle panels : 1 h with nutrient - deprived medium ( 1 h St ) ; right panels : 2 h with nutrient - deprived medium ( 2 h St ) . Insets denote high magnification of the selected area . Scale bar , 5 μm .

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

(a) Confocal analysis of the subcellular localization of HA-PLD1 (green) and endogenous LC3 (red) in HeLa cells in normal medium and under nutrient deprivation conditions. Left panels: normal medium condition (Nm); middle panels: 1 h with nutrient-deprived medium (1 h St); right panels: 2 h with nutrient-deprived medium (2 h St). Insets denote high magnification of the selected area. Scale bar, 5 μm.


( b ) Immuno - gold EM micrographs of HeLa cells transfected with GFP - PLD1 and subjected to 1 h of nutrient deprivation in the presence ( St + B ) or absence ( St ) of bafilomycin ( 50 nM ) . Insets denote high magnification of the selected area . Scale bar , 500 nm . Right panel : Histogram showing morphometric analysis of the relative distribution of GFP - PLD1 - positive gold particles on the outer ( arrows ) and inner ( arrowheads ) membranes of the autophagosomal - like structures . Values denote means ± s . e . m . ( n = 14 ) . * * P0 . 01 .

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

(b) Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and subjected to 1 h of nutrient deprivation in the presence (St+B) or absence (St) of bafilomycin (50 nM). Insets denote high magnification of the selected area. Scale bar, 500 nm. Right panel: Histogram showing morphometric analysis of the relative distribution of GFP-PLD1-positive gold particles on the outer (arrows) and inner (arrowheads) membranes of the autophagosomal-like structures. Values denote means±s.e.m. (n=14). **P0.01.


Confocal analysis of the subcellular localization of HA - PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h . Organelle markers used were as follows ( from top to bottom panels ) : Lamp1 ( late endosomes / lysosomes ) , giantin ( Golgi - TGN ) , Vps35 ( retromer - positive endosomes ) , GFP - Rab5 ( early endosomes ) , GFP - UVRAG ( early and late endosomes ) . Insets show high magnification of the selected areas . The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations . Scale bar , 5 μm .

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

Confocal analysis of the subcellular localization of HA-PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h. Organelle markers used were as follows (from top to bottom panels): Lamp1 (late endosomes/lysosomes), giantin (Golgi-TGN), Vps35 (retromer-positive endosomes), GFP-Rab5 (early endosomes), GFP-UVRAG (early and late endosomes). Insets show high magnification of the selected areas. The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.


( a ) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h . Cells were transfected with HA - PLD1 and GFP - ATG14L ( upper panels ) or with HA - PLD1 alone ( lower panels ) . Upper panels : HA - PLD1 ( red ) , GFP - ATG14L ( green ) , endogenous LC3 ( blue ) . Lower panels : HA - PLD1 ( green ) , endogenous ATG16L ( red ) and LC3 ( blue ) . Insets show high magnification of the selected areas . The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations . Scale bar , 5 μm .

(a) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h. Cells were transfected with HA-PLD1 and GFP-ATG14L (upper panels) or with HA-PLD1 alone (lower panels). Upper panels: HA-PLD1 (red), GFP-ATG14L (green), endogenous LC3 (blue). Lower panels: HA-PLD1 (green), endogenous ATG16L (red) and LC3 (blue). Insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.


( b ) Confocal analysis showing lack of co - localization of HA - PLD1 ( green ) with the isolation membrane marker ATG16L ( blue ) in HeLa cells expressing the strawberry - ATG4 - C74A mutant ( red ) and subjected to 1 h of nutrient deprivation . Insets denote high magnification of the selected area . Scale bar , 5 μm .

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(b) Confocal analysis showing lack of co-localization of HA-PLD1 (green) with the isolation membrane marker ATG16L (blue) in HeLa cells expressing the strawberry-ATG4-C74A mutant (red) and subjected to 1 h of nutrient deprivation. Insets denote high magnification of the selected area. Scale bar, 5 μm.


( c ) EM analysis showing that GFP - PLD1 does not localize to the isolation membrane ( arrows ) in cells expressing the strawberry - ATG4 - C74A mutant . Immuno - gold EM micrographs of HeLa cells transfected with GFP - PLD1 and pStrawberry - ATG4 - C74A and subjected to 1 h of nutrient deprivation . Inset denotes high magnification of the selected area . Scale bar , 250 nm .

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(c) EM analysis showing that GFP-PLD1 does not localize to the isolation membrane (arrows) in cells expressing the strawberry-ATG4-C74A mutant. Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and pStrawberry-ATG4-C74A and subjected to 1 h of nutrient deprivation. Inset denotes high magnification of the selected area. Scale bar, 250 nm.


( a ) Histogram showing relative phospholipid levels in CHO cells treated with 0 . 3 % ethanol for 30 min before cell harvesting . Total PLD activity is measured as phosphatidylethanol ( PEtOH ) levels under the following conditions : normal medium ( Nm ) , serum - free medium ( SFM ) , serum - free medium with 100 nM wortmannin ( SFM + W ) , nutrient deprivation ( 0 . 5 h St ; 1 . 5 h St ; 3 . 0 h St ) , 30 min of nutrient deprivation in presence of 100nM wortmannin ( 0 . 5h St + W ) . Other phospholipids were used as controls : phosphatidylserine ( PS ) , phosphatidylethanolamine ( PE ) , phosphatidylcholine ( PC ) . Values denote means ± s . e . m . ( n = 9 , except for SFM + W and 0 . 5 h St + W , where n = 3 and 5 , respectively ) . * * P0 . 01 ; * , P0 . 05 .

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

(a) Histogram showing relative phospholipid levels in CHO cells treated with 0.3% ethanol for 30 min before cell harvesting. Total PLD activity is measured as phosphatidylethanol (PEtOH) levels under the following conditions: normal medium (Nm), serum-free medium (SFM), serum-free medium with 100 nM wortmannin (SFM+W), nutrient deprivation (0.5 h St; 1.5 h St; 3.0 h St), 30 min of nutrient deprivation in presence of 100nM wortmannin (0.5h St+W). Other phospholipids were used as controls: phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC). Values denote means±s.e.m. (n=9, except for SFM+W and 0.5 h St+W, where n=3 and 5, respectively). **P0.01; *, P0.05.


( b ) Western blot analysis of HA - PLD1 and endogenous LC3 levels in post - nuclear supernatant ( PNS , upper panel ) , as well as in cytosolic / soluble ( S ) and particulate ( P ) fractions prepared from PNS after high - speed centrifugation ( middle panel ) . GAPDH was used as loading control in PNS fractions . HeLa cells were transfected with expression vectors encoding HA - PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin ( 100 nM ) . Lower panel : quantitative analysis of HA - PLD1 immunoreactivity expressed as P / S ratio . Conditions were as follows : normal medium ( Nm ) ; normal medium with wortmannin ( Nm + W ) ; starvation condition ( St ) ; starvation condition + wortmannin ( St + W ) . Values denote means ± s . e . m . ( n = 3 ) .

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

(b) Western blot analysis of HA-PLD1 and endogenous LC3 levels in post-nuclear supernatant (PNS, upper panel), as well as in cytosolic/soluble (S) and particulate (P) fractions prepared from PNS after high-speed centrifugation (middle panel). GAPDH was used as loading control in PNS fractions. HeLa cells were transfected with expression vectors encoding HA-PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin (100 nM). Lower panel: quantitative analysis of HA-PLD1 immunoreactivity expressed as P/S ratio. Conditions were as follows: normal medium (Nm); normal medium with wortmannin (Nm+W); starvation condition (St); starvation condition+wortmannin (St+W). Values denote means±s.e.m. (n=3).


( c ) Confocal analysis of the co - localization between HA - PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence ( ST + W ) or absence ( St ) of wortmannin 100 nM . Insets show high magnification of the selected areas . Scale bar , 5 μm .

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(c) Confocal analysis of the co-localization between HA-PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence (ST+W) or absence (St) of wortmannin 100 nM. Insets show high magnification of the selected areas. Scale bar, 5 μm.


( a ) Western blot analysis showing the effects of siRNA - mediated Vps34 ( siVps34 ) downregulation on the expression levels of HA - PLD1 and Vps34 compared with control cells ( siCtrl ) . Cells were incubated in normal medium ( Nm ) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin ( St + B ) . HeLa cells were transfected for 48 h with siRNA directed to Vps34 ( siVps34 ) or a control sequence ( siCtrl ) and then transfected for 24 h with a plasmid encoding HA - PLD1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Western blot analysis showing the effects of siRNA-mediated Vps34 (siVps34) downregulation on the expression levels of HA-PLD1 and Vps34 compared with control cells (siCtrl). Cells were incubated in normal medium (Nm) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin (St+B). HeLa cells were transfected for 48 h with siRNA directed to Vps34 (siVps34) or a control sequence (siCtrl) and then transfected for 24 h with a plasmid encoding HA-PLD1.


( b ) Confocal imaging showing the effects of Vps34 ( siVps34 ) downregulation on HA - PLD1 ( green ) , endogenous LC3 ( blue ) and Lamp1 ( red ) on 2 h of nutrient deprivation compared with control treatment ( siCtrl ) . Scale bar , 5 μm . ( c ) Confocal analysis showing the subcellular localization of HA - PLD1 mutated in its PX domain ( F120A / R179Q ) , so as to abolish phosphoinositide binding ( HA - PXmut ) . HeLa cells were transiently transfected with a plasmid encoding HA - PXmut and fixed after 2 h of nutrient deprivation . HA - PXmut ( green ) , endogenous LC3 ( blue ) and Lamp1 ( red ) . Scale bar , 10 μm . In b and c , insets show high magnification of the selected areas . The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal imaging showing the effects of Vps34 (siVps34) downregulation on HA-PLD1 (green), endogenous LC3 (blue) and Lamp1 (red) on 2 h of nutrient deprivation compared with control treatment (siCtrl). Scale bar, 5 μm. (c) Confocal analysis showing the subcellular localization of HA-PLD1 mutated in its PX domain (F120A/R179Q), so as to abolish phosphoinositide binding (HA-PXmut). HeLa cells were transiently transfected with a plasmid encoding HA-PXmut and fixed after 2 h of nutrient deprivation. HA-PXmut (green), endogenous LC3 (blue) and Lamp1 (red). Scale bar, 10 μm. In b and c, insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations.


( a ) Confocal analysis showing the effects of PLD inhibition ( FIPI , 750 nM ) on the levels of endogenous LC3 . CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum ( normal medium , Nm ) or in the absence of nutrients ( starvation , St ) . Insets denote high magnification of the selected areas . Scale bar , 10 μm . ( b ) Quantification of the average number , size and surface area of LC3 - positive compartments per cell after FIPI treatment . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 180 - 285 cells for Nm ; n = 449 - 519 cells for St ) . * * P0 . 01 .

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(a) Confocal analysis showing the effects of PLD inhibition (FIPI, 750 nM) on the levels of endogenous LC3. CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum (normal medium, Nm) or in the absence of nutrients (starvation, St). Insets denote high magnification of the selected areas. Scale bar, 10 μm. (b) Quantification of the average number, size and surface area of LC3-positive compartments per cell after FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=180-285 cells for Nm; n=449-519 cells for St). **P0.01.


( c ) Western blot analysis showing levels of LC3 - I and LC3 - II in control - and FIPI - treated CHO cells grown in normal medium ( Nm ) , after nutrient starvation ( St ) or after nutrient starvation in the presence of 50 nM bafilomycin ( St + B ) . Treatments were for 3 h . Right panel : quantification of protein signal intensities showing LC3 - II protein levels in extracts from nutrient - starved and bafilomycin - treated CHO cells following FIPI treatment . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 4 ) .

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

(c) Western blot analysis showing levels of LC3-I and LC3-II in control- and FIPI-treated CHO cells grown in normal medium (Nm), after nutrient starvation (St) or after nutrient starvation in the presence of 50 nM bafilomycin (St+B). Treatments were for 3 h. Right panel: quantification of protein signal intensities showing LC3-II protein levels in extracts from nutrient-starved and bafilomycin-treated CHO cells following FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=4).


( d ) Western blot analysis of the effects of PLD inhibition on the levels of total Tau , phosphorylated Tau ( p - Tau ) and detergent - insoluble Tau . Organotypic brain slices from hTau mice were treated with either DMSO ( D ) or 750nM FIPI ( F ) and harvested after 5 days of treatment . Left panel : western blot analysis showing immunoreactivities for total human Tau ( CP27 ) , hyperphosphorylated tau ( p - Tau , ser202 / ser205 , CP13 ) , p62 and tubulin from the supernatant fraction and aggregated tau ( CP27 ) and p62 from the sarkosyl - insoluble fraction . Two representative pairs are displayed . Right panel , quantification of total proteins ( Tau , p - Tau , p62 and tubulin ) and sarkosyl - insoluble aggregates of tau ( insoluble Tau ) and p62 ( insoluble p62 ) . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 7 ) . * * P0 . 01 ; * , P0 . 05 . For the statistical analysis of FIPI - treated samples , tau and p62 protein levels were compared with tubulin .

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

(d) Western blot analysis of the effects of PLD inhibition on the levels of total Tau, phosphorylated Tau (p-Tau) and detergent-insoluble Tau. Organotypic brain slices from hTau mice were treated with either DMSO (D) or 750nM FIPI (F) and harvested after 5 days of treatment. Left panel: western blot analysis showing immunoreactivities for total human Tau (CP27), hyperphosphorylated tau (p-Tau, ser202/ser205, CP13), p62 and tubulin from the supernatant fraction and aggregated tau (CP27) and p62 from the sarkosyl-insoluble fraction. Two representative pairs are displayed. Right panel, quantification of total proteins (Tau, p-Tau, p62 and tubulin) and sarkosyl-insoluble aggregates of tau (insoluble Tau) and p62 (insoluble p62). Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=7). **P0.01; *, P0.05. For the statistical analysis of FIPI-treated samples, tau and p62 protein levels were compared with tubulin.


a ) Western blot analysis showing reduced levels of LC3 - II in KO MEFs . ( b ) Quantification of protein signal intensities from western blots showing LC3 - II levels in MEFs extracts after normalization to the control protein GAPDH . In a and b , MEFs were grown in normal medium ( Nm ) , treated for 2 h with 50 nM bafilomycin in normal medium ( B ) , or grown in nutrient - deprived medium in the absence ( St ) or presence of 50 nM bafilomycin ( St + B ) . Values denote means ± s . e . m . ( n = 4 ) .

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

a) Western blot analysis showing reduced levels of LC3-II in KO MEFs. (b) Quantification of protein signal intensities from western blots showing LC3-II levels in MEFs extracts after normalization to the control protein GAPDH. In a and b, MEFs were grown in normal medium (Nm), treated for 2 h with 50 nM bafilomycin in normal medium (B), or grown in nutrient-deprived medium in the absence (St) or presence of 50 nM bafilomycin (St+B). Values denote means±s.e.m. (n=4).


( c ) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3 - positive compartment in KO MEFs . The DAPI staining is shown in blue . Cells were deprived of nutrients for 2 h in the absence ( St , upper panels ) or presence of bafilomycin ( St + B , lower panels ) . Scale bar , 5 μm . ( d ) Quantification of the average size ( top panel ) and number of LC3 - positive compartments per cell ( bottom panel ) in Pld1 WT and KO MEFs starved for 2 h . Values denote means ± s . e . m . ( n = 6 , number of cells = 26 - 45 for all the conditions ) . * * P0 . 01 ; * P0 . 05 .

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

(c) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3-positive compartment in KO MEFs. The DAPI staining is shown in blue. Cells were deprived of nutrients for 2 h in the absence (St, upper panels) or presence of bafilomycin (St+B, lower panels). Scale bar, 5 μm. (d) Quantification of the average size (top panel) and number of LC3-positive compartments per cell (bottom panel) in Pld1 WT and KO MEFs starved for 2 h. Values denote means±s.e.m. (n=6, number of cells=26-45 for all the conditions). **P0.01; *P0.05.


( a ) Immunofluorescent detection of endogenous LC3 ( red ) in liver sections from fed or starved Pld1 WT and KO mice . Sections were counterstained with DAPI . Scale bar , 5 μm . ( b ) Quantitative analysis of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT and KO mice . Histograms show the average number , size and surface area of LC3 - positive compartments . Values denote means ± s . e . m . ( ( n = 25 - 28 ) , 4 mice per genotype ) . * * P0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescent detection of endogenous LC3 (red) in liver sections from fed or starved Pld1 WT and KO mice. Sections were counterstained with DAPI. Scale bar, 5 μm. (b) Quantitative analysis of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT and KO mice. Histograms show the average number, size and surface area of LC3-positive compartments. Values denote means±s.e.m. ((n=25-28), 4 mice per genotype). **P0.01.


( c ) Electron microscopic analysis showing a reduction in the size and number of autophagosome / amphisome - like structures ( AP ) in PLD1 - deficient mice after food restriction . Representative electron micrographs from animals analysed in a showing APs ( arrows ) and autophagolysosomes / lysosomes ( AL , arrowheads ) in liver sections from starved WT and KO mice . Insets show high magnification of the selected areas . Scale bar , 1 μm . ( d ) Morphometric analysis of the number ( per 100 μm2 cytoplasmic area ) and relative surface area ( % cytoplasmic area ) of APs and ALs . Values denote means ± s . e . m . ( n = 36 electron micrograms , from a total of 3 , 600 μm2 cytoplasmic area per genotype ) . * * P0 . 01 . ( c ) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1 - deficient mice after food restriction . Values denote means ± s . e . m . ( n = 60 ) . * * P0 . 01 .

O O O O O O 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 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

(c) Electron microscopic analysis showing a reduction in the size and number of autophagosome/amphisome-like structures (AP) in PLD1-deficient mice after food restriction. Representative electron micrographs from animals analysed in a showing APs (arrows) and autophagolysosomes/lysosomes (AL, arrowheads) in liver sections from starved WT and KO mice. Insets show high magnification of the selected areas. Scale bar, 1 μm. (d) Morphometric analysis of the number (per 100 μm2 cytoplasmic area) and relative surface area (% cytoplasmic area) of APs and ALs. Values denote means±s.e.m. (n=36 electron micrograms, from a total of 3,600 μm2 cytoplasmic area per genotype). **P0.01. (c) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1-deficient mice after food restriction. Values denote means±s.e.m. (n=60). **P0.01.


Real - time qRT - PCR analysis of C / EBPβ mRNA in CD4 + naïve T cells inactivated ( naive ) or cultured for 24 hr under conditions as indicated with DMSO or ATRA ( all - trans retinoic acid ) . Data are representative of two independent experiments with consistent results and normalized with β - actin ( mean and s . e . m . of quadruplicates )

O O O O O O O O O O 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 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

Real-time qRT-PCR analysis of C/EBPβ mRNA in CD4+ naïve T cells inactivated (naive) or cultured for 24 hr under conditions as indicated with DMSO or ATRA (all-trans retinoic acid). Data are representative of two independent experiments with consistent results and normalized with β-actin (mean and s.e.m. of quadruplicates)


Immunoblot analysis of C / EBPβ in the nuclear fraction of naïve CD4 + T cells cultured for 2 d under conditions as indicated . The immunoblot was quantified using Image

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

Immunoblot analysis of C/EBPβ in the nuclear fraction of naïve CD4+ T cells cultured for 2 d under conditions as indicated. The immunoblot was quantified using Image J


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean value from 8 ( C are shown on the right Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean value from 8(C are shown on the right Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean values fro 6 ( D are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values fro 6(D are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean values fro E 7 ( F are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values fro E 7(F are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the upper panel and pooled data from 4 ( A are shown in the bottom . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gate . Error bars represent mean ± s . e .

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the upper panel and pooled data from 4 (A are shown in the bottom. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate. Error bars represent mean ± s.e.m


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the upper panel and pooled data fro ) , 3 ( B are shown in the bottom . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gate . Error bars represent mean ± s . e .

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the upper panel and pooled data fro ), 3 (B are shown in the bottom. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate. Error bars represent mean ± s.e.m


Real - time qRT - PCR analysis of FACS - sorted GFP + cells from CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d in the conditions as indicated . Data are representative of two independent experiments with consistent results and normalized with β - actin ( mean ± s . e . m . of triplicates )

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

Real-time qRT-PCR analysis of FACS-sorted GFP+ cells from CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d in the conditions as indicated. Data are representative of two independent experiments with consistent results and normalized with β-actin (mean ± s.e.m. of triplicates)


Body weight of RAG - 2 - / - mice after transfer with 5 x 105 CD4 + CD45RBhigh cells alone ( circles ) or with 2 x 105 FACS - sorted CD4 + GFP + cells transduced with control retrovirus ( MigRI ) ( squares ) or retrovirus encoding C / EBPβ ( triangles ) and cultured for 2 d in the presence of TGF - β and culture supernatant . Each time point contains three ( circles ) or four ( squares and triangles ) mice in each group . Error bars represent mean ± s .

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

Body weight of RAG-2-/- mice after transfer with 5 x 105 CD4+CD45RBhigh cells alone (circles) or with 2 x 105 FACS-sorted CD4+GFP+ cells transduced with control retrovirus (MigRI) (squares) or retrovirus encoding C/EBPβ (triangles) and cultured for 2 d in the presence of TGF-β and culture supernatant. Each time point contains three (circles) or four (squares and triangles) mice in each group. Error bars represent mean ± s.d


Hematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm Histological scores Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m )

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

Hematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm Histological scores Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)


Comparison of the spleen weight Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m )

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

Comparison of the spleen weight Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)


Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb / c mice that received RAG2 - / - DO11 . 10 CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPβ . ( A ) The recipients were immunized via intravenous injection of OVA323 - 339 peptide ( 20 ug ) 1 and 3 d after transfer of transduced cells and analyzed 5 d after the first immunization . ( B ) The recipients were fed 1 % OVA solution in drinking water for 5 consecutive days . Representative experiments are shown in the left panel and pooled data from 4 ( A ) or 3 ( B ) independent experiments with mean values are shown on the right . Dot plots are gated for CD4 + KJ . 1 . 26 + GFP + ( top ) or CD4 + KJ . 1 . 26 + GFP - ( bottom ) and numbers indicate percent Foxp3 + cells in the gate . Statistical analysis was performed using one - way ANOVA . ( * p < 0 . 05 , * * p < 0 . 01 ; ns , not

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

Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb/c mice that received RAG2-/-DO11.10 CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPβ. (A) The recipients were immunized via intravenous injection of OVA323-339 peptide (20 ug) 1 and 3 d after transfer of transduced cells and analyzed 5 d after the first immunization. (B) The recipients were fed 1% OVA solution in drinking water for 5 consecutive days. Representative experiments are shown in the left panel and pooled data from 4 (A) or 3 (B) independent experiments with mean values are shown on the right. Dot plots are gated for CD4+KJ.1.26+GFP+ (top) or CD4+KJ.1.26+GFP- (bottom) and numbers indicate percent Foxp3+ cells in the gate. Statistical analysis was performed using one-way ANOVA. (*p<0.05, **p<0.01; ns, not significant


EMSA of nuclear extracts of Jurkat cells transfected with indicated expression vectors and assessed with a probe covering the putative unmethyl - or methyl - CRE sequence in the

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

EMSA of nuclear extracts of Jurkat cells transfected with indicated expression vectors and assessed with a probe covering the putative unmethyl- or methyl-CRE sequence in the TSDR


ChIP - qPCR assay of C / EBPβ on the Foxp3 TSDR in naïve CD4 + T cells cultured for 2 d under conditions as indicated . Data are representative of two independent experiments with consistent results ( mean ± s . e . m . of triplicates )

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

ChIP-qPCR assay of C/EBPβ on the Foxp3 TSDR in naïve CD4+ T cells cultured for 2 d under conditions as indicated. Data are representative of two independent experiments with consistent results (mean ± s.e.m. of triplicates)


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data from 4 ( C with mean values are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data from 4 (C with mean values are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data fro 5 ( D are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data fro 5 (D are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


CD4 + naïve T cells from Foxp3EGFP mice were transduced with control retrovirus ( MIN ) or retrovirus encoding C / EBPβ ( MIN - C / EBPβ ) and stimulated with anti - CD3 and anti - CD28 for 2 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . GFP + ( Foxp3 + ) hNGFR + T cells were sorted to high purity ( 0 hr ) and restimulated with anti - CD3 and anti - CD28 for 2 d in the presence of IL - 4 , IL - 6 , IL - 12 or none . Foxp3 expression was assessed based on GFP expression . Data are representative of two independent experiments with consistent

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

CD4+ naïve T cells from Foxp3EGFP mice were transduced with control retrovirus (MIN) or retrovirus encoding C/EBPβ (MIN-C/EBPβ) and stimulated with anti-CD3 and anti-CD28 for 2 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. GFP+(Foxp3+) hNGFR+ T cells were sorted to high purity (0 hr) and restimulated with anti-CD3 and anti-CD28 for 2 d in the presence of IL-4, IL-6, IL-12 or none. Foxp3 expression was assessed based on GFP expression. Data are representative of two independent experiments with consistent results


Flow cytometry of CFSE - labeled CD45 . 1 + CD4 + T cells stimulated 3 d alone or together with CD45 . 2 + GFP ( Foxp3 ) + NGFR + - sorted CD4 + naïve T cells from Foxp3EGFP mice transduced with control retrovirus ( Min ) or retrovirus encoding C / EBPβ and cultured for 3 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . Histograms are gated for CD45 . 1 + . The ratios shown are responder to suppressor . Data are representative of two independent experiments with consistent

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

Flow cytometry of CFSE-labeled CD45.1+CD4+ T cells stimulated 3 d alone or together with CD45.2+GFP(Foxp3)+NGFR+-sorted CD4+ naïve T cells from Foxp3EGFP mice transduced with control retrovirus (Min) or retrovirus encoding C/EBPβ and cultured for 3 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. Histograms are gated for CD45.1+. The ratios shown are responder to suppressor. Data are representative of two independent experiments with consistent results


EAE disease course of RAG2 - / - mice that received CD45 . 1 + 2D2CD4 + naive T cells alone or together with CD4 + GFP / Foxp3 + iTreg cells from CD45 . 1 - 2D2CD4 + GFP ( Foxp3 ) + naïve T cells transduced with either control retrovirus ( MIN ) or retrovirus encoding C / EBPβ , cultured in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs and sorted based on GFP / Foxp3 expression . The recipients were immunized with myelin oligodendrocyte glycoprotein peptide ( 100 ug ) 1 d after transfer . Data show mean ± s . e . m of the EAE clinical score of 9 ( day 1 to day 11 ) , 5 ( day 12 to day 16 ) or 4 ( day 17 to day 20 ) mice of each

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

EAE disease course of RAG2-/- mice that received CD45.1+2D2CD4+ naive T cells alone or together with CD4+GFP/Foxp3+ iTreg cells from CD45.1-2D2CD4+GFP(Foxp3)+ naïve T cells transduced with either control retrovirus (MIN) or retrovirus encoding C/EBPβ, cultured in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs and sorted based on GFP/Foxp3 expression. The recipients were immunized with myelin oligodendrocyte glycoprotein peptide (100 ug) 1 d after transfer. Data show mean ± s.e.m of the EAE clinical score of 9 (day 1 to day 11), 5 (day 12 to day 16) or 4 (day 17 to day 20) mice of each group