MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intravenously infected with VSV - GFP ( 2 × 108 PFU / mouse ) , and serum samples were collected at 12 hpi ( hours post - infection ) . ( A ) Virus titer was analyzed in a plaque assay .

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

MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (A) Virus titer was analyzed in a plaque assay.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intravenously infected with VSV - GFP ( 2 × 108 PFU / mouse ) , and serum samples were collected at 12 hpi ( hours post - infection ) . ( B ) Secretion of IFN - β , IL - 6 , IL - 12 , and TNFα was measured in specific ELISAs .

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

MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (B) Secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.


C MARCH2 + / + ( n = 6 ) or MARCH2 - / - ( n = 6 ) mice were intravenously injected with poly ( I : C ) ( 200 μg / mouse ) . Serum samples were collected at 2 hpi and secretion of IFN - β , IL - 6 , IL - 12 , and TNFα was measured in specific ELISAs .

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

C MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were intravenously injected with poly(I:C) (200 μg/mouse). Serum samples were collected at 2 hpi and secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( D ) The percentage of surviving mice ( D , log - rank test , * * P < 0 . 01 )

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

MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (D) The percentage of surviving mice (D, log-rank test, **P < 0.01)


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . body weight changes for each group ( E ) are shown .

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

MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. body weight changes for each group (E) are shown.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( F , G ) At 3 dpc , the bacterial load in the spleen ( F ) and liver ( G ) was examined .

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

MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (F, G) At 3 dpc, the bacterial load in the spleen (F) and liver (G) was examined.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( H - J ) Amount of IL - 6 , IL - 12 , CCL5 , and CXCL10 in serum ( H ) , spleen ( I ) , and liver ( J ) of mice in each group at 24 hpc .

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

MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (H-J) Amount of IL-6, IL-12, CCL5, and CXCL10 in serum (H), spleen (I), and liver (J) of mice in each group at 24 hpc.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . Percentage of surviving mice ( A , log - rank test , * * P < 0 . 01 )

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

MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). Percentage of surviving mice (A, log-rank test, **P < 0.01)


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . body weight changes ( B ) in each groups .

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

MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). body weight changes (B) in each groups.


C MARCH2 + / + ( n = 4 ) or MARCH2 - / - ( n = 4 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . Representing slides of H & E staining of spleen sections from each group . Scale bar , 6mm .

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

C MARCH2+/+ (n = 4) or MARCH2-/- (n = 4) mice were challenged intraperitoneally with LPS (24 mg/kg). Representing slides of H&E staining of spleen sections from each group. Scale bar, 6mm.


D MARCH2 + / + ( n = 6 ) or MARCH2 - / - ( n = 6 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . Levels of IL - 6 , TNF - α , CXCL - 10 , CCL - 5 in serum from mice in each groups were measured at12 hpc by ELISA .

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

D MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were challenged intraperitoneally with LPS (24 mg/kg). Levels of IL-6, TNF-α, CXCL-10, CCL-5 in serum from mice in each groups were measured at12 hpc by ELISA.


E , F MARCH2 + / + ( n = 5 ) or MARCH2 - / - ( n = 5 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . cDNA was prepared from total RNA extracted from spleen and liver of mice . Expression of mRNA encoding IL - 6 , TNF - α , CXCL10 , IL - 1β in spleen ( E ) and liver ( F ) from mice in each group was examined at 6 hpc by qPCR .

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

E, F MARCH2+/+ (n = 5) or MARCH2-/- (n = 5) mice were challenged intraperitoneally with LPS (24 mg/kg). cDNA was prepared from total RNA extracted from spleen and liver of mice. Expression of mRNA encoding IL-6, TNF-α, CXCL10, IL-1β in spleen (E) and liver (F) from mice in each group was examined at 6 hpc by qPCR.


A - C BMDMs isolated from MARCH2 + / + or MARCH2 - / - mice were infected with PR8 - GFP ( A , MOI = 3 ) , VSV - GFP ( B , MOI = 5 ) , or HSV - GFP ( C , MOI = 3 ) , and virus titer was measured in a plaque assay .

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

A-C BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with PR8-GFP (A, MOI=3), VSV-GFP (B, MOI = 5), or HSV-GFP (C, MOI = 3), and virus titer was measured in a plaque assay.


D , E BMDMs isolated from MARCH2 + / + or MARCH2 - / - mice were infected with viruses or treated with poly ( I : C ) ( 80 µg / ml ) or poly ( dA : dT ) ( 1 µg / ml ) . The concentration of secreted IFN - β ( D ) and IL - 6 ( E ) in the supernatants was measured in an ELISA .

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

D, E BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with viruses or treated with poly(I:C) (80 µg/ml) or poly (dA:dT) (1 µg/ml). The concentration of secreted IFN-β (D) and IL-6 (E) in the supernatants was measured in an ELISA.


PBMCs isolated from whole peripheral blood from MARCH2 + / + or MARCH2 - / - mice were infected with VSV - GFP ( MOI = 1 ) . ( F ) The virus titer was measured in a plaque assay .

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

PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (F) The virus titer was measured in a plaque assay.


PBMCs isolated from whole peripheral blood from MARCH2 + / + or MARCH2 - / - mice were infected with VSV - GFP ( MOI = 1 ) . ( G ) Concentration of IFN - β and IL - 6 in the supernatant was measured in an ELISA .

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

PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (G) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.


PMs isolated from MARCH2 + / + or MARCH2 - / - mice infected with CVB - GFP ( MOI = 3 ) . ( H ) Virus titer was measured in a plaque assay .

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

PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (H) Virus titer was measured in a plaque assay.


PMs isolated from MARCH2 + / + or MARCH2 - / - mice infected with CVB - GFP ( MOI = 3 ) . ( I ) Concentration of IFN - β and IL - 6 in the supernatant was measured in an ELISA .

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

PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (I) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.


J , K BMDMs ( J ) or PMs ( K ) isolated from MARCH2 + / + or MARCH2 - / - mice were infected with Salmonella typhimurium or L . monocytogenes , or treated with LPS or zymosan . The concentration of IL - 6 and IL - 12 in supernatant was analyzed in an ELISA .

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

J, K BMDMs (J) or PMs (K) isolated from MARCH2+/+ or MARCH2-/- mice were infected with Salmonella typhimurium or L. monocytogenes, or treated with LPS or zymosan. The concentration of IL-6 and IL-12 in supernatant was analyzed in an ELISA.


L , M RAW 264 . 7 cells transfected with control siRNA ( si - control ) or MARCH2 - specific siRNA ( si - MARCH2 ) were infected with PR8 - GFP ( MOI = 1 ) , VSV - GFP ( MOI = 0 . 5 ) , poly ( I : C ) ( 20 µg / ml ) or HSV - GFP ( MOI = 1 ) poly ( dA : dT ) ( 1 µg / ml ) and IFN - β ( L ) , and IL - 6 ( M ) levels in the supernatant were measured in an ELISA .

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

L, M RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with PR8-GFP (MOI = 1), VSV-GFP (MOI = 0.5), poly (I:C) (20 µg/ml) or HSV-GFP (MOI = 1) poly (dA:dT) (1 µg/ml) and IFN-β (L), and IL-6 (M) levels in the supernatant were measured in an ELISA.


N RAW 264 . 7 cells transfected with control siRNA ( si - control ) or MARCH2 - specific siRNA ( si - MARCH2 ) were infected with S . typhimurium , or L . monocytogenes , or treated with LPS ( 100 ng / ml ) or zymosan ( 100 µg / ml ) and IL - 6 secretion into the cell supernatant was measured in an

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

N RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with S. typhimurium, or L. monocytogenes, or treated with LPS (100 ng/ml) or zymosan (100 µg/ml) and IL-6 secretion into the cell supernatant was measured in an ELISA


HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN - β promoter , plus a TK renilla plasmid and an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for RIG - I 2CARD , RIG - I , MDA - 5 , MAVS or stimulated with poly ( I : C ) , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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

HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter , plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for RIG-I 2CARD, RIG-I, MDA-5, MAVS or stimulated with poly (I:C), for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN - β promoter plus a TK renilla plasmid and an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TBK1 TRIF , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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

HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TBK1 TRIF , for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the the NF - κB promoter plus a TK renilla plasmid and an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TRAF 6 , TRAF 2 , NEMO for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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

HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the the NF-κB promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TRAF 6, TRAF 2, NEMO for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the NF - κB promoter plus a TK renilla plasmid and an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TBK1 TRIF , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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

HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the NF-κB promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TBK1 TRIF , for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


Interaction between MARCH2 and NEMO in response to viral infection . HEK293T cells ( E ) were infected with NDV - GFP in a time - dependent manner in the presence of MG132 ( proteasome inhibitor , 10 μM ) . Cell lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

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

Interaction between MARCH2 and NEMO in response to viral infection. HEK293T cells (E) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


Interaction between MARCH2 and NEMO in response to viral infection . Raw264 . 7 cells ( F ) were infected with NDV - GFP in a time - dependent manner in the presence of MG132 ( proteasome inhibitor , 10 μM ) . Cell lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

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

Interaction between MARCH2 and NEMO in response to viral infection. Raw264.7 cells (F) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


G Confocal microscopy was conducted to examine time - dependent colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection ( MOI = 1 ) in the presence of MG132 ( 10 μM ) . Arrow indicates the co - localized NEMO and MARCH2 protein .

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

G Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.


Interaction between MARCH2 and NEMO in response to bacterial infection . Raw264 . 7 cells were infected with L . monocytogenes ( H ) or S . typhimurium ( I ) in a time - dependent manner in the presence of MG132 . Cell lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

Interaction between MARCH2 and NEMO in response to bacterial infection. Raw264.7 cells were infected with L. monocytogenes (H) or S. typhimurium (I) in a time-dependent manner in the presence of MG132. Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


J Confocal microscopy was conducted to examine time - dependent colocalization of MARCH2 and NEMO in HeLa cells expressing TLR2 upon zymosan treatment ( 100 μg / ml ) in the presence of MG132 ( 10 μM ) . Arrow indicates the co - localized NEMO and MARCH2 protein .

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

J Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells expressing TLR2 upon zymosan treatment (100 μg/ml) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.


NEMO degradation assay . ( A ) HEK293T cells were transfected with different doses of Strep - tagged MARCH2 in the presence HA - tagged NEMO plasmid .

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

NEMO degradation assay. (A) HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence HA-tagged NEMO plasmid.


NEMO degradation assay . ( B ) MARCH2 + / + and MARCH2 - / - HEK293T cells were infected with PR8 - GFP ( MOI = 1 ) in time - dependent manner . Cells were harvested following infection , NEMO expression level , total and phosphorylated IRF - 3 , and TBK - 1 were measured by immunoblotting . β - actin was used to confirm equal loading of protein .

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

NEMO degradation assay. (B) MARCH2+/+ and MARCH2-/- HEK293T cells were infected with PR8-GFP (MOI=1) in time-dependent manner. Cells were harvested following infection, NEMO expression level, total and phosphorylated IRF-3, and TBK-1 were measured by immunoblotting. β-actin was used to confirm equal loading of protein.


C HEK293T cells transfected with a Strep - tagged empty vector or MARCH2 together with GST - tagged NEMO were treated with MG132 ( 10 μM ) , chloroquine , NH4Cl , or Z - VAD for 6 h before harvest . Whole cell lysates were subjected to immunoblotting with indicated antibodies .

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

C HEK293T cells transfected with a Strep-tagged empty vector or MARCH2 together with GST-tagged NEMO were treated with MG132 (10 μM), chloroquine, NH4Cl, or Z-VAD for 6 h before harvest. Whole cell lysates were subjected to immunoblotting with indicated antibodies.


D HEK293T cells were transfected with different doses of Strep - tagged MARCH2 together with and GST - tagged NEMO plasmid . MG132 ( 10 μM ) was added to cells for 6 h prior to harvest . Whole cell lysates were subjected to immunoblotting with the indicated antibodies .

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

D HEK293T cells were transfected with different doses of Strep-tagged MARCH2 together with and GST-tagged NEMO plasmid. MG132 (10 μM) was added to cells for 6 h prior to harvest. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.


E Confocal microscopy was used to examine colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection ( MOI = 1 ) in the presence or absence of MG132 ( 10 μM ) . Scale bar , 10μm . Arrow indicates the co - localized NEMO and MARCH2 protein .

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

E Confocal microscopy was used to examine colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence or absence of MG132 (10 μM). Scale bar, 10μm. Arrow indicates the co-localized NEMO and MARCH2 protein.


F HEK293T cells transfected with Strep - tagged empty vector or MARCH2 with GST - tagged NEMO and each different HA - tagged ubiquitin mutants ( indicated lysine ( K ) only , other lysines ( K ) mutated to arginines ( R ) were treated with MG132 ( 10 μM ) for 6 h before harvest . Lysates were subjected to pull down with GST beads , followed by immunoblotting with an anti - HA antibody . Whole cell lysates were determined by immunoblotting with the indicated antibodies .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293T cells transfected with Strep-tagged empty vector or MARCH2 with GST-tagged NEMO and each different HA-tagged ubiquitin mutants (indicated lysine (K) only, other lysines (K) mutated to arginines (R) were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to pull down with GST beads, followed by immunoblotting with an anti-HA antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.


G NEMO ubiquitination assay . HEK293T cells were transfected with different doses of Strep - tagged MARCH2 in the presence of a HA - tagged NEMO plasmid . Lysates were immunoprecipitated with anti - HA antibody followed by immunoblotting with anti - K48 antibody .

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

G NEMO ubiquitination assay. HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence of a HA-tagged NEMO plasmid. Lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-K48 antibody.


MARCH2 + / + HEK293T cells harboring empty vector or MARCH2 - / - HEK293T cells harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within the catalytic domain : C64S , C67S , and H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . Viral replication was assessed under a fluorescence microscope ( H )

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

MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). Viral replication was assessed under a fluorescence microscope (H)


MARCH2 + / + HEK293T cells harboring empty vector or MARCH2 - / - HEK293T cells harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within the catalytic domain : C64S , C67S , and H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . by measuring GFP expression using a fluorescence

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

MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). by measuring GFP expression using a fluorescence modulator


MARCH2 + / + HEK293T cells harboring empty vector or MARCH2 - / - HEK293T cells harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within the catalytic domain : C64S , C67S , and H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . ( J ) Levels of IFN - β ( top ) and IL - 6 ( bottom ) in cell supernatants were measured in an ELISA .

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

MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). (J) Levels of IFN-β (top) and IL-6 (bottom) in cell supernatants were measured in an ELISA.


K In vitro ubiquitination assay for NEMO . HEK293T cells were transfected with GST - tagged NEMO , Strep - tagged MARCH2 or MARCH2CCH individually . Immunoprecipitates were incubated with a reaction mixture containing ubiquitin , E1 , and UbcH6 ( E2 ) , followed by immunoblotting with an anti - K48 - linkage specific polyubiquitin antibody .

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

K In vitro ubiquitination assay for NEMO. HEK293T cells were transfected with GST-tagged NEMO, Strep-tagged MARCH2 or MARCH2CCH individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.


( B ) HEK293T cells were transfected with Strep - tagged MARCH2 domain constructs together with GST - tagged NEMO . Whole cell lysates were subjected to a Strep - PD assay , followed by immunoblotting with an anti - GST antibody .

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

(B) HEK293T cells were transfected with Strep-tagged MARCH2 domain constructs together with GST-tagged NEMO. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.


( D ) HEK293T cells were transfected with GST - tagged NEMO domain constructs together with Strep - tagged MARCH2 - TM . Whole cell lysates were subjected to a Strep - PD assay , followed by immunoblotting with an anti - GST antibody .

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

(D) HEK293T cells were transfected with GST-tagged NEMO domain constructs together with Strep-tagged MARCH2-TM. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.


( F ) HEK293T cells were transfected with HA - tagged NEMO domain constructs together with GST - tagged MARCH2 . Whole cell lysates were subjected to a GST - PD assay , followed by immunoblotting with an anti - HA antibody .

(F) HEK293T cells were transfected with HA-tagged NEMO domain constructs together with GST-tagged MARCH2. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-HA antibody.


G HEK293T cells transfected with His - tagged ubiquitin and HA - tagged NEMO WT or NEMO lacking the CC1 domain together with Strep - tagged empty vector or MARCH2 were treated with MG132 ( 10 μM ) for 6 h before harvest . Lysates were subjected to immunoprecipitation with an anti - HA antibody , followed by immunoblotting with an anti - His antibody . WCL were subjected to immunoblotting with anti - HA , anti - Strep , or anti - β - actin antibody .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293T cells transfected with His-tagged ubiquitin and HA-tagged NEMO WT or NEMO lacking the CC1 domain together with Strep-tagged empty vector or MARCH2 were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-His antibody. WCL were subjected to immunoblotting with anti-HA, anti-Strep, or anti-β-actin antibody.


( I ) HEK293T cells transfected with GST - tagged NEMO mutants together with Strep - tagged empty vector or MARCH2 were subjected to immunoblotting with anti - GST , anti - Strep , or anti - β - actin antibody .

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

(I) HEK293T cells transfected with GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to immunoblotting with anti-GST, anti-Strep, or anti-β-actin antibody.


J HEK293T cells transfected with HA - tagged ubiquitin and GST - tagged NEMO mutants together with Strep - tagged empty vector or MARCH2 were subjected to pulldown with GST beads , followed by immunoblotting with anti - Lys48 ( K48 ) - linkage specific polyubiquitin ( K48 - ub ) , anti - Lys63 ( K63 ) - linkage specific polyubiquitin ( K63 - ub ) , or anti - Strep antibody . Whole cell lysates were determined by immunoblotting with the indicated antibodies .

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

J HEK293T cells transfected with HA-tagged ubiquitin and GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to pulldown with GST beads, followed by immunoblotting with anti-Lys48 (K48)-linkage specific polyubiquitin (K48-ub), anti-Lys63 (K63)-linkage specific polyubiquitin (K63-ub), or anti-Strep antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.


A HEK293T cells were transfected with GST - tagged NEMO - WT and its point mutants with or without Strep - tagged MARCH2 . Whole cell lysates were subjected to immunoblotting with the indicated antibodies .

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

A HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants with or without Strep-tagged MARCH2. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.


B HEK293T cells were transfected with GST - tagged NEMO - WT and its point mutants together with Strep - tagged MARCH2 in the presence of MG132 . Whole cell lysates were subjected to a GST - PD assay , followed by immunoblotting with an anti - K48 linkage specific polyubiquitin antibody .

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

B HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants together with Strep-tagged MARCH2 in the presence of MG132. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-K48 linkage specific polyubiquitin antibody.


C In vitro ubiquitination assay for NEMO - K326R . HEK293T cells were transfected with GST - tagged NEMO , GST - tagged NEMO - K326R or Strep - tagged MARCH2 individually . Immunoprecipitates were incubated with a reaction mixture containing ubiquitin , E1 , and UbcH6 ( E2 ) , followed by immunoblotting with an anti - K48 - linkage specific polyubiquitin antibody .

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

C In vitro ubiquitination assay for NEMO-K326R. HEK293T cells were transfected with GST-tagged NEMO, GST-tagged NEMO-K326R or Strep-tagged MARCH2 individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.


HEK293T cells transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . ( D - F ) Viral replication was determined by fluorescence microscopy ( D )

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

HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (D-F) Viral replication was determined by fluorescence microscopy (D)


HEK293T cells transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . , fluorescence absorbance ( E )

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

HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. , fluorescence absorbance (E)


HEK293T cells transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . plaque assay ( F ) .

HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. plaque assay (F).


HEK293T cells transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . ( G ) IFN - β and IL - 6 in the cell supernatants were measured in an ELISA .

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

HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (G) IFN-β and IL-6 in the cell supernatants were measured in an ELISA.


H , I IFN - β ( H ) and NF - κB ( I ) luciferase reporter assay .

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

H, I IFN-β (H) and NF-κB (I) luciferase reporter assay.


HUVECs transfected with control or CEP41 siRNAs were scratched ( 0 h ) to induce wounding and then incubated for 12 h to allow wound closure . The wound margins were observed every 4 h in the CEP41 # 1 and # 2 siRNA - transfected cells and compared to those of control cells . Representative images of cells subjected to the wound closure assay in ( A ) . Scale bars , 600 μm . Quantification of the extent of wound closure in ( B ) presented graphically by measuring the distance between the dotted lines at each time point .

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

HUVECs transfected with control or CEP41 siRNAs were scratched (0 h) to induce wounding and then incubated for 12 h to allow wound closure. The wound margins were observed every 4 h in the CEP41#1 and #2 siRNA-transfected cells and compared to those of control cells. Representative images of cells subjected to the wound closure assay in (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (B) presented graphically by measuring the distance between the dotted lines at each time point.


The siRNA - transfected HUVECs were plated inside a transwell chamber and incubated with serum for 18 h . The cells that invaded were observed after staining with crystal violet ( CV ) solution . Scale bars , 600 μm . The numbers of cells that invaded in each field of view was counted with the ImageJ software in ( D ) .

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

The siRNA-transfected HUVECs were plated inside a transwell chamber and incubated with serum for 18 h. The cells that invaded were observed after staining with crystal violet (CV) solution. Scale bars, 600 μm. The numbers of cells that invaded in each field of view was counted with the ImageJ software in (D).


Tubulogenesis of control and CEP41 - knockdown cells for 18 h was compared via an in vitro angiogenesis assay . Scale bars , 600 μm . Quantification of tube node numbers in ( F ) and tube length in ( G ) from each field of view using the ImageJ angiogenesis analyzer at the indicated time points . The graph compares the relative length of control cells and CEP41 - knockdown cells . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

Tubulogenesis of control and CEP41-knockdown cells for 18 h was compared via an in vitro angiogenesis assay. Scale bars, 600 μm. Quantification of tube node numbers in (F) and tube length in (G) from each field of view using the ImageJ angiogenesis analyzer at the indicated time points. The graph compares the relative length of control cells and CEP41-knockdown cells. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


Blood vessels were observed in MOs ( control , cep41 AUG ( 2 . 5 ng ) , or cep41 SB ( 2 ng ) ) - injected or cep41 - mutated Tg ( kdrl : eGFP ) zebrafish at 40 hours post - fertilization ( hpf ) by fluorescent microscopy . Asterisks and arrowheads indicate impaired ISVs and DLAVs , respectively . The representative images for analysis of ISV lumen diameter are indicated by dotted rectangles . A , anterior ; P , posterior ; DLAV , dorsal longitudinal anastomotic vessel ; ISV , intersegmental vessel . Scale bars , 100 μm . Quantification of ISV lumen diameter in ( B ) , the numbers of defective ISVs in ( C ) , the numbers of embryos with aberrant DLAVs in ( D ) , and the numbers of ruptured DLAVs in ( E ) from data observed in equivalent fields of view ( within eight somites ) . The severity of blood vessel defects in cep41 - deficient zebrafish : B ( narrowed ISVs ) < C ( shorten , fused , and missing ISVs ) < D and E ( ruptured DLAVs ) . Data are shown as mean ± SD of three independent experiments with ≥ 20 embryos per condition .

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

Blood vessels were observed in MOs (control, cep41 AUG (2.5 ng), or cep41 SB (2 ng))-injected or cep41-mutated Tg(kdrl:eGFP) zebrafish at 40 hours post-fertilization (hpf) by fluorescent microscopy. Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. The representative images for analysis of ISV lumen diameter are indicated by dotted rectangles. A, anterior; P, posterior; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel. Scale bars, 100 μm. Quantification of ISV lumen diameter in (B), the numbers of defective ISVs in (C), the numbers of embryos with aberrant DLAVs in (D), and the numbers of ruptured DLAVs in (E) from data observed in equivalent fields of view (within eight somites). The severity of blood vessel defects in cep41-deficient zebrafish: B (narrowed ISVs) < C (shorten, fused, and missing ISVs) < D and E (ruptured DLAVs). Data are shown as mean ± SD of three independent experiments with ≥ 20 embryos per condition.


CV protrusions ( arrowheads ) and vascular loops ( asterisks ) were observed in zebrafish at 35 hpf . The images within the dotted rectangles are magnified in the right panels . A , anterior ; P , posterior ; CA , caudal artery ; CV , caudal vein . Scale bars , 40 μm . Quantification of the length of the CV protrusions in ( G ) presented graphically by measuring them in equivalent fields of view ( within five somites ) .

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

CV protrusions (arrowheads) and vascular loops (asterisks) were observed in zebrafish at 35 hpf. The images within the dotted rectangles are magnified in the right panels. A, anterior; P, posterior; CA, caudal artery; CV, caudal vein. Scale bars, 40 μm. Quantification of the length of the CV protrusions in (G) presented graphically by measuring them in equivalent fields of view (within five somites).


Control or CEP41 siRNA - transfected HUVECs were immunostained with Ac - Tub - or GT335 - specific antibodies in ( A ) . The rectangles indicate the representative cells from each immunostaining experiment presented as magnified images in the right panels . Scale bars , 20 μm . Quantification of the ciliated cell numbers in the images in ( A ) from the results of three independent experiments with ≥ 200 cells per condition ( mean ± SD ) in ( B ) .

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

Control or CEP41 siRNA-transfected HUVECs were immunostained with Ac-Tub- or GT335-specific antibodies in (A). The rectangles indicate the representative cells from each immunostaining experiment presented as magnified images in the right panels. Scale bars, 20 μm. Quantification of the ciliated cell numbers in the images in (A) from the results of three independent experiments with ≥ 200 cells per condition (mean ± SD) in (B).


Wild - type and cep41 - deficient Tg ( kdrl : eGFP ) zebrafish embryos were fixed for immunostaining with Ac - Tub - or GT335 - specific antibodies at 28 hpf in ( C ) . The rectangles indicate the arterial cilia ( AC ) and venous cilia ( VC ) in zebrafish endothelial cells ( EC ) . Magnified representative images are displayed in the right panels . Scale bars , 40 μm . Quantification of the labeled cilia observed in equivalent fields of view are presented graphically in ( D ) . Data are shown as median of three independent experiments ( n ≥ 20 embryos per condition ) .

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

Wild-type and cep41-deficient Tg(kdrl:eGFP) zebrafish embryos were fixed for immunostaining with Ac-Tub- or GT335-specific antibodies at 28 hpf in (C). The rectangles indicate the arterial cilia (AC) and venous cilia (VC) in zebrafish endothelial cells (EC). Magnified representative images are displayed in the right panels. Scale bars, 40 μm. Quantification of the labeled cilia observed in equivalent fields of view are presented graphically in (D). Data are shown as median of three independent experiments (n ≥ 20 embryos per condition).


HUVECs transfected with CCP5 and / or CEP41 siRNAs were scratched and incubated to evaluate wound closure . The extent of wound closure in co - transfected cells with CCP5 and CEP41 siRNAs was compared with that in control or CEP41 siRNA - transfected cells at the indicated time points ( A ) . Scale bars , 600 μm . Quantification of the extent of wound closure in ( A ) is presented graphically by measuring the distance between the dotted lines at each time point in ( B ) . Data are shown as mean ± SD of three independent experiments ( n ≥ 3 scratches per experimental condition ) .

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

HUVECs transfected with CCP5 and/or CEP41 siRNAs were scratched and incubated to evaluate wound closure. The extent of wound closure in co-transfected cells with CCP5 and CEP41 siRNAs was compared with that in control or CEP41 siRNA-transfected cells at the indicated time points (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) is presented graphically by measuring the distance between the dotted lines at each time point in (B). Data are shown as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition).


The indicated siRNA - transfected HUVECs were subjected to in vitro angiogenesis assays . Tubulogenesis of HUVECs depleted for CEP41 and CCP5 was compared to that of control , CEP41 single - knockdown , or CCP5 single - knockdown cells ( C ) . Scale bars , 600 μm . Quantification of tube node number in ( D ) and tube length in ( E ) from data examined within equivalent fields of view at the indicated time points using the ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

The indicated siRNA-transfected HUVECs were subjected to in vitro angiogenesis assays. Tubulogenesis of HUVECs depleted for CEP41 and CCP5 was compared to that of control, CEP41 single-knockdown, or CCP5 single-knockdown cells (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at the indicated time points using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


The blood vessels in the trunks of cep41 - mutant / morphant zebrafish were compared to those of zebrafish co - injected with ccp5 MOs ( 2 ng ) at 40 hpf in ( F ) . Asterisks and arrowheads indicate impaired ISVs and DLAVs , respectively . A , anterior ; P , posterior . Scale bars , 100 μm . Quantification of the number of defective ISVs and DLAVs from data observed in equivalent fields of view ( within eight somites ) . Data are median of three independent experiments with ≥ 20 embryos per condition .

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

The blood vessels in the trunks of cep41-mutant/morphant zebrafish were compared to those of zebrafish co-injected with ccp5 MOs (2 ng) at 40 hpf in (F). Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. A, anterior; P, posterior. Scale bars, 100 μm. Quantification of the number of defective ISVs and DLAVs from data observed in equivalent fields of view (within eight somites). Data are median of three independent experiments with ≥ 20 embryos per condition.


HUVECs co - transfected with CEP41 and CCP5 siRNAs were immunostained with ARL13b - and GT335 - specific antibodies . The staining results were compared to those of control , single CEP41 , or CCP5 siRNA - transfected cells . Representative images indicated with rectangles appear in magnified images in the lower panels . Scale bars , 20 μm . Quantification of relative intensity of GT335 / ARL13b signals in the cilia ciliary length ( J ) in images ( H ) are the result of three independent experiments with ≥ 200 cells per condition ( median ) . * *

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

HUVECs co-transfected with CEP41 and CCP5 siRNAs were immunostained with ARL13b- and GT335-specific antibodies. The staining results were compared to those of control, single CEP41, or CCP5 siRNA-transfected cells. Representative images indicated with rectangles appear in magnified images in the lower panels. Scale bars, 20 μm. Quantification of relative intensity of GT335/ARL13b signals in the cilia ciliary length (J) in images (H) are the result of three independent experiments with ≥ 200 cells per condition (median). **


Control or CEP41 siRNA - transfected HUVECs cultivated under either static or shear stress states and examined in a wound healing assay in ( A ) . Scale bars , 600 μm . Quantification of the extent of wound closure in ( A ) displayed graphically by measuring the distance between the lines at the indicated time points in ( B ) . Data are presented as mean ± SD of three independent experiments ( n ≥ 3 scratches per experimental condition ) . Statistical significance was determined using the two - way ANOVA followed by Tukey ' s post hoc test ( * * * P < 0 . 01 , ns : non - significant ) .

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

Control or CEP41 siRNA-transfected HUVECs cultivated under either static or shear stress states and examined in a wound healing assay in (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) displayed graphically by measuring the distance between the lines at the indicated time points in (B). Data are presented as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition). Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (***P < 0.01, ns: non-significant).


in vitro angiogenesis assay was performed in static or shear stress - exposed HUVECs transfected with the indicated siRNAs for 18 h in ( C ) .

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

in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C).


in vitro angiogenesis assay was performed in static or shear stress - exposed HUVECs transfected with the indicated siRNAs for 18 h in ( C ) . Scale bars , 600 μm . Quantification of tube node number in ( D ) and tube length in ( E ) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


Control or CEP41 - deficient HUVECs cultivated under laminar shear stress and immunostained with ARL13b - specific antibodies to measure deciliation . Representative images are indicated by rectangles with numbers and shown in magnified images in the right panels . The resulting data were compared to those of cells under static conditions in ( F ) . Scale bars , 20 μm . Quantification of ARL13b - positive ciliary cells ( G ) and of relative intensity of GT335 / ARL13b signals in the cilia ( H ) in images ( F ) is the results of three independent experiments with ≥ 200 cells per condition ( mean ± SD ( G ) , median ( H ) ) . *

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

Control or CEP41-deficient HUVECs cultivated under laminar shear stress and immunostained with ARL13b-specific antibodies to measure deciliation. Representative images are indicated by rectangles with numbers and shown in magnified images in the right panels. The resulting data were compared to those of cells under static conditions in (F). Scale bars, 20 μm. Quantification of ARL13b-positive ciliary cells (G) and of relative intensity of GT335/ARL13b signals in the cilia (H) in images (F) is the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (G), median (H)). *


Control , CEP41 - , or CCP5 - depleted hTERT - RPE1 cells cultured under serum starvation for 48 h and fixed at 18 h after serum retrieval for immunostaining with both ARL13b - and GT335 - specific antibodies in ( I ) . Images of the results 18 h after serum addition . Representative cells are indicated by rectangles . Scale bars , 20 μm . Quantification of ARL13b - positive ciliated cells ( J ) and relative intensity of GT335 / ARL13b signals in the cilia ( K ) under either serum starvation or serum retrieval ( 18 h ) conditions are the results of three independent experiments with ≥ 200 cells per condition ( mean ± SD ( J ) , median ( K ) ) .

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

Control, CEP41-, or CCP5-depleted hTERT-RPE1 cells cultured under serum starvation for 48 h and fixed at 18 h after serum retrieval for immunostaining with both ARL13b- and GT335-specific antibodies in (I). Images of the results 18 h after serum addition. Representative cells are indicated by rectangles. Scale bars, 20 μm. Quantification of ARL13b-positive ciliated cells (J) and relative intensity of GT335/ARL13b signals in the cilia (K) under either serum starvation or serum retrieval (18 h) conditions are the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (J), median (K)).


VEGFA ( A ) and VEGFR2 ( B ) mRNA levels quantified by qRT - PCR in control or CEP41 - knockdown HUVECs under static or shear stress conditions . The expression of GAPDH was quantified for the normalization of the qRT - PCR results . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined with the Brown - Forsythe ANOVA followed by Dunnett ' s T3 post hoc test ( * * P < 0 . 01 , * * * P < 0 . 001 , ns : non - significant ) .

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

VEGFA (A) and VEGFR2 (B) mRNA levels quantified by qRT-PCR in control or CEP41-knockdown HUVECs under static or shear stress conditions. The expression of GAPDH was quantified for the normalization of the qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined with the Brown-Forsythe ANOVA followed by Dunnett's T3 post hoc test (**P < 0.01, ***P < 0.001, ns: non-significant).


Immunoblot assays for phospho - AURKA and AURKA were performed in HUVECs transfected with the indicated siRNAs . Protein levels were normalized against β - ACTIN in the same blots .

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

Immunoblot assays for phospho-AURKA and AURKA were performed in HUVECs transfected with the indicated siRNAs. Protein levels were normalized against β-ACTIN in the same blots.


Immunoblot assays for phospho - AURKA and AURKA were performed in hTERT - RPE1 cells ( D ) transfected with the indicated siRNAs . Protein levels were normalized against β - ACTIN in the same blots .

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

Immunoblot assays for phospho-AURKA and AURKA were performed in hTERT-RPE1 cells (D) transfected with the indicated siRNAs. Protein levels were normalized against β-ACTIN in the same blots.


The mRNA levels of zebrafish vegfa ( E ) and vegfr2 ( F ) were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish at 18 hpf ( low shear stress ) and 26 hpf ( high shear stress ) . The expression of vegfa and vegfr2 was quantified in neuronal cells from control MO - injected zebrafish for comparisons with that of ECs . The expression of zebrafish β - actin was quantified for the normalization of those qRT - PCR results . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined using the one - way ANOVA followed by Tukey ' s post hoc test ( * * * P < 0 . 001 , ns : non - significant ) .

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

The mRNA levels of zebrafish vegfa (E) and vegfr2 (F) were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish at 18 hpf (low shear stress) and 26 hpf (high shear stress). The expression of vegfa and vegfr2 was quantified in neuronal cells from control MO-injected zebrafish for comparisons with that of ECs. The expression of zebrafish β-actin was quantified for the normalization of those qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).


Tg ( kdrl : eGFP ) zebrafish were injected with control - or cep41 - MOs and subjected to immunostaining with phospho - AURKA - specific antibodies ( red ) and DAPI at 26 hpf . The insets indicate the representative area from each immunostaining experiment and the red dots indicate EC aurka activation . Scale bars , 40 μm . Quantification of the phospho - aurka - positive ECs ( H ) in equivalent fields of view for each MO - injected zebrafish in images in ( G ) are the result of three independent experiments with ≥ 10 embryos per condition . The top and bottom whiskers represent the maximum and minimum values , respectively . * * P < 0 . 01 ( Unpaired Student ' s t - test with Welch ' s correction ) .

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

Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 26 hpf. The insets indicate the representative area from each immunostaining experiment and the red dots indicate EC aurka activation. Scale bars, 40 μm. Quantification of the phospho-aurka-positive ECs (H) in equivalent fields of view for each MO-injected zebrafish in images in (G) are the result of three independent experiments with ≥ 10 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. **P < 0.01 (Unpaired Student's t-test with Welch's correction).


CEP41 - depleted hTERT - RPE1 cells were transfected with vectors encoding nothing ( MOCK ) , AURKA - T288D , or AURKA - K162R and then immunostained with ARL13b - specific antibodies after serum starvation or serum retrieval . Images were taken from the results of staining done 18 h after serum retrieval . Rectangles indicate the representative cells that appear in the magnified images in the right panels . Scale bars , 20 μm . A quantification of ARL13b - labeled ciliated cells under the indicated conditions in ( J ) . These are the results of three independent experiments with ≥ 200 cells per condition ( mean ± SD ) . * P < 0 . 05 , * * * P < 0 . 01 , ns : non - significant ( Two - way ANOVA with Tukey ' s post hoc test ) .

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

CEP41-depleted hTERT-RPE1 cells were transfected with vectors encoding nothing (MOCK), AURKA-T288D, or AURKA-K162R and then immunostained with ARL13b-specific antibodies after serum starvation or serum retrieval. Images were taken from the results of staining done 18 h after serum retrieval. Rectangles indicate the representative cells that appear in the magnified images in the right panels. Scale bars, 20 μm. A quantification of ARL13b-labeled ciliated cells under the indicated conditions in (J). These are the results of three independent experiments with ≥ 200 cells per condition (mean ± SD). *P < 0.05, ***P < 0.01, ns: non-significant (Two-way ANOVA with Tukey's post hoc test).


Control and CEP41 - deficient HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , or AURKA - K162R and subjected to an in vitro angiogenesis assay for 18 h under static ( K , L ) or shear stress ( M , N ) states . Quantification of tube node number in ( K , M ) and tube length in ( L , N ) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition . Statistical significance was determined using the two - way ANOVA followed by Tukey ' s post hoc test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : non - significant ) .

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

Control and CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, or AURKA-K162R and subjected to an in vitro angiogenesis assay for 18 h under static (K, L) or shear stress (M, N) states. Quantification of tube node number in (K, M) and tube length in (L, N) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant).


The control or CEP41 siRNA - transfected HUVECs were cultivated under hypoxia ( 1 % O2 ) and their whole cell lysates were used for immunoblot assays of CEP41 , phospho - AURKA , and AURKA . The resulting data were also compared to those of normoxic cells . Protein levels were normalized against β - ACTIN in the same blots .

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

The control or CEP41 siRNA-transfected HUVECs were cultivated under hypoxia (1% O2) and their whole cell lysates were used for immunoblot assays of CEP41, phospho-AURKA, and AURKA. The resulting data were also compared to those of normoxic cells. Protein levels were normalized against β-ACTIN in the same blots.


VEGFA ( B ) and VEGFR2 ( C ) mRNA levels were measured by qRT - PCR using cDNA from normoxic and hypoxic controls or CEP41 - depleted cells . The expression of GAPDH was quantified for the normalization of those qRT - PCR results .

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

VEGFA (B) and VEGFR2 (C) mRNA levels were measured by qRT-PCR using cDNA from normoxic and hypoxic controls or CEP41-depleted cells. The expression of GAPDH was quantified for the normalization of those qRT-PCR results.


Tg ( kdrl : eGFP ) zebrafish were injected with control - or cep41 - MOs and then incubated under hypoxia at 28 hpf ( a stage of high shear stress ) for 2 h . They were subjected to immunostaining with phospho - AURKA - specific antibodies ( red ) and DAPI at 30 hpf . The insets indicate the representative areas from each immunostaining and red dots indicate activated aurka within ECs . Scale bars , 40 μm . Quantification of phospho - aurka - positive ECs ( E ) in equivalent fields of view for each MO - injected zebrafish in ( D ) are the result of three independent experiments with ≥ 20 embryos per condition . The top and bottom whiskers represent the maximum and minimum values , respectively . * P < 0 . 05 ( Unpaired Student ' s t - test ) .

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

Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and then incubated under hypoxia at 28 hpf (a stage of high shear stress) for 2 h. They were subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 30 hpf. The insets indicate the representative areas from each immunostaining and red dots indicate activated aurka within ECs. Scale bars, 40 μm. Quantification of phospho-aurka-positive ECs (E) in equivalent fields of view for each MO-injected zebrafish in (D) are the result of three independent experiments with ≥ 20 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. *P < 0.05 (Unpaired Student's t-test).


Zebrafish vegfa mRNA levels were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish subjected to either normoxia or hypoxia at 30 hpf . The expression of zebrafish β - actin was quantified for the normalization of these qRT - PCR results .

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

Zebrafish vegfa mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results.


vegfr2 ( G ) mRNA levels were quantified by qRT - PCR in eGFP - positive ECs of control - or cep41 - MO - injected Tg ( kdrl : eGFP ) zebrafish subjected to either normoxia or hypoxia at 30 hpf . The expression of zebrafish β - actin was quantified for the normalization of these qRT - PCR results .

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

vegfr2 (G) mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results.


Control and CEP41 - deficient HUVECs , transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , HIF1α , or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h . Scale bars , 400 μm .

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

Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm.


Control and CEP41 - deficient HUVECs , transfected with expression vectors encoding nothing ( MOCK ) , AURKA , AURKA - T288D , HIF1α , or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h . Scale bars , 400 μm . Quantification of tube node number in ( I ) and tube length in ( J ) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm. Quantification of tube node number in (I) and tube length in (J) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


HUVECs transfected with control or CEP41 siRNAs were cultivated under hypoxia and their whole cell lysates were used for immunoblot assays of HIF1α . The HIF1α expression of hypoxic cells was compared to that of normoxic cells and the protein levels were normalized against β - ACTIN in the same blot .

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

HUVECs transfected with control or CEP41 siRNAs were cultivated under hypoxia and their whole cell lysates were used for immunoblot assays of HIF1α. The HIF1α expression of hypoxic cells was compared to that of normoxic cells and the protein levels were normalized against β-ACTIN in the same blot.


Co - immunoprecipitation assays using HIF1α - , and CEP41 - specific antibodies were performed in HEK 293T cells expressing GFP - CEP41 under normoxia or hypoxia . An IP using IgG - specific antibodies was performed as a negative control . NOR , normoxia ; HYP , hypoxia .

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

Co-immunoprecipitation assays using HIF1α-, and CEP41-specific antibodies were performed in HEK 293T cells expressing GFP-CEP41 under normoxia or hypoxia. An IP using IgG-specific antibodies was performed as a negative control. NOR, normoxia; HYP, hypoxia.


The CEP41 - depleted HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , or HIF1α and cultivated under hypoxia . Whole cell lysates were used for immunoblot assays for phospho - AURKA and AURKA , and the resulting data were compared to those of normoxic and hypoxic control cells . Protein levels were normalized against β - ACTIN in the same blots . NOR , normoxia ; HYP , hypoxia .

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

The CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, or HIF1α and cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for phospho-AURKA and AURKA, and the resulting data were compared to those of normoxic and hypoxic control cells. Protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.


The CEP41 - deficient HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , HIF1α , or HIF1α with CEP41 and exposed to hypoxia . VEGFA ( E ) and VEGFR2 ( F ) mRNA levels were analyzed by qRT - PCR using cDNA from transfected cells . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined using the one - way ANOVA followed by Tukey ' s post hoc test ( * * * P < 0 . 001 , ns : non - significant ) . NOR , normoxia ; HYP , hypoxia .

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

The CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, HIF1α, or HIF1α with CEP41 and exposed to hypoxia. VEGFA (E) and VEGFR2 (F) mRNA levels were analyzed by qRT-PCR using cDNA from transfected cells. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant). NOR, normoxia; HYP, hypoxia.


Control or CEP41 - depleted HUVECs were transfected with expression vectors encoding nothing ( MOCK ) or HIF1α and treated with MG132 or not , and then the cells were cultivated under hypoxia . Whole cell lysates were used for immunoblot assays for HIF1α , phospho - AURKA , and AURKA , and the protein levels were normalized against β - ACTIN in the same blots . NOR , normoxia ; HYP , hypoxia .

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

Control or CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK) or HIF1α and treated with MG132 or not, and then the cells were cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for HIF1α, phospho-AURKA, and AURKA, and the protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.


Quantification of tube node number in ( H ) and tube length in ( I ) from in vitro angiogenesis assays with HIF1α # 1 or # 2 siRNAs - transfected HUVECs overexpressing nothing ( MOCK ) or CEP41 under hypoxia . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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

Quantification of tube node number in (H) and tube length in (I) from in vitro angiogenesis assays with HIF1α #1 or #2 siRNAs-transfected HUVECs overexpressing nothing (MOCK) or CEP41 under hypoxia. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


VEGFA ( J ) and VEGFR2 ( K ) mRNA levels were analyzed by qRT - PCR using cDNA from hypoxic HIF1α - depleted HUVECs transfected with control or CEP41 expression vectors . The results were compared to those of control and normoxic HIF1α - depleted cells . Data are shown as mean ± SD of three independent experiments .

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

VEGFA (J) and VEGFR2 (K) mRNA levels were analyzed by qRT-PCR using cDNA from hypoxic HIF1α-depleted HUVECs transfected with control or CEP41 expression vectors. The results were compared to those of control and normoxic HIF1α-depleted cells. Data are shown as mean ± SD of three independent experiments.


( A ) Analysis of the single - cell transcriptomics dataset for the mouse visual cortex ( Tasic et al , 2016 ) revealed that the expression of the metabolism - associated gene Cox6a2 is highly restricted to PV + interneurons . Cox6a2 is expressed in all subtypes of PV + interneurons ( marked in blue color ) but rarely detected in other neuronal subtypes from the mouse visual cortex . Cox6a1 , which encodes the other protein isoform of COX6A , is expressed in all subtypes .

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

(A) Analysis of the single-cell transcriptomics dataset for the mouse visual cortex (Tasic et al, 2016) revealed that the expression of the metabolism-associated gene Cox6a2 is highly restricted to PV+ interneurons. Cox6a2 is expressed in all subtypes of PV+ interneurons (marked in blue color) but rarely detected in other neuronal subtypes from the mouse visual cortex. Cox6a1, which encodes the other protein isoform of COX6A, is expressed in all subtypes.


( B RT - PCR showing expression of Cox6a2 in the brain and heart .

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

(B RT-PCR showing expression of Cox6a2 in the brain and heart.


C ) Western - blot showing expression of Cox6a2 in the brain and heart .

C) Western-blot showing expression of Cox6a2 in the brain and heart.


( D ) Western - blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 ( P0 ) . Then , COX6A2 protein levels gradually increase until P60 .

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 B-MEASURED_VAR O O O O O O O

(D) Western-blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 (P0). Then, COX6A2 protein levels gradually increase until P60.


COX6A2 immunoreactivity is detected in PV + interneurons in different brain areas of adult mice , including the cortex ( general overview in E , and single PV + interneuron magnification in F ) White arrowheads indicate PV / Cox6a2 double positive cells .

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

COX6A2 immunoreactivity is detected in PV+ interneurons in different brain areas of adult mice, including the cortex (general overview in E, and single PV+ interneuron magnification in F) White arrowheads indicate PV/Cox6a2 double positive cells.


COX6A2 immunoreactivity is detected in PV + interneurons in hippocampus ( G ) and striatum ( H ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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

COX6A2 immunoreactivity is detected in PV+ interneurons in hippocampus (G) and striatum (H). White arrowheads indicate PV/Cox6a2 double positive cells.


( I , J ) Quantifications showing almost 100 % of co - localization of COX6A2 + cells and PV + cells in the somatosensory ( SS ) and motor ( MT ) cortices , and the hippocampus ( HI ) of adult mice ( n animals = 4 , 2 months old , n sections = 24 SS , 16 MT , 8 HI ) .

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

(I,J) Quantifications showing almost 100% of co-localization of COX6A2+ cells and PV+ cells in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult mice (n animals=4, 2 months old, n sections=24 SS, 16 MT, 8 HI).


( K ) COX6A2 immunoreactivity in the cortex at P9 ( n animals = 2 , n sections = 12 SS , 8 MT , 4 HI ) . White arrowheads indicate Cox6a2 positive cells .

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 B-MEASURED_VAR O O O

(K) COX6A2 immunoreactivity in the cortex at P9 (n animals=2, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate Cox6a2 positive cells.


( L - N ) As soon as parvalbumin immunoreactivity becomes visible at P13 , almost 100 % of PV + cells are co - labelled with COX6A2 immunoreactivity in the SS and MT cortices , and the HI of P13 mice ( n = 2 ) .

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

(L-N) As soon as parvalbumin immunoreactivity becomes visible at P13, almost 100% of PV+ cells are co-labelled with COX6A2 immunoreactivity in the SS and MT cortices, and the HI of P13 mice (n=2).


( A , B ) COX6A2 immunoreactivity is virtually restricted to PV + interneurons in the somatosensory ( SS ) and motor ( MT ) cortices , and the hippocampus ( HI ) of adult rat ( n animals = 1 , 2 . 5 months old , n sections = 12 SS , 8 MT , 4 HI ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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

(A,B) COX6A2 immunoreactivity is virtually restricted to PV+ interneurons in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult rat (n animals=1, 2.5 months old, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate PV/Cox6a2 double positive cells.


( C , D ) In the SS of adult rhesus monkey , COX6A2 immunoreactivity is present exclusively in PV + cells ( n animals = 1 , 1 - year - old , n sections = 6 ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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

(C,D) In the SS of adult rhesus monkey, COX6A2 immunoreactivity is present exclusively in PV+ cells (n animals=1, 1-year-old, n sections=6). White arrowheads indicate PV/Cox6a2 double positive cells.


( E , F ) In adult humans , all cortical PV + interneurons from Brodmann areas 9 and 10 ( BA9 and BA10 ) are co - labelled with COX6A2 immunoreactivity . However , PV + interneurons only represent 60 % of the cells in which COX6A2 is detected ( n human subjects = 2 , 59 - and 69 - year - old , n sections = 6 BA9 , 6 BA10 ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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

(E,F) In adult humans, all cortical PV+ interneurons from Brodmann areas 9 and 10 (BA9 and BA10) are co-labelled with COX6A2 immunoreactivity. However, PV+ interneurons only represent 60% of the cells in which COX6A2 is detected (n human subjects=2, 59- and 69-year-old, n sections=6 BA9, 6 BA10). White arrowheads indicate PV/Cox6a2 double positive cells.


( A ) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2 - / - mice .

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

(A) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2-/- mice.


( B , C ) Parvalbumin immunoreactivity in PV + interneurons was significantly decreased in the somatosensory cortex of Cox6a2 - / - mice compared to wild - types ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . C : two - tailed unpaired Student ' s t - test .

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

(B,C) Parvalbumin immunoreactivity in PV+ interneurons was significantly decreased in the somatosensory cortex of Cox6a2-/- mice compared to wild-types (n=4 WT, 4 Cox6a2-/-; 2-month-old). C: two-tailed unpaired Student's t-test.


( D ) In Cox6a2 - / - mice , cortical PV + interneurons showed a fewer number of synapses innervating principal neurons ( n = 3 WT , 3 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Student ' s t - test .

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

(D) In Cox6a2-/- mice, cortical PV+ interneurons showed a fewer number of synapses innervating principal neurons (n=3 WT, 3 Cox6a2-/-; 2-month-old). Two-tailed unpaired Student's t-test.


( E ) To analyze changes in oxidative stress in PV + interneurons , the somatosensory and motor cortices of WT and Cox6a2 - / - mice were stained with an antibody against 8 - OHdG , a marker of oxidative DNA damage . ( F ) 8 - OHdG immunoreactivity was significantly higher in PV + null in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Mann - Whitney tests .

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

(E) To analyze changes in oxidative stress in PV+ interneurons, the somatosensory and motor cortices of WT and Cox6a2-/- mice were stained with an antibody against 8-OHdG, a marker of oxidative DNA damage. (F) 8-OHdG immunoreactivity was significantly higher in PV+ null in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( G ) The density of PNNs around PV + interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs . ( H ) WFA immunoreactivity around PV + cells was lower in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Mann - Whitney tests .

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

(G) The density of PNNs around PV+ interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs. (H) WFA immunoreactivity around PV+ cells was lower in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( A ) PV + interneurons were sorted from cortices of PV - EGFP WT and Cox6a2 - / - mice at P15 and processed for bulk RNA sequencing ( n = 4 WT , 6 Cox6a2 - / - ; P15 ) . Heatmap shows expression for top down - ( red ) and upregulated ( blue ) genes in Cox6a2 - / - PV + interneurons relative to their WT counterparts .

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

(A) PV+ interneurons were sorted from cortices of PV-EGFP WT and Cox6a2-/- mice at P15 and processed for bulk RNA sequencing (n= 4 WT, 6 Cox6a2-/-; P15). Heatmap shows expression for top down- (red) and upregulated (blue) genes in Cox6a2-/- PV+ interneurons relative to their WT counterparts.


( B ) Cox6a2 expression was effectively abolished in Cox6a2 - / - PV + interneurons . In addition , Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes ( n = 4 WT , 6 Cox6a2 - / - ) . Two - tailed unpaired Mann - Whitney tests .

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

(B) Cox6a2 expression was effectively abolished in Cox6a2-/- PV+ interneurons. In addition, Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes (n= 4 WT, 6 Cox6a2-/-). Two-tailed unpaired Mann-Whitney tests.


( C ) Changes in expression of oxidative stress - related genes ( green ) , genes coding for AMPA receptors ( orange ) , sodium channels ( red ) , potassium channels ( magenta ) , and internal calcium signaling proteins ( blue ) . Color scale bars show normalized read counts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Changes in expression of oxidative stress-related genes (green), genes coding for AMPA receptors (orange), sodium channels (red), potassium channels (magenta), and internal calcium signaling proteins (blue). Color scale bars show normalized read counts.


( D ) Representative images of PV + interneurons from PV - CRE and PV - CRE ; Cox6a2fl / fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm . White arrowheads indicate PercevalHR expressing cells . Scale bar : 20 μm .

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

(D) Representative images of PV+ interneurons from PV-CRE and PV-CRE;Cox6a2fl/fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm. White arrowheads indicate PercevalHR expressing cells. Scale bar: 20 μm.


( E ) PV + interneurons from PV - CRE ; Cox6a2fl / fl mice exhibited significantly lower ATP - to - ADP ratios than PV + interneurons from PV - CRE mice throughout the entire duration of the recordings ( 23 cells from 4 PV - CRE mice ; 15 cells from 6 PV - CRE ; Cox6a2 - / - mice ) .

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

(E) PV+ interneurons from PV-CRE;Cox6a2fl/fl mice exhibited significantly lower ATP-to-ADP ratios than PV+ interneurons from PV-CRE mice throughout the entire duration of the recordings (23 cells from 4 PV-CRE mice; 15 cells from 6 PV-CRE;Cox6a2-/- mice).


( A , B ) The resting membrane potential and amplitude of action potentials in WT and Cox6a2 - / - PV + interneurons , respectively . Two - tailed unpaired Student ' s t - tests .

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

(A,B) The resting membrane potential and amplitude of action potentials in WT and Cox6a2-/- PV+ interneurons, respectively. Two-tailed unpaired Student's t-tests.


( C , D ) The threshold for action potentials increased over maturation in WT but not in Cox6a2 - / - PV + interneurons . As a result , the threshold for action potentials was significantly lower in P34 - 45 Cox6a2 - / - PV + interneurons when compared to their WT counterparts . D : two - tailed unpaired Student ' s t - tests .

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

(C,D) The threshold for action potentials increased over maturation in WT but not in Cox6a2-/- PV+ interneurons. As a result, the threshold for action potentials was significantly lower in P34-45 Cox6a2-/- PV+ interneurons when compared to their WT counterparts. D: two-tailed unpaired Student's t-tests.


( E , F ) Trains of action potentials were evoked by 2 sec depolarizing current pulses . The amplitude difference ( difference between the mean amplitude of 1st - 10th action potentials and 190th - 200th action potentials ) was significantly higher in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . In addition , the amplitude difference decreased with maturation in WT but not in Cox6a2 - / - PV + interneurons . F : two - tailed unpaired Mann - Whitney tests .

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

(E,F) Trains of action potentials were evoked by 2 sec depolarizing current pulses. The amplitude difference (difference between the mean amplitude of 1st-10th action potentials and 190th-200th action potentials) was significantly higher in P34-45 Cox6a2-/- compared to WT PV+ interneurons. In addition, the amplitude difference decreased with maturation in WT but not in Cox6a2-/- PV+ interneurons. F: two-tailed unpaired Mann-Whitney tests.


( G ) While in WT PV + interneurons the maximal firing frequency increased during maturation , such increase was abolished by Cox6a2 knockout . Thus , maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . Two - tailed unpaired Student ' s t - tests .

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

(G) While in WT PV+ interneurons the maximal firing frequency increased during maturation, such increase was abolished by Cox6a2 knockout. Thus, maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34-45 Cox6a2-/- compared to WT PV+ interneurons. Two-tailed unpaired Student's t-tests.


( H ) Recorded PV + interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software . Scale bars : 30 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

(H) Recorded PV+ interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software. Scale bars: 30 μm.