( I ) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3 , ASC , pro - caspase - 1 and pro - IL - 1β together with NS1 .

O O O O O O O O O O O O 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) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3, ASC, pro-caspase-1 and pro-IL-1β together with NS1.


( J ) Immunoblot analysis of protein extracts of Flag - tagged NS1 - inducible THP - 1 cells treated with increasing doses of doxycycline ( Dox ) for 24 hrs .

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

(J) Immunoblot analysis of protein extracts of Flag-tagged NS1-inducible THP-1 cells treated with increasing doses of doxycycline (Dox) for 24 hrs.


( K ) ELISA of IL - 1β in the supernatants of Flag - NS1 - inducible THP - 1 cells left unprimed then treated with ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) , or pre - treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) stimulation .

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

(K) ELISA of IL-1β in the supernatants of Flag-NS1-inducible THP-1 cells left unprimed then treated with ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs), or pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.


( L ) Immunoblot analysis of supernatants ( Sup ) and cell extracts ( Lys ) of Flag - NS1 - inducible THP - 1 cells pre - treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) stimulation .

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

(L) Immunoblot analysis of supernatants (Sup) and cell extracts (Lys) of Flag-NS1-inducible THP-1 cells pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.


( A ) Relative qRT - PCR analysis of ZIKV RNA in THP - 1 cells left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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

(A) Relative qRT-PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.


( B ) Relative qRT - PCR analysis of ZIKV RNA in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(B) Relative qRT-PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( C ) Immunoblot analysis of extracts of WT or Nlrp3 - / - BMDMs by the indicated antibodies .

(C) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs by the indicated antibodies.


( D ) Relative qRT - PCR analysis of ZIKV RNA in WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(D) Relative qRT-PCR analysis of ZIKV RNA in WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.


( E ) Plaque titration of ZIKV in supernatants of WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(E) Plaque titration of ZIKV in supernatants of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.


( F ) Immunoblot analysis of extracts of WT or caspase - 1 KO THP - 1 cells by the indicated antibodies .

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

(F) Immunoblot analysis of extracts of WT or caspase-1 KO THP-1 cells by the indicated antibodies.


( G ) Relative qRT - PCR analysis of ZIKV RNA in WT or caspase - 1 KO THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(G) Relative qRT-PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( H ) Relative qRT - PCR analysis of ZIKV RNA in NS - 1 - inducible THP - 1 cells treated with or without doxycycline ( Dox ) , then left untreated or treated with Ac - YVAD - cmk ( 20 μΜ ) for 3 hrs followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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

(H) Relative qRT-PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μΜ) for 3 hrs followed by ZIKV infection (MOI=1) for the indicated time points.


( I ) Relative qRT - PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors .

(I) Relative qRT-PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors.


( J ) Relative qRT - PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs .

(J) Relative qRT-PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.


( K ) qRT - PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

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

(K) qRT-PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.


( A ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA - NS1 together with Flag - NLRP3 , Flag - ASC , Flag - caspase - 1 and Flag - IL - 1β . WCL , whole cell lysates .

O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-NS1 together with Flag-NLRP3, Flag-ASC, Flag-caspase-1 and Flag-IL-1β. WCL, whole cell lysates.


( B ) Co - immunoprecipitation and immunoblot analysis of extracts of NS1 - inducible THP - 1 cells , which expressed Flag - tagged NS1 , treated with or without doxycycline ( Dox ) ( 100 ng / ml ) .

O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of NS1-inducible THP-1 cells, which expressed Flag-tagged NS1, treated with or without doxycycline (Dox) (100 ng/ml).


( C ) Immunoblot analysis of extracts of THP - 1 cells treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) followed by increasing doses of Dox treatment .

O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of extracts of THP-1 cells treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs) followed by increasing doses of Dox treatment.


( D ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with increasing amount of HA - NS1 . Below , caspase - 1 mRNA RT - PCR ; RPL13A mRNA RT - PCR serves as a loading control .

(D) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-NS1. Below, caspase-1 mRNA RT-PCR; RPL13A mRNA RT-PCR serves as a loading control.


( E ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with increasing amount of HA - DENV - 2 - NS1 .

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

(E) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-DENV-2-NS1.


( F ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with empty vector or HA - NS1 followed by cycloheximide ( CHX ) treatment ( 100 μg / ml ) for the indicated time points .

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

(F) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 followed by cycloheximide (CHX) treatment (100 μg/ml) for the indicated time points.


( G ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with empty vector or HA - NS1 then treated with MG132 ( 10 mM ) , Lactacystin ( 5 μM ) , Carfilzomib ( 100 mM ) or CQ ( 50 mM ) for 6 hrs .

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

(G) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 then treated with MG132 (10 mM), Lactacystin (5 μM), Carfilzomib (100 mM) or CQ (50 mM) for 6 hrs.


( H ) Co - immunoprecipitation and immunoblot analysis of extracts of Flag - NS1 - inducible THP - 1 cells treated with or without Dox ( 100 ng / ml ) .

O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml).


( I ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag - caspase - 1 and indicated HA - tagged WT ubiquitin or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - and K63 - Only ) , together with empty vector or Myc - NS1 , followed by MG132 ( 10 mM ) treatment for 6 hrs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-NS1, followed by MG132 (10 mM) treatment for 6 hrs.


( A ) Immunoblot analysis of extracts of wild type ( WT ) and USP8 knockout ( KO ) 293T cells transfected with Flag - caspase - 1 together with empty vector or Myc - NS1 .

O O O O O O O O O O O O O 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) Immunoblot analysis of extracts of wild type (WT) and USP8 knockout (KO) 293T cells transfected with Flag-caspase-1 together with empty vector or Myc-NS1.


( B ) Immunoblot analysis of extracts of transfected with Flag - caspase - 1 together with empty vector or Myc - USP8 then treated with or without MG132 ( 10 mM ) for 6 hrs .

O O O O 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

(B) Immunoblot analysis of extracts of transfected with Flag-caspase-1 together with empty vector or Myc-USP8 then treated with or without MG132 (10 mM) for 6 hrs.


( C ) Co - immunoprecipitation and immunoblot analysis of extracts of Flag - NS1 - inducible THP - 1 cells treated with or without Dox ( 100 ng / ml ) then transfected with or without poly ( I : C ) ( 2 mg / ml , 6 hrs ) .

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

(C) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml) then transfected with or without poly (I:C) (2 mg/ml, 6 hrs).


( D ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 and Myc - USP8 together with HA - NS1 followed by MG132 treatment ( 10 mM ) for 6 hrs .

(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 and Myc-USP8 together with HA-NS1 followed by MG132 treatment (10 mM) for 6 hrs.


( E ) Co - immunoprecipitation and immunoblot analysis of extracts of THP - 1 cells infected with ZIKV ( MOI = 1 ) for 24 hrs .

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

(E) Co-immunoprecipitation and immunoblot analysis of extracts of THP-1 cells infected with ZIKV (MOI=1) for 24 hrs.


( F ) Co - immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP - 1 cells infected with ZIKV ( MOI = 1 ) for 24 hrs .

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

(F) Co-immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP-1 cells infected with ZIKV (MOI=1) for 24 hrs.


( G ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag - caspase - 1 and indicated HA - tagged WT ubiquitin ( Ub ) or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - and K63 - Only ) , together with empty vector or Myc - USP8 , followed by MG132 ( 10 mM ) treatment for 6 hrs .

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

(G) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin (Ub) or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-USP8, followed by MG132 (10 mM) treatment for 6 hrs.


( H ) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag - caspase - 1 , HA - K11 - Ub together with empty vector or Myc - NS1 , followed by two - step immunoprecipitation with anti - Flag beads and immunoblot analysis with anti - HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag-caspase-1, HA-K11-Ub together with empty vector or Myc-NS1, followed by two-step immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA.


( I ) Co - immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag - caspase - 1 and HA - K11 - Ub together with WT or inactive mutant C786A of USP8 .

O O O O O O O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag-caspase-1 and HA-K11-Ub together with WT or inactive mutant C786A of USP8.


( A ) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA - NS1 together with Flag - caspae - 1 wild type ( WT ) and mutants ( K37R , K44R or K134R ) . Lower panel shows the intensity analysis of the bands from the three independent experiments .

(A) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA-NS1 together with Flag-caspae-1 wild type (WT) and mutants (K37R, K44R or K134R). Lower panel shows the intensity analysis of the bands from the three independent experiments.


( B ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA - K11 - ubiquitin ( Ub ) together with Flag - caspase - 1 ( WT , K37R , K44R , K134R ) .

(B) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-K11-ubiquitin (Ub) together with Flag-caspase-1 (WT, K37R, K44R, K134R).


( C ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 ( WT or K134R ) and HA - K11 - Ub , together with empty vector or Myc - NS1 . Lower panel shows the intensity analysis of the bands from the three independent experiments .

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

(C) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-NS1. Lower panel shows the intensity analysis of the bands from the three independent experiments.


( D ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 ( WT or K134R ) and HA - K11 - Ub , together with empty vector or Myc - USP8 . Lower panel shows the intensity analysis of the bands from the three independent experiments .

(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-USP8. Lower panel shows the intensity analysis of the bands from the three independent experiments.


( A ) Relative qRT - PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in wild type ( WT ) or caspase - 1 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative qRT-PCR analysis of IFNB, IFIT2 and IFIT1 mRNA levels in wild type (WT) or caspase-1 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( B ) Relative qRT - PCR analysis of IFNB mRNA levels in THP - 1 cells left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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

(B) Relative qRT-PCR analysis of IFNB mRNA levels in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.


( C ) Relative qRT - PCR analysis of IFNB mRNA levels in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

O O O O O O O O O O O O O O O O O O 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) Relative qRT-PCR analysis of IFNB mRNA levels in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( D ) Relative qRT - PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag - NS1 - inducible THP - 1 cells treated with or without Dox then left untreated or treated with YVAD ( 20 μM ) for 3 hrs , followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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

(D) Relative qRT-PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag-NS1-inducible THP-1 cells treated with or without Dox then left untreated or treated with YVAD (20 μM) for 3 hrs, followed by ZIKV infection (MOI=1) for the indicated time points.


( E ) Relative qRT - PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs .

(E) Relative qRT-PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.


( F ) Relative qRT - PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

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

(F) Relative qRT-PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.


( G ) ELISA of IFN - β in the supernatants of PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

(G) ELISA of IFN-β in the supernatants of PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.


( H ) ELISA of IL - 1β production in brain of WT or Nlrp3 - / - 1 - day - old neonatal mice ( n = 3 per group ) intracerebrally injected with 1 × 105 plaque - forming units ( PFU ) of ZIKV for 5 days .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA of IL-1β production in brain of WT or Nlrp3-/- 1-day-old neonatal mice (n=3 per group) intracerebrally injected with 1×105 plaque-forming units (PFU) of ZIKV for 5 days.


( I ) Relative qRT - PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV

(I) Relative qRT-PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection


( J ) ELISA of IFN - β production in brains of WT or Nlrp3 - / - neonatal

(J) ELISA of IFN-β production in brains of WT or Nlrp3-/- neonatal mice


( K ) Relative qRT - PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV

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

(K) Relative qRT-PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection


( L ) Plaque titration of ZIKV in brain tissues of WT or Nlrp3 - / - neonatal

(L) Plaque titration of ZIKV in brain tissues of WT or Nlrp3-/- neonatal mice


( M ) Body weight gain of WT and Nlrp3 - / - neonatal mice ( n = 4 per group ) inoculated with ZIKV ( 1 × 105 PFUs ) .

(M) Body weight gain of WT and Nlrp3-/- neonatal mice (n=4 per group) inoculated with ZIKV (1×105 PFUs).


( A ) Relative qRT - PCR analysis of ZIKV RNA levels in wild type ( WT ) or cGAS knockout ( KO ) THP - 1 cells after ZIKV infection ( MOI = 1 ) for indicated time points .

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

(A) Relative qRT-PCR analysis of ZIKV RNA levels in wild type (WT) or cGAS knockout (KO) THP-1 cells after ZIKV infection (MOI=1) for indicated time points.


( B ) Relative qRT - PCR analysis of IFNΒ , IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP - 1 cells after ZIKV infection ( MOI = 1 ) for indicated time points .

O O O O O O O O O O O O O O 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) Relative qRT-PCR analysis of IFNΒ, IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP-1 cells after ZIKV infection (MOI=1) for indicated time points.


( C ) Relative qRT - PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP , cGAS or NLRP3 and infected with ZIKV ( MOI = 1 ) for 36 hrs .

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

(C) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP, cGAS or NLRP3 and infected with ZIKV (MOI=1) for 36 hrs.


( D ) Relative qRT - PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP - 1 cells treated with or without YVAD ( 20 μM ) , followed by ZIKV infection ( MOI = 1 ) for indicated times points .

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

(D) Relative qRT-PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP-1 cells treated with or without YVAD (20 μM), followed by ZIKV infection (MOI=1) for indicated times points.


( E ) Immunoblot analysis of extracts of Flag - cGAS - overexpressed THP - 1 cells treated with or without YVAD ( 20 μM ) then infected with ZIKV ( MOI = 5 ) for 24 hrs or infected with HSV - 1 ( MOI = 1 ) for 24 hrs .

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(E) Immunoblot analysis of extracts of Flag-cGAS-overexpressed THP-1 cells treated with or without YVAD (20 μM) then infected with ZIKV (MOI=5) for 24 hrs or infected with HSV-1 (MOI=1) for 24 hrs.


Immunoblot analysis of WT , NLRP3 KO ( F ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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Immunoblot analysis of WT, NLRP3 KO (F) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


Immunoblot analysis of WT , caspase - 1 KO ( G ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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Immunoblot analysis of WT, caspase-1 KO (G) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( H ) Immunoblot analysis of extracts of WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points .

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(H) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.


( I ) Immunoblot analysis of PBMCs from Donor # 1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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(I) Immunoblot analysis of PBMCs from Donor#1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for the indicated time points.


( J ) Immunoblot analysis of extracts of 293T cells transfected with Myc - cGAS ( tagged on C - terminal ) , GFP - ASC , Flag - caspase - 1 , together with empty vector or HA - NS1 .

(J) Immunoblot analysis of extracts of 293T cells transfected with Myc-cGAS (tagged on C-terminal), GFP-ASC, Flag-caspase-1, together with empty vector or HA-NS1.


( K ) Relative qRT - PCR analysis of ZIKV RNA and IFNB mRNA levels in WT , cGAS KO THP - 1 cells as well as cGAS KO THP - 1 cells reconstituted with WT or D140A / D157A mutant of cGAS , followed by ZIKV infection ( MOI = 1 ) for 24 hrs .

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(K) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in WT, cGAS KO THP-1 cells as well as cGAS KO THP-1 cells reconstituted with WT or D140A/D157A mutant of cGAS, followed by ZIKV infection (MOI=1) for 24 hrs.


Sexing the established ESCs by detecting the Y - linked Zfy gene using genomic DNA ( gDNA ) - qPCR , normalized to Gapdh . n . d . : not detected . Average of presumed males was set to 1 .

Sexing the established ESCs by detecting the Y-linked Zfy gene using genomic DNA (gDNA)-qPCR, normalized to Gapdh. n.d.: not detected. Average of presumed males was set to 1.


DNA - FISH detection of the number of X - linked Atrx gene foci . Representative images of the DNA - FISH are shown . Nuclei are surrounded by white dotted lines . Magenta : Atrx ( white arrowheads ) , cyan : DAPI . N > 100 .

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DNA-FISH detection of the number of X-linked Atrx gene foci. Representative images of the DNA-FISH are shown. Nuclei are surrounded by white dotted lines. Magenta: Atrx (white arrowheads), cyan: DAPI. N>100.


Heatmap analysis from RNA - seq . Each log10 ( FPKM + 1 ) value was used for clustering . Genes with high or low expression are shown in red and blue , respectively . FPKM : fragments per kilobases per million reads .

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Heatmap analysis from RNA-seq. Each log10(FPKM+1) value was used for clustering. Genes with high or low expression are shown in red and blue, respectively. FPKM: fragments per kilobases per million reads.


X and Y chromosome mapping . Genes with high expression ( up ) are shown in orange and low expression ( down ) are shown in blue . The defined ratio of up or down genes is > 1 . 5 and < two - thirds , respectively . The comparison is ( each female vs the average for males ) or ( each male vs the average for females ) . The location controls for the expressed genes ( exp . genes ) on the X - chromosome and Y - chromosome are plotted ( red squares ) for genes whose FPKM averages were above 1 for females or males .

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X and Y chromosome mapping. Genes with high expression (up) are shown in orange and low expression (down) are shown in blue. The defined ratio of up or down genes is > 1.5 and < two-thirds, respectively. The comparison is (each female vs the average for males) or (each male vs the average for females). The location controls for the expressed genes (exp. genes) on the X-chromosome and Y-chromosome are plotted (red squares) for genes whose FPKM averages were above 1 for females or males.


Clonogenic assay for ESCs in response to etoposide ( A ) or camptothecin ( B ) . Non - treated cells were set to 1 .

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Clonogenic assay for ESCs in response to etoposide (A) or camptothecin (B). Non-treated cells were set to 1.


Nanog HR assay . The percentage of GFP - positive colonies after gene targeting to the Nanog locus is shown . The results for each cell line are shown at right .

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Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown. The results for each cell line are shown at right.


Nanog expression confirmed by RNA - seq .

Nanog expression confirmed by RNA-seq.


Detection of the number of X - linked Atrx gene foci by DNA - FISH in GFP - positive subclones of the Nanog - targeting assay ( nine independent clones , N > 100 each ) , performed as described in Figure 1C .

Detection of the number of X-linked Atrx gene foci by DNA-FISH in GFP-positive subclones of the Nanog-targeting assay (nine independent clones, N> 100 each), performed as described in Figure 1C.


Sexing of established ESCs ( gDNA - qPCR , Zfy / Gapdh ) , performed as described in Figure 1B .

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Sexing of established ESCs (gDNA-qPCR, Zfy/Gapdh), performed as described in Figure 1B.


Detection of the number of X - linked Atrx gene foci by DNA - FISH with or without dox addition for 96 h ( N > 100 ) , performed as described in Figure 1C .

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Detection of the number of X-linked Atrx gene foci by DNA-FISH with or without dox addition for 96 h (N >100), performed as described in Figure 1C.


Detection of the Xist cloud in # B3 by RNA - FISH , with or without dox addition for 96 h ( N > 200 each ) . A representative image is shown [ magenta : Xist ( white arrowhead ) , cyan : DAPI . Scale bar : 10 μm ] .

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Detection of the Xist cloud in #B3 by RNA-FISH, with or without dox addition for 96 h ( N >200 each). A representative image is shown [magenta: Xist (white arrowhead), cyan: DAPI. Scale bar: 10 μm].


Allele - distinguishable RT - PCR for detecting Hprt , G6pdx , and Rnf12 X - linked genes . Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown .

Allele-distinguishable RT-PCR for detecting Hprt, G6pdx, and Rnf12 X-linked genes. Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown.


Nanog HR assay . The percentage of GFP - positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C . # B2 or # B3 were cultured with or without dox addition for 96 h . The combined number of GFP - positive cells and total colonies from all experiments are listed and summarized at right .

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Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C. #B2 or #B3 were cultured with or without dox addition for 96 h. The combined number of GFP-positive cells and total colonies from all experiments are listed and summarized at right.


Detection of the Xist foci by RNA - FISH , with or without dox addition for 96 h ( N > 200 each ) . A representative image with percentage of Xist - positive cells is shown [ magenta : Xist , cyan : DAPI . ] .

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Detection of the Xist foci by RNA-FISH, with or without dox addition for 96 h (N >200 each). A representative image with percentage of Xist-positive cells is shown [magenta: Xist, cyan: DAPI.].


Detection of the number of X - linked Atrx gene foci by DNA - FISH , with or without dox addition for 96 h ( N > 100 each ) , performed as described in Figure 1C .

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

Detection of the number of X-linked Atrx gene foci by DNA-FISH, with or without dox addition for 96 h (N >100 each), performed as described in Figure 1C.


Estimated repair events . The flow cytometry plots are shown in Figure EV4 . The percentage of GFP - positive cells in TX - DRGFP # 11 and TX - EJ5GFP # 23 was normalized to the transfection efficiency detected by the VENUS expression vector . Dox ( - ) was set to 1 and the ratio of relative GFP - positive cells is shown ( n = 3 ) .

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Estimated repair events. The flow cytometry plots are shown in Figure EV4. The percentage of GFP-positive cells in TX-DRGFP#11 and TX-EJ5GFP#23 was normalized to the transfection efficiency detected by the VENUS expression vector. Dox (-) was set to 1 and the ratio of relative GFP-positive cells is shown (n=3).


Volcano plot of DEGs in female ESCs ( # A4 , # A10 , # A11 ) versus males ( # A6 , # A13 , # A17 ) . The definition of up ( red dots ) or down ( blue dots ) genes is as follows : the ratio of up or down genes is > 1 . 5 and < two - thirds , respectively , with q - value < 0 . 05 .

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Volcano plot of DEGs in female ESCs (#A4, #A10, #A11) versus males (#A6, #A13, #A17). The definition of up (red dots) or down (blue dots) genes is as follows: the ratio of up or down genes is > 1.5 and < two-thirds, respectively, with q-value< 0.05.


Brcc3 expression confirmed by RNA - seq [ biological replicates ( n = 3 ) with three different cell lines ( male : # A6 , # A13 , # A17 ; female : # A4 , # A10 , # A11 ) ] .

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Brcc3 expression confirmed by RNA-seq [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Brcc3 expression confirmed by RT - qPCR [ biological replicates ( n = 3 ) with three different cell lines ( male : # A6 , # A13 , # A17 ; female : # A4 , # A10 , # A11 ) ] .

Brcc3 expression confirmed by RT-qPCR [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Brcc3 expression confirmed by western blotting [ biological replicates ( n = 3 ) with three different cell lines ( male : # A6 , # A13 , # A17 ; female : # A4 , # A10 , # A11 ) ] .

Brcc3 expression confirmed by western blotting [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].


Confirmation of Brcc3 expression from knockdown samples in by RT - qPCR ( technical duplicates )

Confirmation of Brcc3 expression from knockdown samples in by RT-qPCR (technical duplicates)


Confirmation of Brcc3 expression from knockdown samples in western blotting


Nanog HR assay with Brcc3 knockdown ( three biological replicates ) . The percentage of GFP - positive colonies after gene targeting to the Nanog locus is shown , as shown in Figure 2C . The combined number of GFP - positive cells and total number of colonies are shown ( please note that total cell number of # A13 for drug screening was 3 . 67 × 107 because of contamination of 1 out of 12 dishes , compared with the other colonies for which 4 × 107 cells were used ) .

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Nanog HR assay with Brcc3 knockdown (three biological replicates). The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown, as shown in Figure 2C. The combined number of GFP-positive cells and total number of colonies are shown (please note that total cell number of #A13 for drug screening was 3.67×107 because of contamination of 1 out of 12 dishes, compared with the other colonies for which 4×107 cells were used).


Kaplan - Meier curves for the overall survival of breast cancer patients based on BRCC3 ( A ) , XIST ( B ) , and BRCC3 / XIST ( C ) expression levels . HR : hazard ratio .

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Kaplan-Meier curves for the overall survival of breast cancer patients based on BRCC3 (A), XIST (B), and BRCC3/XIST(C) expression levels. HR: hazard ratio.


Trajectory plots of siRNA - transfected HUVECs under VEGF and / or Sema3E gradients for 12 h . Red trajectories indicate cells with displacement to the negative y - axis at the end of analyses .

Trajectory plots of siRNA-transfected HUVECs under VEGF and/or Sema3E gradients for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses.


Rose plots of cell trajectories in ( A ) . Leaflets are grouped into 10 ° segments . The radius of each leaflet represents the cumulative number of cells migrating in the corresponding angular bin . P values for the Rayleigh test represent non - random distributions of cell endpoints .

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Rose plots of cell trajectories in (A). Leaflets are grouped into 10° segments. The radius of each leaflet represents the cumulative number of cells migrating in the corresponding angular bin. P values for the Rayleigh test represent non-random distributions of cell endpoints.


Forward migration index ( FMI ) along the y - and x - axes during cell migration . n = 90 cells per group . Data represent mean ± SEM . * * p < 0 . 01 ; * * * p < 0 . 001 ; NS , not significant , by Tukey - Kramer test .

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Forward migration index (FMI) along the y- and x-axes during cell migration. n = 90 cells per group. Data represent mean ± SEM. **p < 0.01; ***p < 0.001; NS, not significant, by Tukey-Kramer test.


Confocal ( left three columns ) and super - resolution ( right - most columns ) images of subcellular distributions of ectopic RhoJ ( red ) , endogenous VEGFR2 ( green ) and PlexinD1 ( left , white ; right , blue ) in cultured HUVECs 6 h after transfection with the indicated RhoJ constructs . Note the colocalization of WT - RhoJ and CA - RhoJ ( Q79L ) with PlexinD1 in perinuclear endosomes and DN - RhoJ ( T35N ) with VEGFR2 in cytoplasmic and nuclear puncta . Light - blue asterisks in the PlexinD1 panels indicate nuclei . See also Appendix Fig S2 for the full images of super - resolution microscopy . Scale bar , 5 µm ( left ) ; 1 µm ( right ) .

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Confocal (left three columns) and super-resolution (right-most columns) images of subcellular distributions of ectopic RhoJ (red), endogenous VEGFR2 (green) and PlexinD1 (left, white; right, blue) in cultured HUVECs 6 h after transfection with the indicated RhoJ constructs. Note the colocalization of WT-RhoJ and CA-RhoJ (Q79L) with PlexinD1 in perinuclear endosomes and DN-RhoJ (T35N) with VEGFR2 in cytoplasmic and nuclear puncta. Light-blue asterisks in the PlexinD1 panels indicate nuclei. See also Appendix Fig S2 for the full images of super-resolution microscopy. Scale bar, 5 µm (left); 1 µm (right).


Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 and / or PlexinD1 . n = 10 cells per group . Data represent mean .

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Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 and/or PlexinD1. n = 10 cells per group. Data represent mean.


Co - immunoprecipitation ( co - IP ) from 293T cells 48 h after transfection . WT - and CA - RhoJ associated with PlexinD1 , while DN - RhoJ associated with VEGFR2 .

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Co-immunoprecipitation (co-IP) from 293T cells 48 h after transfection. WT- and CA-RhoJ associated with PlexinD1, while DN-RhoJ associated with VEGFR2.


Co - IP from 293T cells 48 h after transfection . RhoJ - PlexinD1 binding was disrupted by deleting the RBD or ECD of PlexinD1 and by 30 - min stimulation with Sema3E . Arrows indicate PD1 Full ( upper , 250 kDa ) and PD1 △ RBD ( lower , 1 . 7 kDa smaller than PD1 Full ) .

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

Co-IP from 293T cells 48 h after transfection. RhoJ-PlexinD1 binding was disrupted by deleting the RBD or ECD of PlexinD1 and by 30-min stimulation with Sema3E. Arrows indicate PD1 Full (upper, 250 kDa) and PD1△RBD (lower, 1.7 kDa smaller than PD1 Full).


PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation . Scale bar , 10 µm .

PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation. Scale bar, 10 µm.


Super - resolution images of subcellular distributions of ectopic WT - RhoJ ( red ) and endogenous PlexinD1 ( green ) in HUVECs 24 h after transfection . RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation . Scale bar , 1 µm .

Super-resolution images of subcellular distributions of ectopic WT-RhoJ (red) and endogenous PlexinD1 (green) in HUVECs 24 h after transfection. RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation. Scale bar, 1 µm.


Labeling of siRNA - transfected HUVECs with phalloidin ( white ) 30 min after Sema3E stimulation . Scale bar , 50 µm .

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Labeling of siRNA-transfected HUVECs with phalloidin (white) 30 min after Sema3E stimulation. Scale bar, 50 µm.


Quantification of areas of individual HUVECs . n > 100 cells per group . Data represent mean ± SEM . * * * p < 0 . 001 ; NS , not significant , by Tukey - Kramer test .

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Quantification of areas of individual HUVECs. n > 100 cells per group. Data represent mean ± SEM. ***p < 0.001; NS, not significant, by Tukey-Kramer test.


Co - IP from HUVECs . Blots for β - actin shown in ( A ) were used to confirm equal loading in ( B ) , as the same cell lysates were analyzed in both experiments .

Co-IP from HUVECs. Blots for β-actin shown in (A) were used to confirm equal loading in (B), as the same cell lysates were analyzed in both experiments.


Immunoblots in siRNA - transfected HUVECs .

Immunoblots in siRNA-transfected HUVECs.


Co - IP from siRNA - transfected HUVECs . The graph shows relative levels of PlexinD1 - VEGFR2 association , in which the values at 0 min are set to 1 . n = 3 per group .

Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.


Super - resolution images of receptor colocalization in siRNA - transfected HUVECs 30 min after Sema3E stimulation . Left panels show immunolabeling of VEGFR2 ( green ) , PlexinD1 ( red ) , and Nrp1 ( white ) , with light - blue asterisks indicating nuclei . Middle panels show magnified views of white boxes in the left panels . Right panels represent fluorescence intensity profiles along 5 μm arbitrary lines in the middle panels . Scale bar , 10 µm . The graph shows the proportion of receptor complexes in PlexinD1 - positive vesicles . n = 10 cells per group .

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Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after Sema3E stimulation. Left panels show immunolabeling of VEGFR2 (green), PlexinD1 (red), and Nrp1 (white), with light-blue asterisks indicating nuclei. Middle panels show magnified views of white boxes in the left panels. Right panels represent fluorescence intensity profiles along 5 μm arbitrary lines in the middle panels. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in PlexinD1-positive vesicles. n = 10 cells per group.


Trajectory plots ( left ) and rose plots ( right ) of HUVECs pre - treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h . Red trajectories indicate cells with displacement to the negative y - axis at the end of analyses . P values for the Rayleigh test represent non - random distributions of cell endpoints . The graphs show FMI along the y - and x - axes during cell migration . n = 90 cells per group .

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Trajectory plots (left) and rose plots (right) of HUVECs pre-treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses. P values for the Rayleigh test represent non-random distributions of cell endpoints. The graphs show FMI along the y- and x-axes during cell migration. n = 90 cells per group.


Super - resolution images of receptor colocalization in siRNA - transfected HUVECs 30 min after VEGF stimulation . VEGFR2 ( green ) , PlexinD1 ( red ) , and Nrp1 ( white ) are shown as in Fig 4E . Scale bar , 10 µm . The graph shows the proportion of receptor complexes in the PlexinD1 - positive vesicles . Data of " no stimuli " are the same from Fig 4E . n = 10 cells per group .

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Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after VEGF stimulation. VEGFR2 (green), PlexinD1 (red), and Nrp1 (white) are shown as in Fig 4E. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in the PlexinD1-positive vesicles. Data of "no stimuli" are the same from Fig 4E. n = 10 cells per group.


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Immunoblots in siRNA - transfected HUVECs . Blots for β - actin shown in ( C ) were used to confirm equal loading in ( D ) , as the same cell lysates were analyzed in these experiments . The graph shows changes of total VEGFR2 protein levels as a percentage of the values at 0 min . n = 3 per group .

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Immunoblots in siRNA-transfected HUVECs. Blots for β-actin shown in (C) were used to confirm equal loading in (D), as the same cell lysates were analyzed in these experiments. The graph shows changes of total VEGFR2 protein levels as a percentage of the values at 0 min. n = 3 per group.


Immunoblots in siRNA - transfected HUVECs . Note the early and transient activation of PLCγ , Erk1 / 2 , and Akt , but not p38 MAPK , in RhoJ - knockdown ECs .

Immunoblots in siRNA-transfected HUVECs. Note the early and transient activation of PLCγ, Erk1/2, and Akt, but not p38 MAPK, in RhoJ-knockdown ECs.


Immunoblots for ectopic RhoJ and endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ - expressing vectors . The graph shows protein levels of VEGFR2 relative to those of the control set as 100 % . n = 3 per group .

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Immunoblots for ectopic RhoJ and endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ-expressing vectors. The graph shows protein levels of VEGFR2 relative to those of the control set as 100%. n = 3 per group.


Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model . Upper panels are snapshots of time - lapse imaging in which EC nuclei are labeled by SYTO dye ( green ) . In analyzed regions framed by dotted lines , the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan . Initial discrete labeling becomes intermixed over time ( see also Movie EV1 ) . Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation . Scale bar , 100

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

Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model. Upper panels are snapshots of time-lapse imaging in which EC nuclei are labeled by SYTO dye (green). In analyzed regions framed by dotted lines, the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan. Initial discrete labeling becomes intermixed over time (see also Movie EV1). Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation. Scale bar, 100 µ


Quantification of the kinetics of EC migration . Values of forward and reverse migration for RhojWT / WT are normalized to 1 . n = 33 vessel branches per group .

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

Quantification of the kinetics of EC migration. Values of forward and reverse migration for RhojWT/WT are normalized to 1. n = 33 vessel branches per group.


Quantification of vessel elongation ( left graph ) . n = 33 vessel branches per group . Scatter plot shows relationship between " vessel elongation " and " reverse migration " in RhojGFP / GFP aortic rings as evaluated by a Pearson correlation coefficient .

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

Quantification of vessel elongation (left graph). n = 33 vessel branches per group. Scatter plot shows relationship between "vessel elongation" and "reverse migration" in RhojGFP/GFP aortic rings as evaluated by a Pearson correlation coefficient.


Labeling for YFP / GFP ( green ) , CD31 ( red ) , and ETS transcription factor ERG ( blue ) in retinal vascular fronts of P5 CAG - MerCreMer : R26R - EYFPflox / WT and CAG - MerCreMer : Rhojfloxflox mice after intraperitoneal ( i . p . ) injections of 10 µg of 4 - hydroxytamoxifen ( 4OHT ) at P1 . Scale bar , 50 µm . Relative contribution of YFP / GFP - positive ECs to the tip positions . n = 16 per group . Values for CAG - MerCreMer : R26R - EYPflox / WT are normalized to 1 .

Labeling for YFP/GFP (green), CD31 (red), and ETS transcription factor ERG (blue) in retinal vascular fronts of P5 CAG-MerCreMer:R26R-EYFPflox/WT and CAG-MerCreMer:Rhojfloxflox mice after intraperitoneal (i.p.) injections of 10 µg of 4-hydroxytamoxifen (4OHT) at P1. Scale bar, 50 µm.   Relative contribution of YFP/GFP-positive ECs to the tip positions. n = 16 per group. Values for CAG-MerCreMer:R26R-EYPflox/WT are normalized to 1.


Labeling for GFP ( green ) and CD31 ( red ) in retina of P4 RhojGFP / GFP mouse . Scale bar , 200 µm ( left ) ; 20 µm ( right ) .

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

Labeling for GFP (green) and CD31 (red) in retina of P4 RhojGFP/GFP mouse. Scale bar, 200 µm (left); 20 µm (right).


Labeling for EdU ( green ) , ERG ( red ) , and CD31 ( gray ) in retinas of P4 RhojWT / WT and RhojGFP / GFP mice 2 h after i . p . EdU injections . Scale bar , 20 µm . The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts . n = 12 per group .

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

Labeling for EdU (green), ERG (red), and CD31 (gray) in retinas of P4 RhojWT/WT and RhojGFP/GFP mice 2 h after i.p. EdU injections. Scale bar, 20 µm. The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts. n = 12 per group.


Labeling for CD31 in retinas of P4 and P7 RhojWT / WT and RhojGFP / GFP mice . Scale bars , 500 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

Labeling for CD31 in retinas of P4 and P7 RhojWT/WT and RhojGFP/GFP mice. Scale bars, 500 µm.


Morphometric analyses of retinal vessels in RhojWT / WT ( P4 , n = 18 ; P7 , n = 8 ) and RhojGFP / GFP ( P4 , n = 16 ; P7 , n = 11 ) mice .

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

Morphometric analyses of retinal vessels in RhojWT/WT (P4, n = 18; P7, n = 8) and RhojGFP/GFP (P4, n = 16; P7, n = 11) mice.


Labeling for CD31 in retinas of P4 Rhojflox / flox and Pdgfb - iCreERT2 : Rhojflox / flox ( RhojiΔEC ) mice after single i . p . injection of 100 µg of 4OHT at P1 . Scale bar , 500 µm .

Labeling for CD31 in retinas of P4 Rhojflox/flox and Pdgfb-iCreERT2:Rhojflox/flox (RhojiΔEC) mice after single i.p. injection of 100 µg of 4OHT at P1. Scale bar, 500 µm.


Morphometric analyses of retinal vessels in P4 Rhojflox / flox ( n = 7 ) and Rhoji ∆ EC ( n = 6 ) mice .

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

Morphometric analyses of retinal vessels in P4 Rhojflox/flox (n = 7) and Rhoji∆EC (n = 6) mice.


Relative expression levels of Vegfa and Sema3e mRNA from WT mouse retinas . n = 3 per group .

Relative expression levels of Vegfa and Sema3e mRNA from WT mouse retinas. n = 3 per group.


Labeling for GFP ( green ) and CD31 ( red ) in retina of P18 OIR - RhojGFP / WT mouse . Scale bar , 200

Labeling for GFP (green) and CD31 (red) in retina of P18 OIR-RhojGFP/WT mouse. Scale bar, 200 µ


Labeling for CD31 in retinas of P18 OIR - Rhojflox / flox and OIR - Rhoji ∆ EC mice after daily i . p . injection of 200 µg of 4OHT from P12 . Scale bar , 500 µm . Quantification of areas of neovascular tufts in P18 OIR - Rhojflox / flox and OIR - Rhoji ∆ EC mice . n = 6 per group .

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

Labeling for CD31 in retinas of P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice after daily i.p. injection of 200 µg of 4OHT from P12. Scale bar, 500 µm. Quantification of areas of neovascular tufts in P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice. n = 6 per group.


A - D LSM images of proximity ligation assay ( PLA ) dots composed of VEGFR3 and phosphorylated tyrosine ( p - Tyr ) on cross - sections through the jugular lymph sac / primordial thoracic duct ( jls / pTD ) of E13 . 5 control and ILK K . O . embryos . Scale bars : 10 µm . E - H LSM images of PLA dots composed of VEGFR2 and phosphorylated tyrosine ( p - Tyr ) on cross - sections through the jls / pTD of E13 . 5 control and ILK K . O . embryos . Scale bars : 10 µm . I Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK K . O . embryos ( n = 9 embryos per genotype ) , * P = 0 . 022 . J Quantification of the PLA dots indicating VEGFR2 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK K . O . embryos ( n = 3 embryos per genotype ) . K Quantification of the total number of PLA dots indicating both VEGFR2 ( left ) and VEGFR3 ( right ) with phosphorylated tyrosine ( p - Tyr ) per jls / pTD section of E13 . 5 control or ILK K . O . embryos ( n = 3 embryos per genotype ) , * P = 0 . 005 ( VEGFR2 / p - Tyr in ILK K . O . versus VEGFR3 / p - Tyr in ILK K . O . ) , * P = 0 . 013 ( VEGFR3 / p - Tyr in control versus VEGFR3 / p - Tyr in ILK K . O . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D LSM images of proximity ligation assay (PLA) dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. E-H LSM images of PLA dots composed of VEGFR2 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. I Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 9 embryos per genotype), *P = 0.022. J Quantification of the PLA dots indicating VEGFR2 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype). K Quantification of the total number of PLA dots indicating both VEGFR2 (left) and VEGFR3 (right) with phosphorylated tyrosine (p-Tyr) per jls/pTD section of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype), *P = 0.005 (VEGFR2/p-Tyr in ILK K.O. versus VEGFR3/p-Tyr in ILK K.O.), *P = 0.013 (VEGFR3/p-Tyr in control versus VEGFR3/p-Tyr in ILK K.O.)


LSM images of cross - sections through the jugular lymph sac / primordial thoracic duct ( jls / pTD ) of an E13 . 5 control embryo stained for surface VEGFR3 and β1 integrin . Scale bar : 10

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

LSM images of cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 µm


LSM images of proximity ligation assay ( PLA ) dots ( arrows ) composed of VEGFR3 and β1 integrin on cross - sections through the jls / pTD of E13 . 5 control and ILK K . O . embryos . Scale bars : 10 µm Quantification of VEGFR3 / β1 integrin PLA dots normalised to Lyve1 - positive area of control or ILK K . O . embryos ( n = 6 embryos per genotype ) ,

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

LSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1-positive area of control or ILK K.O. embryos (n = 6 embryos per genotype),


Bright - field image of an E13 . 5 Flk1 - Cre ; Ilk ∆ / + ; Itgb1 ∆ / + mouse embryo ( referred to as " Control " ) with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells , and a LSM image of a stained cross - section through its jugular lymph sac / primordial thoracic duct ( jls / pTD ) . Scale bars : 500 and 100 µm , respectively . C , D Bright - field image of an E13 . 5 Flk1 - Cre ; Ilk ∆ / ∆ ; Itgb1 ∆ / + embryo ( referred to as " ILK & β1 integrin K . O . " ) with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells , and a LSM image of a stained cross - section through its jls / pTD . Scale bars : 500 and 100 µm , respectively . E - H LSM images of cross - sections through the jls / pTD of E13 . 5 control and ILK & β1 integrin K . O . embryos stained for the proliferation marker phospho - Histone H3 . Arrows point to phospho - Histone H3 - positive

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

Bright-field image of an E13.5 Flk1-Cre;Ilk∆/+;Itgb1∆/+ mouse embryo (referred to as "Control") with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jugular lymph sac / primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 µm, respectively. C, D Bright-field image of an E13.5 Flk1-Cre;Ilk∆/∆;Itgb1∆/+ embryo (referred to as "ILK & β1 integrin K.O.") with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jls/pTD. Scale bars: 500 and 100 µm, respectively. E-H LSM images of cross-sections through the jls/pTD of E13.5 control and ILK & β1 integrin K.O. embryos stained for the proliferation marker phospho-Histone H3. Arrows point to phospho-Histone H3-positive LEC


LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine ( p - Tyr ) on stained cross - sections through the jls / pTD of control and ILK & β1 integrin K . O . embryos . Arrows point to PLA dots within the Lyve1 stained area . Scale bars : 10

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

LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on stained cross-sections through the jls/pTD of control and ILK & β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1 stained area. Scale bars: 10 µm


Number of LECs per jls / pTD section in E13 . 5 control or ILK & β1 integrin K . O .

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

Number of LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryos


LEC proliferation as determined by the number of phospho - Histone H3 - positive LECs per jls / pTD section in E13 . 5 control or ILK & β1 integrin K . O .

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

LEC proliferation as determined by the number of phospho-Histone H3-positive LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryos


Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK & β1 integrin K . O .

Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK & β1 integrin K.O. embryos


LSM images of a whole mount stained adult Prox1 - CreERT2 ; Ilk + / + mouse ear ( referred to as " Adult Control " ) and Prox1 - CreERT2 ; Ilk ∆ / ∆ mouse ear ( referred to as " Adult ILK K . O . " ) , with higher magnification images indicated by dashed lines . Scale bars : 500 and LSM images of a whole - mount stained adult control and ILK K . O . ear , with higher magnification images indicated by dashed lines . Arrows point to proliferating LECs . Scale bars : 100 and 20 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

LSM images of a whole mount stained adult Prox1-CreERT2;Ilk+/+ mouse ear (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse ear (referred to as "Adult ILK K.O."), with higher magnification images indicated by dashed lines. Scale bars: 500 and LSM images of a whole-mount stained adult control and ILK K.O. ear, with higher magnification images indicated by dashed lines. Arrows point to proliferating LECs. Scale bars: 100 and 20 µm, respectively


Dermal lymph vessel density as determined by VEGFR3 - positive area normalised to the total analysed area of adult control or ILK K . O . mice ( n = 4 control and n = 5 ILK K . O . mice ) ,

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

Dermal lymph vessel density as determined by VEGFR3-positive area normalised to the total analysed area of adult control or ILK K.O. mice (n = 4 control and n = 5 ILK K.O. mice),


LEC proliferation as determined by phospho - Histone H3 - positive LECs normalised to analysed VEGFR3 - positive area in the skin of adult control or ILK K . O . mice ( with n = 7 control and n = 5 ILK K . O . mice ) ,

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

LEC proliferation as determined by phospho-Histone H3-positive LECs normalised to analysed VEGFR3-positive area in the skin of adult control or ILK K.O. mice (with n = 7 control and n = 5 ILK K.O. mice),


VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K . O . mice ( n = 3 mice per genotype )

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

VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K.O. mice (n = 3 mice per genotype)


LSM images of a whole mount stained adult control and ILK K . O . cornea , with detailed images on their right ( L " , M " ) . Arrows point to lymph vessels protruding into the cornea ( encircled by a dotted line ) . Scale bars : 500 µm and 100 µm , re Number of lymph vessels in control or ILK K . O . corneas ( n = 5 and n = 7 corneas ) ,

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

LSM images of a whole mount stained adult control and ILK K.O. cornea, with detailed images on their right (L", M"). Arrows point to lymph vessels protruding into the cornea (encircled by a dotted line). Scale bars: 500 µm and 100 µm, re Number of lymph vessels in control or ILK K.O. corneas (n = 5 and n = 7 corneas),


LSM images of cross - sections through the heart of an adult Prox1 - CreERT2 ; Ilk + / + mouse ( referred to as " Adult Control " ) and Prox1 - CreERT2 ; Ilk ∆ / ∆ mouse ( referred to as " Adult ILK K . O . " ) showing the outer lateral region of the left ventricle ( LV ) Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1 - positive and CD31 - positive area ( normalised to total analysed myocardial area ) , respectively ( n = 4 control and n = 5 ILK K . O . mice )

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

LSM images of cross-sections through the heart of an adult Prox1-CreERT2;Ilk+/+ mouse (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse (referred to as "Adult ILK K.O.") showing the outer lateral region of the left ventricle (LV) Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively (n = 4 control and n = 5 ILK K.O. mice)


LSM images of cross - sections through the heart of an adult control and ILK K . O . mouse four weeks after myocardial ischemia and reperfusion ( MI / R ) , showing the outer lateral region of the LV . Scale bars : 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1 - positive and CD31 - positive area ( normalised to total analysed myocardial area ) , respectively . ( n = 5 control and n = 7 ILK K . O . mice ) , P = 0 . 051 ( cardiac lymph vessel density in adult control versus adult ILK K . O . ) , * P = 0 . 025 ( cardiac blood vessel density in adult control versus adult ILK K . O . )

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

LSM images of cross-sections through the heart of an adult control and ILK K.O. mouse four weeks after myocardial ischemia and reperfusion (MI/R), showing the outer lateral region of the LV. Scale bars: 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively. (n = 5 control and n = 7 ILK K.O. mice), P = 0.051 (cardiac lymph vessel density in adult control versus adult ILK K.O.), *P = 0.025 (cardiac blood vessel density in adult control versus adult ILK K.O.)


Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA . Scale bars : 50

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

Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 µm


LEC proliferation as determined by the number of BrdU - positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF - C Cys156Ser ( n = 3 independent transfections per siRNA ) , * P = 0 . 032 ( control versus ILK - 1 ) , * P = 0 . 005 ( control versus ILK - 2 ) , * P = 0 . 0003 ( control versus ILK - 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 O

LEC proliferation as determined by the number of BrdU-positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 3 independent transfections per siRNA), *P = 0.032 (control versus ILK-1), *P = 0.005 (control versus ILK-2), *P = 0.0003 (control versus ILK-3)


VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF - C Cys156Ser ( n = 4 ( control siRNA , ILK - 1 siRNA , and ILK - 3 siRNA ) , or n = 8 ( ILK - 2 siRNA ) independent transfections per siRNA ) , * P = 0 . 0001 ( control versus each siRNA )

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

VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 4 (control siRNA, ILK-1 siRNA, and ILK-3 siRNA), or n = 8 (ILK-2 siRNA) independent transfections per siRNA), *P = 0.0001 (control versus each siRNA)


LSM images of VEGFR3 / β1 integrin PLA dots in human LECs transfected with control or ILK siRNA . Scale bars : 10 µm Quantification of VEGFR3 / β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA ) , P = 0 . 234 ( control versus ILK - 1 ) , * P = 0 . 024 ( control versus ILK - 2 ) , * P = 0 . 001 ( control versus ILK - 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 O O O O O O O O

LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs (n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK-1), *P = 0.024 (control versus ILK-2), *P = 0.001 (control versus ILK-3)


LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes . Red dots are PLA dots composed of VEGFR3 and β1 integrin . Scale bars : 10 µm Quantification of VEGFR3 / β1 integrin PLA dots per human LEC with ( + ) or without ( - ) mechanical stretch ( n = 6 independent stretch chambers ) ,

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

LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (-) mechanical stretch (n = 6 independent stretch chambers),


Western blot ( WB ) image of human LECs that were either kept unstretched or stretched for 30 minutes , and used for immunoprecipitation ( IP ) of HA - tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with ( + ) or without ( - ) mechanical stretch ; normalised to the respective amount of HA - tagged β1 integrin ( n = 3 ( unstretched ) or n = 5 ( stretched ) independent stretch

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Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 minutes, and used for immunoprecipitation (IP) of HA-tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (-) mechanical stretch; normalised to the respective amount of HA-tagged β1 integrin (n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers


C , D Two types of TNTs were detected in H1299 ( C ) and A549 ( D ) cells , Type 1 , containing F - actin with little or no detectable microtubules ( white box ) and Type2 , containing both microtubules and F - actin ( yellow box ) . Anti - β - tubulin antibody ( green ) and phalloidin ( red ) were used for fluorescent staining . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) . Boxed areas are enlarged and shown in panels on the right .

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C, D Two types of TNTs were detected in H1299 (C) and A549 (D) cells, Type 1, containing F-actin with little or no detectable microtubules (white box) and Type2, containing both microtubules and F-actin (yellow box). Anti-β-tubulin antibody (green) and phalloidin (red) were used for fluorescent staining. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right.


C , D TNT formation ( marked by black triangles and white arrowheads ) was imaged in control and shM2 H1299 ( C ) and shM2 A549 ( D ) cells by confocal microscopy . Scale bar : 60 μm . Boxed areas are enlarged and shown at the bottom of the corresponding images . E , F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 ( E ) and shM2 A549 ( F ) cells . 13 images per group ( E ) and 10 per group ( F ) were captured for quantification . Three independent experiments were conducted . G Data information : data were analyzed by one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * * * P < 0 . 001 . Three independent experiments were

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C, D TNT formation (marked by black triangles and white arrowheads) was imaged in control and shM2 H1299 (C) and shM2 A549 (D) cells by confocal microscopy. Scale bar: 60 μm. Boxed areas are enlarged and shown at the bottom of the corresponding images. E, F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 (E) and shM2 A549 (F) cells. 13 images per group (E) and 10 per group (F) were captured for quantification. Three independent experiments were conducted. G Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed


H , I H1299 cells stably expressing the Ctr or GFP - M2 were cultured in the presence of 20 μM 2 - DG for 24 h to induce TNT formation . TNTs were imaged by confocal microscopy ( A ) and the percentage of cells forming TNTs was quantified ( B ) . n = 21 , 20 and 19 images were captured from Ctr , GFP - M2 # 1 and GFP - M2 # 2 , respectively . Three independent experiments were performed . Scale bar : 60 μm . Data information : data were analyzed by one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * * * P < 0 . 001 . Three independent experiments were

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H,I H1299 cells stably expressing the Ctr or GFP-M2 were cultured in the presence of 20 μM 2-DG for 24 h to induce TNT formation. TNTs were imaged by confocal microscopy (A) and the percentage of cells forming TNTs was quantified (B). n=21, 20 and 19 images were captured from Ctr, GFP-M2#1 and GFP-M2#2, respectively. Three independent experiments were performed. Scale bar: 60 μm. Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed


A Time - lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito - GFP were connected by TNTs containing mitochondria . Cells were seeded in collagen - coated dishes and cultured for 24 h before time - lapse life cell images were acquired by confocal microscopy ( also see Movies EV1 and EV2 ) . Fluorescent images ( with bright field ) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells . Scale bar : 20 μm . The moving mitochondria are marked by the white arrows . Boxed areas ( shCtr in yellow dotted boxes ; shM2 in red dotted boxes ) are enlarged and shown in the middle panel . B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti - Tom20 antibody ( green ) and staining using phalloidin ( red ) . The white arrows marked TNTs , whereas the white triangles marked mitochondria inside TNTs . Scale bar : 25 μm . C Data information : nuclei were stained with Hoechst 33342 ( blue ) .

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A Time-lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito-GFP were connected by TNTs containing mitochondria. Cells were seeded in collagen-coated dishes and cultured for 24 h before time-lapse life cell images were acquired by confocal microscopy (also see Movies EV1 and EV2). Fluorescent images (with bright field) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells. Scale bar: 20 μm. The moving mitochondria are marked by the white arrows. Boxed areas (shCtr in yellow dotted boxes; shM2 in red dotted boxes) are enlarged and shown in the middle panel. B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). The white arrows marked TNTs, whereas the white triangles marked mitochondria inside TNTs. Scale bar: 25 μm. C Data information: nuclei were stained with Hoechst 33342 (blue).


C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells . The percentage of TNTs containing mitochondria was determined . n = 168 , 187 and 192 for shCtr , shM2 # 1 and shM2 # 2 groups , respectively ; three independent experiments . Data information : data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or

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C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells. The percentage of TNTs containing mitochondria was determined. n=168, 187 and 192 for shCtr, shM2#1 and shM2#2 groups, respectively; three independent experiments. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f


E Representative flow cytometry plot of shCtr and shM2 cells following co - culture for 2 days . Experiments were conducted under three conditions : co - cultured in direct contact in normal glucose medium or in galactose medium or co - cultured through a filter .

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E Representative flow cytometry plot of shCtr and shM2 cells following co-culture for 2 days. Experiments were conducted under three conditions: co-cultured in direct contact in normal glucose medium or in galactose medium or co-cultured through a filter.


F The percentage of mCherry - expressing recipient cells containing Mito - GFP was quantified . Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or fewer recipient cells contained Mito - GFP , and this group is not included in the graph to avoid an overcrowded figure . Three independent experiments were conducted . Data information : data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or

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F The percentage of mCherry-expressing recipient cells containing Mito-GFP was quantified. Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fewer recipient cells contained Mito-GFP, and this group is not included in the graph to avoid an overcrowded figure. Three independent experiments were conducted. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f


A Down - regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton . Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti - Tom20 antibody ( green ) and staining using phalloidin ( red ) . Scale bar : 25 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

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A Down-regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton. Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). Scale bar: 25 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


C Down - regulating MICAL2PV enhances the mitochondrial localization of Miro1 , Miro2 and KIF5B . Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting . The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β - tubulin protein .

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C Down-regulating MICAL2PV enhances the mitochondrial localization of Miro1, Miro2 and KIF5B. Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting. The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β-tubulin protein.


E MICAL2PV interacts with the C - terminal domain of Miro2 containing the transmembrane region . H1299 cells were co - transfected with plasmids encoding Flag - MICAL2PV and Myc - tagged Miro2 mutants as indicated . Following transfection , cells were lysed for immunoprecipitation using the anti - Flag antibody and Western blotting . Please note the Myc - Miro2 ( 410 - 618 ) protein band in lane 4 migrated as a faint band , slightly faster than the IgG light chain .

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E MICAL2PV interacts with the C-terminal domain of Miro2 containing the transmembrane region. H1299 cells were co-transfected with plasmids encoding Flag-MICAL2PV and Myc-tagged Miro2 mutants as indicated. Following transfection, cells were lysed for immunoprecipitation using the anti-Flag antibody and Western blotting. Please note the Myc-Miro2 (410-618) protein band in lane 4 migrated as a faint band, slightly faster than the IgG light chain.


F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration . H1299 cells were co - transfected with Myc - tagged Miro2 and GFP - tagged MCAL2PV ( GFP - M2 ) or control vector . Following transfection , cells were fixed and stained with anti - Tom20 ( red ) and anti - Myc antibodies ( magenta ) . Confocal microscopic images were taken under the same objective and the same microscope settings . In cells co - expressing the GFP vector and Myc - Miro2 ( upper panels ) , most of Miro2 signals ( magenta ) are co - colocalized with mitochondria ( red Tom20 signals ) . However , a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co - expressing GFP - M2 and Myc - Miro2 ( marked by the yellow arrowheads ) . Mitochondria in cells expressing GFP - M2 show the perinuclear distribution pattern , whereas mitochondria in cells transfected by Myc - Miro2 without GFP - M2 ( marked by the yellow stars ) exhibit an infiltration pattern into the cortical cytoplasm . Scale bar : 20 μm .

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F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration. H1299 cells were co-transfected with Myc-tagged Miro2 and GFP-tagged MCAL2PV (GFP-M2) or control vector. Following transfection, cells were fixed and stained with anti-Tom20 (red) and anti-Myc antibodies (magenta). Confocal microscopic images were taken under the same objective and the same microscope settings. In cells co-expressing the GFP vector and Myc-Miro2 (upper panels), most of Miro2 signals (magenta) are co-colocalized with mitochondria (red Tom20 signals). However, a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co-expressing GFP-M2 and Myc-Miro2 (marked by the yellow arrowheads). Mitochondria in cells expressing GFP-M2 show the perinuclear distribution pattern, whereas mitochondria in cells transfected by Myc-Miro2 without GFP-M2 (marked by the yellow stars) exhibit an infiltration pattern into the cortical cytoplasm. Scale bar: 20 μm.


A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2 . Following transfection , cells were stained with anti - Tom20 antibody ( green ) and phalloidin ( red ) . Confocal microscopic images are shown . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

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A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. Following transfection, cells were stained with anti-Tom20 antibody (green) and phalloidin (red). Confocal microscopic images are shown. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


F Down - regulating Miro2 inhibits mitochondrial transport in shM2 cells . Mito - GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2 . These cells were then co - cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito - GFP was quantified . Twenty thousand events were analyzed for each condition Data information : Data were analyzed using two - way ANOVA with Bonferroni post - test and presented as mean ± SEM ; * P < 0 . 05 , * * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant . Three independent experiments were conducted .

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F Down-regulating Miro2 inhibits mitochondrial transport in shM2 cells. Mito-GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. These cells were then co-cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito-GFP was quantified. Twenty thousand events were analyzed for each condition Data information: Data were analyzed using two-way ANOVA with Bonferroni post-test and presented as mean ± SEM; * P < 0.05, *** P < 0.01, *** P < 0.001, ns, not significant. Three independent experiments were conducted.


A Stable H1299 cells ( shCtr or shM2 ) were treated by 5 - FU ( 50 μM , 24 h ) and then cultured alone or co - cultured with non - drug treated vehicle - treated cells stably expressing mito - mCherry in the absence or presence of Cyto B ( 500 nM , 24 h ) . The percentage of dead cells in each group was quantified . Three independent experiments were conducted . Data information : data were analyzed using two - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant .

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A Stable H1299 cells (shCtr or shM2) were treated by 5-FU (50 μM, 24 h) and then cultured alone or co-cultured with non-drug treated vehicle-treated cells stably expressing mito-mCherry in the absence or presence of Cyto B (500 nM, 24 h). The percentage of dead cells in each group was quantified. Three independent experiments were conducted. Data information: data were analyzed using two-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.


D Confocal microscopic images of TNTs in each group , white arrowheads and yellow triangle in enlarged views ( zoom - in panels , zoom ) marked the TNTs formed between non - drug treated ( mCherry positive ) and drug - treated cells . Scale bar : 25 μm .

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D Confocal microscopic images of TNTs in each group, white arrowheads and yellow triangle in enlarged views (zoom-in panels, zoom) marked the TNTs formed between non-drug treated (mCherry positive) and drug-treated cells. Scale bar: 25 μm.


D MICAL2PV mutants containing the MO domain are active in depolymerizing F - actin . Following transfection of plasmids expressing different HA - MICAL2PV mutants ( # 1 , # 2 , or # 4 ) into shM2 cells , confocal microscopy was performed following staining with phalloidin ( red ) and anti - HA antibody ( green ) . F - actin signals were reduced in cells expressing different mutants ( HA - positive green cells marked by dashed lines ) . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

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D MICAL2PV mutants containing the MO domain are active in depolymerizing F-actin. Following transfection of plasmids expressing different HA-MICAL2PV mutants (#1, #2, or #4) into shM2 cells, confocal microscopy was performed following staining with phalloidin (red) and anti-HA antibody (green). F-actin signals were reduced in cells expressing different mutants (HA-positive green cells marked by dashed lines). Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


G , H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation . Stable shM2 H1299 ( G ) or A549 ( H ) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site ( MOmut : amino acid residues 93 - 98 GxGxxG mutated to WxWxxW ) or the control vector ( Ctr ) . Following transfection , Western blotting and quantification of TNT formation were carried out as in panel E . 10 images were captured for quantification in each group . Three independent experiments were performed . Data information : Data were analyzed using a one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant .

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G, H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation. Stable shM2 H1299 (G) or A549 (H) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site (MOmut: amino acid residues 93-98 GxGxxG mutated to WxWxxW) or the control vector (Ctr). Following transfection, Western blotting and quantification of TNT formation were carried out as in panel E. 10 images were captured for quantification in each group. Three independent experiments were performed. Data information: Data were analyzed using a one-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.


( A ) Quantification of the amount of LC3 ‐ II protein in mouse Rab ‐ knockdown cells . Representative blots are shown in supplementary Fig S1C online . Error bars represent the means and s . e . m . of three independent experiments .

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(A) Quantification of the amount of LC3‐II protein in mouse Rab‐knockdown cells. Representative blots are shown in supplementary Fig S1C online. Error bars represent the means and s.e.m. of three independent experiments.


( B ) Quantification of the amount of p62 protein in candidates Rab ‐ knockdown cells . Representative blots are shown in supplementary Fig S1D online . Error bars represent the means and s . e . m . of three independent experiments .

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(B) Quantification of the amount of p62 protein in candidates Rab‐knockdown cells. Representative blots are shown in supplementary Fig S1D online. Error bars represent the means and s.e.m. of three independent experiments.


( C , D ) Control and Rab12 ‐ knockdown MEFs were cultured under N or S conditions , fixed and then immunostained with the antibodies indicated . Representative images are shown in supplementary Fig S2 online . The mean numbers of LC3 ‐ positive ( C ) or Atg16L1 ‐ positive ( D ) dots per cell are shown . Error bars represent the means and s . e . m . of representative data ( n ≥ 80 ) from three independent experiments .

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(C,D) Control and Rab12‐knockdown MEFs were cultured under N or S conditions, fixed and then immunostained with the antibodies indicated. Representative images are shown in supplementary Fig S2 online. The mean numbers of LC3‐positive (C) or Atg16L1‐positive (D) dots per cell are shown. Error bars represent the means and s.e.m. of representative data (n≥80) from three independent experiments.