Metagene plot of RNAPII signal by plaNET - seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes ( n = 5753 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrow denotes the direction of transcriptional read - through . Pink dashed line rectangle indicates the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction . Metagene plot of RNAPII signal by plaNET - seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in " tail to tail " orientation ( n = 1384 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrows denote the directions of transcriptional read - through from both PASs . Pink dashed line rectangles indicate the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction .

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

Metagene plot of RNAPII signal by plaNET-seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes (n=5753) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrow denotes the direction of transcriptional read-through. Pink dashed line rectangle indicates the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction. Metagene plot of RNAPII signal by plaNET-seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in "tail to tail" orientation (n=1384) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrows denote the directions of transcriptional read-through from both PASs. Pink dashed line rectangles indicate the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction.


A ) ELISA quantification of PD - L1 , pTau 181 and Aβx - 42 in the CSF of control and AD patients ( n = 10 + / - SEM , Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , PD - L1 : t = 2 . 47 df = 18 , Aβx - 42 : t = 4 . 83 df = 18 , pTau 181 : t = 5 , 768 df = 18 ) .

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

A) ELISA quantification of PD-L1, pTau 181 and Aβx-42 in the CSF of control and AD patients (n=10 +/-SEM, Student's t-test, * p<0.05, *** p<0.001, PD-L1: t=2.47 df = 18, Aβx-42: t=4.83 df=18, pTau 181: t=5,768 df=18).


B , C ) ( B ) Colocalization of PD - L1 and GFAP in AD and ( C ) in control patients ( MX - O4 = amyloid deposits , upper picture : bar = 100 µm , lower picture : bar = 30 µm ) . D ) Immunohistochemical detection of PD - L1 in 9 - month - old female APP / PS1 mice ( bar = 50 µM ) . E ) Immunohistochemical detection of PD - L1 in 4 - month - old female APP / PS1 mice ( bar = 20 µm ) .

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

B, C) (B) Colocalization of PD-L1 and GFAP in AD and (C) in control patients (MX-O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar=30 µm). D) Immunohistochemical detection of PD-L1 in 9-month-old female APP/PS1 mice (bar = 50 µM). E) Immunohistochemical detection of PD-L1 in 4-month-old female APP/PS1 mice (bar = 20 µm).


A ) C6 cells expressing PD - L1 - myc treated with the γ - secretase inhibitor DAPT for 18 h . PD - L1 was detected using a myc - antibody .

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A) C6 cells expressing PD-L1-myc treated with the γ-secretase inhibitor DAPT for 18 h. PD-L1 was detected using a myc-antibody.


B ) Immunoblot of HEK293 cells expressing PD - L1 - myc and presenilin 1 ( hPS1 ) or a presenilin 1 dominant negative mutant ( hPS1 D246A ) .

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

B) Immunoblot of HEK293 cells expressing PD-L1-myc and presenilin 1 (hPS1) or a presenilin 1 dominant negative mutant (hPS1 D246A).


D ) 18 h 1 μM PMA treatment of C6 cells expressing PD - L1 - myc . Secretion of PD - L1 in the conditioned medium ( CM ) was detected using antibody AF1019 .

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D) 18 h 1 μM PMA treatment of C6 cells expressing PD-L1-myc. Secretion of PD-L1 in the conditioned medium (CM) was detected using antibody AF1019.


E ) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg / ml TNFα / 100 U / μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019 .

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E) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg/ml TNFα / 100 U/μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019.


A ) Colocalization of PD - 1 and CD11b ( MX - 04 = methoxy - XO4 , stains amyloid deposits ) in AD by immunohistochemistry ( bar = 50 µm ) . B ) Colocalization of PD - 1 and Iba1 or PD - 1 and Aβ in a female APP / PS1 mouse at 9 months of age by immunohistochemistry ( bar = 20 µm ) .

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

A) Colocalization of PD-1 and CD11b (MX-04=methoxy-XO4, stains amyloid deposits) in AD by immunohistochemistry (bar=50 µm). B) Colocalization of PD-1 and Iba1 or PD-1 and Aβ in a female APP/PS1 mouse at 9 months of age by immunohistochemistry (bar=20 µm).


E ) ELISA analysis of TNFα secretion by wild type ( wt ) and PD - 1 - / - microglia induced by 0 . 5 µM Aβ1 - 42 for 6 h in vitro ( n = 3 biological replicates , one - way ANOVA ( DF = 1 , F = 8 . 29 , p = 0 . 021 ) , Tukey ' s HSD , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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

E) ELISA analysis of TNFα secretion by wild type (wt) and PD-1-/- microglia induced by 0.5 µM Aβ1-42 for 6 h in vitro (n=3 biological replicates, one-way ANOVA (DF = 1, F = 8.29, p= 0.021), Tukey's HSD, * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


F - H ) Microglia from methoxy - XO4 - injected , 8 - month - old female , wild type ( wt ) and APP / PS1 mice were analyzed ( F ) for PD - 1 ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 13 . 99 , p = 0 . 0013 ) , Tukey ' s HSD , * * p < 0 . 01 ) , ( G ) for CD11b ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 30 . 06 , p = 5 . 47x10 - 05 ) , Tukey ' s HSD , * * * p < 0 . 001 ) and ( H ) CD45 by flow cytometry ( n = 5 biological replicates per group , one - way ANOVA ( DF = 1 , F = 24 . 96 , p = 0 . 00013 ) , Tukey ' s HSD , * * * p < 0 . 001 ) and normalized to the expression of wild type mice ( represented by dashed lines ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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

F-H) Microglia from methoxy-XO4-injected, 8-month-old female, wild type (wt) and APP/PS1 mice were analyzed (F) for PD-1 (n=5 biological replicates per group, one-way ANOVA (DF = 2, F = 13.99, p=0.0013), Tukey's HSD, ** p<0.01), (G) for CD11b (n=5 biological replicates per group, one- way ANOVA (DF = 2, F = 30.06, p=5.47x10-05), Tukey's HSD, *** p<0.001) and (H) CD45 by flow cytometry (n=5 biological replicates per group, one-way ANOVA (DF = 1, F = 24.96, p=0.00013), Tukey's HSD, *** p<0.001) and normalized to the expression of wild type mice (represented by dashed lines). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


A , B ) ELISA of the ( A ) RIPA soluble fraction of female APP / PS1 and APP / PS1 PD - 1 - / - mice for Aβx - 40 ( biological replicates with n = 6 for APP / PS1 and n = 5 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 0 . 6 , p = 0 . 45 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) and ( B ) of the SDS soluble fraction ( biological replicates with n = 6 for APP / PS1 and n = 5 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 32 . 8 , p = 2x10 - 5 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

A, B) ELISA of the (A) RIPA soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for Aβx-40 (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA (DF = 1, F = 0.6, p=0.45), Tukey's HSD, ** p<0.01, *** p<0.001) and (B) of the SDS soluble fraction (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA(DF=1, F= 32.8, p=2x10-5), Tukey's HSD, ** p<0.01, *** p<0.001).


D , E ) Mice were analyzed for ( D ) percentage of the covered areas ( 5 sections per mouse analyzed with the mean representing an individual data point ; biological replicates with n = 6 for APP / PS1 and n = 9 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 0 . 905 , p = 0 . 90 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) and ( E ) the number of plaques per mm2 ( 5 sections per mouse analyzed with the mean representing an individual data point ; biological replicates with n = 6 for APP / PS1 and n = 9 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 4 . 08 , p = 0 . 054 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

D, E) Mice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=0.905, p=0.90), Tukey's HSD, ** p<0.01, *** p<0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=4.08, p=0.054), Tukey's HSD, ** p<0.01, *** p<0.001).


G ) Time needed to reach the hidden platform ( latency in seconds ) in the Morris water maze test ( mean + / - SEM of biological replicates with n = 12 for wt , n = 5 for PD - 1 - / - , n = 7 for APP / PS1 , and n = 8 for APP / PS1 PD - 1 - / - , one - way ANOVA ( DF = 3 , F = 17 . 72 , p = 1x10 - 9 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) .

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

G) Time needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean +/-SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=7 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, one-way ANOVA (DF=3, F=17.72, p=1x10-9), Tukey's HSD, * p<0.05, *** p<0.001).


A ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type or PD - 1 - / - microglia for up to 6 h ( mean + / - SEM of a technical quadruplicate , Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate ) . B ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type microglia cultured in astrocyte - conditioned medium ( ACM ) from wild type or PD - L1 - / - astroglial cultures ( mean + / - SEM of a technical quadruplicate , Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate )

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

A) In vitro phagocytosis of FAM-Aβ1-42 by wild type or PD-1-/- microglia for up to 6 h (mean +/-SEM of a technical quadruplicate, Student's t-test, ** p<0.01, *** p<0.001, representative result of an n=2 biological replicate). B) In vitro phagocytosis of FAM-Aβ1-42 by wild type microglia cultured in astrocyte-conditioned medium (ACM) from wild type or PD-L1-/- astroglial cultures (mean +/-SEM of a technical quadruplicate, Student's t-test, * p<0.05, *** p<0.001, representative result of an n=2 biological replicate)


D ) Quantification of microglial in vivo phagocytosis in female APP / PS1 and APP / PS1 PD - 1 - / - mice at 8 . 5 month of age after intraperitoneal injection of methoxy - XO4 ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice , Student ' s t - test ( t = 2 . 1808 , df = 6 . 9829 ) , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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

D) Quantification of microglial in vivo phagocytosis in female APP/PS1 and APP/PS1 PD-1-/- mice at 8.5 month of age after intraperitoneal injection of methoxy-XO4 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, Student's t-test (t = 2.1808, df = 6.9829), * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


E - G ) Relative expression of the cell surface marker ( E ) CD36 ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice , ANOVA ( DF = 1 , F = 0 . 002 , p = 0 . 94 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) , ( F ) CD11b ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice ) and ( G ) CD45 on methoxy - XO4 + versus methoxy - XO4 - microglia in APP / PS1 and APP / PS1 PD - 1 - / - mice normalized to methoxy - X04 - negative cells from APP / PS1 mice ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice ) Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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E-G) Relative expression of the cell surface marker (E) CD36 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, ANOVA (DF=1, F= 0.002, p=0.94), Tukey's HSD, * p<0.05, *** p<0.001), (F) CD11b (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) and (G) CD45 on methoxy-XO4+ versus methoxy-XO4- microglia in APP/PS1 and APP/PS1 PD-1-/- mice normalized to methoxy-X04-negative cells from APP/PS1 mice (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


B ) Different expression of inflammasome - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia . C ) Different expression of complement system - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia .

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

B) Different expression of inflammasome-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini- Hochberg adjusted) in PD-1-/- vs. wild type microglia. C) Different expression of complement system-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini-Hochberg adjusted) in PD-1-/- vs. wild type microglia.


Immunoblot analysis of p62 and LC3 I / II in KRIT1 wt and KRIT1 - KO endothelial cells . Actin was used as a loading control . Quantification of total LC3 on actin is reported ( * P = 0 . 02712 ) . The results are representative of three independent experiments .

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

Immunoblot analysis of p62 and LC3 I/II in KRIT1 wt and KRIT1-KO endothelial cells. Actin was used as a loading control. Quantification of total LC3 on actin is reported (*P = 0.02712). The results are representative of three independent experiments.


Representative images of p62 dots in KRIT1 wt and KRIT1 - KO endothelial cells . Scale bar , 20 μm . Magnifications in insets . Right , quantitative analysis of p62 distribution of dots is reported ( four independent experiments ; n = 35 cells per group ) . * P = 0 . 00542 ( dotted ) ; * P = 0 . 00014 ( nuclear ) .

O O O B-MEASURED_VAR 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 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

Representative images of p62 dots in KRIT1 wt and KRIT1-KO endothelial cells. Scale bar, 20 μm. Magnifications in insets. Right, quantitative analysis of p62 distribution of dots is reported (four independent experiments; n = 35 cells per group). *P = 0.00542 (dotted); *P = 0.00014 (nuclear).


Immunoblot analysis of p62 and LC3 I / II in KRIT1 - KO and KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) MEFs . Left , immunoblot showing KRIT1 levels in KRIT1 - KO and KO + KRIT1 cells . Right , immunoblots for p62 and LC3 I / II . Actin was used as a loading marker . Quantification of total LC3 on actin is reported ( * P = 0 . 01248 ) . The results are representative of three independent experiments .

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

Immunoblot analysis of p62 and LC3 I/II in KRIT1-KO and KRIT1-KO re-expressing KRIT1 (KO+KRIT1) MEFs. Left, immunoblot showing KRIT1 levels in KRIT1-KO and KO+KRIT1 cells. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.01248). The results are representative of three independent experiments.


Representative images of p62 dots in KO + KRIT1 ( top ) and KRIT1 - KO cells ( bottom ) . Scale bar , 50 μm . Magnifications in insets . Right , quantitative analysis of the number of p62 dots per cell is shown ( four independent experiments ; n = 50 cells per group ) . * P = 7 . 18e − 14 .

O O O B-MEASURED_VAR 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 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

Representative images of p62 dots in KO+KRIT1 (top) and KRIT1-KO cells (bottom). Scale bar, 50 μm. Magnifications in insets. Right, quantitative analysis of the number of p62 dots per cell is shown (four independent experiments; n = 50 cells per group). *P = 7.18e−14.


Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA . Left , evaluation of siRNA efficiency with antibody directed against KRIT1 . Right , immunoblots for p62 and LC3 I / II . Actin was used as a loading marker . Quantification of total LC3 on actin is reported ( * P = 0 . 03071 ) . The results are representative of three independent experiments .

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

Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.03071). The results are representative of three independent experiments.


Immunoblot analysis of EA . hy926 cells transiently transfected with control siRNA or KRIT1 siRNA . Left , evaluation of siRNA efficiency with antibody directed against KRIT1 . Right , immunoblot for p62 and LC3 I / II . Actin was used as a loading marker . Quantification of total LC3 on actin is reported ( * P = 0 . 02527 ) . The results are representative of three independent experiments .

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

Immunoblot analysis of EA.hy926 cells transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblot for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.02527). The results are representative of three independent experiments.


Immunofluorescence analysis of p62 ( green ) and ProteoStat Aggresome staining detection reagent ( red ) in KRIT1 wt and KRIT1 - KO lung endothelial cells . The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence . Magnification in insets . Scale bar , 50 μm . The images are representative of four independent experiments .

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

Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1 wt and KRIT1-KO lung endothelial cells. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.


Immunofluorescence analysis of p62 ( green ) and ProteoStat Aggresome staining detection reagent ( red ) in KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) and KRIT1 - KO MEFs . The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence . Magnification in insets . Scale bar , 50 μm . The images are representative of four independent experiments .

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 B-CONTROLLED_VAR 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

Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.


Immunoblot analysis with antibodies directed against phosphorylated mTOR ( Ser 2448 ) , total mTOR , phosphorylated p70 S6 Kinase ( Ser 371 ) , total p70 S6 Kinase , phosphorylated 4E - BP1 ( Thr 37 / 46 ) , and total 4E - BP1 ; actin was used as a loading marker . Where indicated , KRIT1 wt and KRIT1 - KO endothelial cells were treated with 100 nM Torin1 for 4 h . The results are representative of three independent experiments .

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

Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1 - KO endothelial cells . Where indicated , cells were treated with 100 nM Torin1 for 4 h . The results are representative of three independent experiments .

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

Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1-KO endothelial cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot analysis of p62 , LC3 I / II , and actin in KRIT1 wt and KRIT1 - KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h . The results are representative of three independent experiments .

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

Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


Immunoblot analysis with antibodies directed against phosphorylated mTOR ( Ser 2448 ) , total mTOR , phosphorylated p70 S6 Kinase ( Ser 371 ) , total p70 S6 Kinase , phosphorylated 4E - BP1 ( Thr 37 / 46 ) , and total 4E - BP1 ; actin was used as a loading marker . Where indicated , KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) and KRIT1 - KO MEFs were treated with 100 nM Torin1 for 4 h . The results are representative of three independent experiments .

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

Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot analysis of phosphorylated ULK1 ( Ser 757 ) , total ULK1 , and actin in KRIT1 KO + KRIT1 , and KRIT1 KO MEFs . Where indicated , cells were treated with 100 nM Torin1 for 4 h . The results are representative of three independent experiments .

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

Immunoblot analysis of phosphorylated ULK1 (Ser 757), total ULK1, and actin in KRIT1 KO+KRIT1, and KRIT1 KO MEFs. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot analysis of p62 , actin , LC3 I / II in KO + KRIT1 and KRIT1 - KO cells . Where indicated , cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h . The results are representative of three independent experiments .

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

Immunoblot analysis of p62, actin, LC3 I/II in KO+KRIT1 and KRIT1-KO cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


KRIT1 wt and KRIT1 - KO endothelial cells were transiently transfected with mRFP - GFP - LC3 . Where indicated , the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h . The differences in the autophagic flux were evaluated by counting the yellow LC3 I / II dots / cell ( RFP + GFP + ) and red LC3 dots / cell ( RFP + GFP − ) for each condition . Yellow dots : autophagosomes ; red dots : autophagolysosomes . * P = 5 . 74e − 5 ( red dots , WT ctrl vs . WT Tor1 ) ; * P = 9 . 62e − 5 ( red dots , WT ctrl vs . WT xesto ) ; * P = 0 . 00727 ( red dots , WT ctrl vs . KO ctrl ) ; # P = 0 . 00046 ( red dots , KO ctrl vs . KO Tor1 ) . The data are expressed as the mean ± s . e . m .

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

KRIT1 wt and KRIT1-KO endothelial cells were transiently transfected with mRFP-GFP-LC3. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 5.74e−5 (red dots, WT ctrl vs. WT Tor1); *P = 9.62e−5 (red dots, WT ctrl vs. WT xesto); *P = 0.00727 (red dots, WT ctrl vs. KO ctrl); #P = 0.00046 (red dots, KO ctrl vs. KO Tor1). The data are expressed as the mean ± s.e.m.


KO + KRIT1 and KRIT1 - KO MEFs were transiently transfected with the mRFP - GFP - LC3 tandem construct . Where indicated , the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h . The differences in the autophagic flux were evaluated by counting the yellow LC3 I / II dots / cell ( RFP + GFP + ) and red LC3 dots / cell ( RFP + GFP − ) for each condition . Yellow dots : autophagosomes ; red dots : autophagolysosomes . * P = 0 . 00023 ( red dots , KO + KRIT1 ctrl vs . KO + KRIT1 Tor1 ) ; * P = 0 . 00045 ( red dots , KO + KRIT1 ctrl vs . KO + KRIT1 xesto ) ; # P = 3 . 08e − 6 ( red dots , KO ctrl vs . KO Tor1 ) ; # # P = 6 . 73e − 5 ( yellow dots , KO + KRIT1 ctrl vs . KO ctrl ) . The data are expressed as the mean ± s . e . m . of four independent experiments .

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

KO+KRIT1 and KRIT1-KO MEFs were transiently transfected with the mRFP-GFP-LC3 tandem construct. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 0.00023 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 Tor1); *P = 0.00045 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 xesto); #P = 3.08e−6 (red dots, KO ctrl vs. KO Tor1); ##P = 6.73e−5 (yellow dots, KO+KRIT1 ctrl vs. KO ctrl). The data are expressed as the mean ± s.e.m. of four independent experiments.


Cd44 , PAI1 ( also known as Serpine1 ) , and Id1 mRNA expression levels in KRIT1 wt and KRIT1 - KO endothelial cells were assessed by quantitative real - time PCR . Where indicated , KRIT1 wt and KRIT1 - KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h . The data are expressed as the mean ± s . e . m . Cd44 : * P = 0 . 02848 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 02605 ( KO ctrl vs . KO Tor1 ) . PAI1 : * P = 0 . 04446 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 03996 ( KO ctrl vs . KO Tor1 ) . Id1 : * P = 0 . 00266 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 01554 ( KO ctrl vs . KO Tor1 ) . n = 3 independent experiments .

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

Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1-KO endothelial cells were assessed by quantitative real-time PCR. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO ctrl vs. KO Rapa); *P = 0.01554 (KO ctrl vs. KO Tor1). n = 3 independent experiments.


Immunoblot analysis of CD31 / Pecam - 1 , vascular endothelial cadherin ( VE - cadherin ) , and actin in KRIT1 - KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h . The results are representative of three independent experiments .

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

Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), and actin in KRIT1-KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h. The results are representative of three independent experiments.


Immunoblot analysis of CD31 / Pecam - 1 , vascular endothelial cadherin ( VE - cadherin ) , N - cadherin , alpha - smooth muscle actin ( alpha - SMA ) , and actin in HUVECs transfected with control siRNA or ATG7 siRNA .

Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), N-cadherin, alpha-smooth muscle actin (alpha-SMA), and actin in HUVECs transfected with control siRNA or ATG7 siRNA.


Formation of capillary - like structures measured by tube formation assays . HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h . Representative phase - contrast microscopy ( Scale bar , 100 μm ) and calcein - fluorescent ( Scale bar , 50 μm ) images were reported . All data are presented as percentage ± s . e . m from three different experiments performed in duplicate . * P = 1 . 29e − 11 .

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

Formation of capillary-like structures measured by tube formation assays. HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h. Representative phase-contrast microscopy (Scale bar, 100 μm) and calcein-fluorescent (Scale bar, 50 μm) images were reported. All data are presented as percentage ± s.e.m from three different experiments performed in duplicate. *P = 1.29e−11.


Immunoblot analysis of p62 , LC3I / II , and rapamycin in CCM3 wt and CCM3 - KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h . The results are representative of three independent experiments .

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

Immunoblot analysis of p62, LC3I/II, and rapamycin in CCM3 wt and CCM3-KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


Representative immunostaining of retina sections from wt and a model of inducible and endothelial - specific CCM3 - KO ( CCM3 - ECKO ) mice at postnatal day 14 . Endothelium was stained with isolectin B4 ( ISOB4 ) ( blue ) . A , artery ; V , vein . p62 aggregates can be observed in endothelial cells forming retinal lesions in CCM3 - ECKO animals ( scale bar : 200 μm ) . Scale bar of magnifications : 100 μm .

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

Representative immunostaining of retina sections from wt and a model of inducible and endothelial-specific CCM3-KO (CCM3-ECKO) mice at postnatal day 14. Endothelium was stained with isolectin B4 (ISOB4) (blue). A, artery; V, vein. p62 aggregates can be observed in endothelial cells forming retinal lesions in CCM3-ECKO animals (scale bar: 200 μm). Scale bar of magnifications: 100 μm.


Representative immunostaining of brain sections from wt and a model of inducible and endothelial - specific CCM3 - knockout mice ( CCM3 - ECKO ) at postnatal day 9 . p62 aggregates can be observed in the proximity of CCM lesions ( arrows ) . Cell nuclei ( DAPI ) are in blue . Scale bar , 30 μm . Smaller panel shows the magnifications of blood vessels ( green ) . Scale bar , 10 μm .

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 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

Representative immunostaining of brain sections from wt and a model of inducible and endothelial-specific CCM3-knockout mice (CCM3-ECKO) at postnatal day 9. p62 aggregates can be observed in the proximity of CCM lesions (arrows). Cell nuclei (DAPI) are in blue. Scale bar, 30 μm. Smaller panel shows the magnifications of blood vessels (green). Scale bar, 10 μm.


p62 immunohistochemistry ( IHC ) staining in humanbrain tissue . A , B Normal vascular endothelium of autoptic brain parenchyma samples is lacking the typical autophagic p62 granules as shown by the negative staining for p62 . Scale bars : ( A ) 200 μm ; ( B ) 100 μm .

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

p62 immunohistochemistry (IHC) staining in humanbrain tissue.A, B Normal vascular endothelium of autoptic brain parenchyma samples is lacking the typical autophagic p62 granules as shown by the negative staining for p62. Scale bars: (A) 200 μm; (B) 100 μm.


p62immunohistochemistry ( IHC ) staining in humanbrain tissue . C - F Two different representative samples of CCM lesions with a thin , single layer brain endothelium displaying either moderate ( C , D ) or marked ( E , F ) positive perinuclear " pearl necklace - like " immunostaining for p62 granules . ( C , D ) , case n ° 4 ( p62 + + ) , and ( E , F ) , case n ° 8 ( p62 + + + ) are representative of CCM cases listed in Appendix Table S1 . Scale bars : ( C , E ) 200 μm ; ( D , F ) 100 μm . Arrows indicate endothelial p62 positive staining .

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 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 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 B-MEASURED_VAR O O O

p62immunohistochemistry (IHC) staining in humanbrain tissue.C-F Two different representative samples of CCM lesions with a thin, single layer brain endothelium displaying either moderate (C, D) or marked (E, F) positive perinuclear "pearl necklace-like" immunostaining for p62 granules. (C, D), case n° 4 (p62++), and (E, F), case n° 8 (p62+++) are representative of CCM cases listed in Appendix Table S1. Scale bars: (C, E) 200 μm; (D, F) 100 μm. Arrows indicate endothelial p62 positive staining.


p62immunohistochemistry ( IHC ) staining in humanbrain tissue . G - I Hematoxylin and eosin ( H & amp ; amp ; E ) staining ( G ) and p62immunohistochemistry analysis ( H , I ) of a CCM surgical sample ( case n ° 6 in Appendix Table S1 ) containing normal vessels in the peri - lesional area , which served as an internal control . Notice marked p62 - positive staining in endothelial cells lining a CCM lesion ( H , arrows ) and p62 - negative staining in endothelial cells lining a normal peri - lesional vessel ( I , arrows ) . Scale bars : ( G ) 300 μm ; ( H , I ) 100 μm . Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells .

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p62immunohistochemistry (IHC) staining in humanbrain tissue.G-I Hematoxylin and eosin (H&amp;E) staining (G) and p62immunohistochemistry analysis (H, I) of a CCM surgical sample (case n° 6 in Appendix Table S1) containing normal vessels in the peri-lesional area, which served as an internal control. Notice marked p62-positive staining in endothelial cells lining a CCM lesion (H, arrows) and p62-negative staining in endothelial cells lining a normal peri-lesional vessel (I, arrows). Scale bars: (G) 300 μm; (H, I) 100 μm. Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells.


Depletion of RBM14 induces ectopic formation of centriolar protein complexesA - E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) ( A ) , Centrobin ( green ) and C - Nap1 ( magenta ) ( B ) , centrin - 2 ( green ) and CPAP ( magenta ) ( C ) , acetylated - tubulin ( green ) and CP110 ( magenta ) ( D ) , and centrin - 2 ( green ) and γ - tubulin ( magenta ) ( E ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition . Values are mean percentages ± standard error of mean ( SEM ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) . Note that we counted the number of C - Nap1 foci after anaphase in mitosis when C - Nap1 signals recover from the reduction in prometaphase .

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Depletion of RBM14 induces ectopic formation of centriolar protein complexesA-E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta) (A), Centrobin (green) and C-Nap1 (magenta) (B), centrin-2 (green) and CPAP (magenta) (C), acetylated-tubulin (green) and CP110 (magenta) (D), and centrin-2 (green) and γ-tubulin (magenta) (E). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01, n.s., not significant (one-tailed t-test). Note that we counted the number of C-Nap1 foci after anaphase in mitosis when C-Nap1 signals recover from the reduction in prometaphase.


A U2OS cells or U2OS cells expressing FLAG - RBM14 full length ( FL , aa1 - 669 ) , N - terminal fragment ( RBM14 [ N ] , aa1 - 150 ) or C - terminal fragment ( RBM14 [ C ] , aa151 - 669 ) and treated with control siRNA or siRNA against 3 ′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin - 2 . Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition . Values are mean percentages ± SEM from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) .

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A U2OS cells or U2OS cells expressing FLAG-RBM14 full length (FL, aa1-669), N-terminal fragment (RBM14[N], aa1-150) or C-terminal fragment (RBM14[C], aa151-669) and treated with control siRNA or siRNA against 3′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin-2. Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).


C , D Cytoplasmic RBM14 could suppress centriole amplification in HU - treated cells . ( C ) U2OS cells or U2OS cells expressing FLAG - RBM14 FL or [ C ] and treated with or without HU were stained with antibodies against centrin - 2 ( green ) and FLAG ( magenta ) . ( D ) U2OS cells or U2OS cells expressing FLAG - RBM14 FL , FLAG - RBM14 - NES or GFP - RBM14 - PACT and treated with or without HU were stained with antibodies against FLAG ( magenta , left ) or GFP ( magenta , right ) as well as centrin - 2 ( green ) . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 10 μm . Histograms represent frequency of cells in interphase with excess centrin foci in each condition . The percentages of U2OS cells with centrosomal localization of the RBM14 full - length protein or mutants are shown below the immunofluorescence images . We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . Please note that cytoplasmic expression levels of GFP - RBM14 - PACT are less than those of FLAG - RBM14 FL ( ˜ 0 . 5 - fold ) or FLAG - RBM14 - NES ( ˜ 0 . 7 - fold ) .

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C, D Cytoplasmic RBM14 could suppress centriole amplification in HU-treated cells. (C) U2OS cells or U2OS cells expressing FLAG-RBM14 FL or [C] and treated with or without HU were stained with antibodies against centrin-2 (green) and FLAG (magenta). (D) U2OS cells or U2OS cells expressing FLAG-RBM14 FL, FLAG-RBM14-NES or GFP-RBM14-PACT and treated with or without HU were stained with antibodies against FLAG (magenta, left) or GFP (magenta, right) as well as centrin-2 (green). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 10 μm. Histograms represent frequency of cells in interphase with excess centrin foci in each condition. The percentages of U2OS cells with centrosomal localization of the RBM14 full-length protein or mutants are shown below the immunofluorescence images. We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Please note that cytoplasmic expression levels of GFP-RBM14-PACT are less than those of FLAG-RBM14 FL (˜0.5-fold) or FLAG-RBM14-NES (˜0.7-fold).


A HeLa cells immunoprecipitated with control IgG or STIL antibodies . Soluble cytosolic fractions ( input ) and immunoprecipitates ( IPs ) were analyzed by Western blotting using RBM14 , STIL or CPAP antibodies .

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A HeLa cells immunoprecipitated with control IgG or STIL antibodies. Soluble cytosolic fractions (input) and immunoprecipitates (IPs) were analyzed by Western blotting using RBM14, STIL or CPAP antibodies.


B GST pull - down assay testing interactions between purified STIL [ N ] ( ˜ 5 μg , aa1 - 1018 ) and GST - RBM14 [ N ] or [ C ] . The asterisks indicate non - specific bands .

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B GST pull-down assay testing interactions between purified STIL[N] (˜5 μg, aa1-1018) and GST-RBM14 [N] or [C]. The asterisks indicate non-specific bands.


D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin - 2 ( green ) and STIL ( magenta ) . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess STIL foci co - localized with centrin foci . Values are mean percentages ± SEM from three independent samples ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) .

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D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin-2 (green) and STIL (magenta). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess STIL foci co-localized with centrin foci. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01 (one-tailed t-test).


E In vitro competitive binding assay . GST pull - down experiment was performed as in ( B ) , with purified STIL [ N ] and GST - RBM14 [ C ] in the presence of the indicated amount of purified His - CPAP [ SBD ] , His - tagged STIL - Binding Domain of CPAP . The fraction of STIL [ N ] bound to GST - RBM14 [ C ] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N - terminal region of STIL . Input materials were analyzed by Western blotting using the STIL or CPAP antibodies . The precipitated GST - RBM14 [ C ] was analyzed by SDS - PAGE , stained with SimplyBlue ™ Safe ( Invitrogen ) .

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E In vitro competitive binding assay. GST pull-down experiment was performed as in (B), with purified STIL[N] and GST-RBM14[C] in the presence of the indicated amount of purified His-CPAP[SBD], His-tagged STIL-Binding Domain of CPAP. The fraction of STIL[N] bound to GST-RBM14[C] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N-terminal region of STIL. Input materials were analyzed by Western blotting using the STIL or CPAP antibodies. The precipitated GST-RBM14[C] was analyzed by SDS-PAGE, stained with SimplyBlue™ Safe (Invitrogen).


F Interaction between STIL and GFP - CPAP in the presence of FLAG - RBM14 FL or fragments . U2OS cells expressing GFP - CPAP were transfected with empty vector as a control , FLAG - RBM14 [ N ] , FLAG - RBM14 [ C ] or FLAG - RBM14 FL constructs , immunoprecipitated with STIL antibodies , and the resulting IPs were analyzed by Western blotting using STIL , CPAP or FLAG antibodies . Quantification of relative protein amounts of co - immunoprecipitated GFP - CPAP in each STIL - IP fraction , normalized with the amount of STIL precipitated , is shown below the panels . Means ± SEM were calculated from three independent experiments . * * P < 0 . 01 ( one - tailed t - test ) . Note that we did not find any significant effect of exogenous expression of RBM14 full - length protein or deletion mutants on the expression levels of GFP - CPAP or endogenous STIL proteins in this experiment .

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F Interaction between STIL and GFP-CPAP in the presence of FLAG-RBM14 FL or fragments. U2OS cells expressing GFP-CPAP were transfected with empty vector as a control, FLAG-RBM14[N], FLAG-RBM14[C] or FLAG-RBM14 FL constructs, immunoprecipitated with STIL antibodies, and the resulting IPs were analyzed by Western blotting using STIL, CPAP or FLAG antibodies. Quantification of relative protein amounts of co-immunoprecipitated GFP-CPAP in each STIL-IP fraction, normalized with the amount of STIL precipitated, is shown below the panels. Means ± SEM were calculated from three independent experiments. **P < 0.01 (one-tailed t-test). Note that we did not find any significant effect of exogenous expression of RBM14 full-length protein or deletion mutants on the expression levels of GFP-CPAP or endogenous STIL proteins in this experiment.


G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin - 2 ( green ) and C - Nap1 ( magenta ) . Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) . Representative mitotic cells treated with siRNAs against RBM14 alone , RBM14 + STIL or RBM14 + CPAP are shown below . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm .

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G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta). Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Representative mitotic cells treated with siRNAs against RBM14 alone, RBM14 + STIL or RBM14 + CPAP are shown below. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm.


A U2OS cells treated with control siRNA , RBM14 siRNA alone or RBM14 + HsSAS - 6 siRNAs were stained with antibodies against centrin - 2 ( green ) and HsSAS - 6 ( magenta ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . n . s . , not significant ( one - tailed t - test ) .

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A U2OS cells treated with control siRNA, RBM14 siRNA alone or RBM14 + HsSAS-6 siRNAs were stained with antibodies against centrin-2 (green) and HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). n.s., not significant (one-tailed t-test).


B Live imaging of cycling RBM14 - depleted HeLa cells expressing GFP - centrin1 ( green ) and RFP - H2B ( magenta ) . Insets show approximately 1 . 5 - fold magnified images of fluorescent foci . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the onset of excess centrin foci formation . Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time . Means ± SEM are shown ( n = 5 ) .

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B Live imaging of cycling RBM14-depleted HeLa cells expressing GFP-centrin1 (green) and RFP-H2B (magenta). Insets show approximately 1.5-fold magnified images of fluorescent foci. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of excess centrin foci formation. Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time. Means ± SEM are shown (n = 5).


C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase . Time - lapse recording across the cell cycle of HeLa cells expressing GFP - centrin ( green ) and RFP - H2B ( magenta ) and treated with RBM14 siRNA . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the metaphase onset .

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C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase. Time-lapse recording across the cell cycle of HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the metaphase onset.


D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment . Insets show threefold magnified images of fluorescent foci . Scale bar , 5 μm . Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - tailed t - test ) .

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D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment. Insets show threefold magnified images of fluorescent foci. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).


E Amorphous electron - dense structures containing microtubules are formed in RBM14 - depleted cells ( see also Supplementary Fig S5I ) . Electron micrographs from U2OS cells depleted of RBM14 . Squares indicate the ectopic electron - dense structures , whereas circles indicate the pre - existing centrioles . Scale bar , 500 nm and 200 nm ( magnified views ) . Average diameter of pre - existing centrioles and the amorphous electron - dense structures are 225 ± 8 nm ( n = 10 ) and 130 + 11 nm ( n = 16 ) , respectively . Insets show magnified views of the squares . Note that microtubules ( black arrowheads ) are observed within the electron - dense structures ( white arrowheads ) .

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E Amorphous electron-dense structures containing microtubules are formed in RBM14-depleted cells (see also Supplementary Fig S5I). Electron micrographs from U2OS cells depleted of RBM14. Squares indicate the ectopic electron-dense structures, whereas circles indicate the pre-existing centrioles. Scale bar, 500 nm and 200 nm (magnified views). Average diameter of pre-existing centrioles and the amorphous electron-dense structures are 225 ± 8 nm (n = 10) and 130 + 11 nm (n = 16), respectively. Insets show magnified views of the squares. Note that microtubules (black arrowheads) are observed within the electron-dense structures (white arrowheads).


F Histograms represent frequency of the cells with ectopic foci of GFP - CPAP in the indicated conditions . U2OS cells expressing GFP - CPAP were fixed and stained 24 h after RNAi treatment and induction of GFP - CPAP . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - tailed t - test ) .

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F Histograms represent frequency of the cells with ectopic foci of GFP-CPAP in the indicated conditions. U2OS cells expressing GFP-CPAP were fixed and stained 24 h after RNAi treatment and induction of GFP-CPAP. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test).


G U2OS cells expressing GFP - CPAP ( green ) and treated with RBM14 siRNA were analyzed using CLEM . Separate z - plane images of the cell with GFP - CPAP foci and the threefold magnified images are aligned from bottom to top along z - axis ( left LM panels ) . White broken lines represent cell shapes , and blue broken lines represent nuclear shapes . Corresponding regions of EM images are shown on the right . White squares ( a - d ) represent the region around GFP - CPAP foci . Note that other than pre - existing centrioles ( a , 1 - 4 ) , ectopic small CPAP foci are recognizable as amorphous electron - dense structures ( b - d ) . One of pre - existing centrioles ( 1 ) is elongated because of exogenous GFP - CPAP expression . Scale bar , 10 μm ( LM ) , 2 μm ( EM , low magnified image ) , 500 nm [ magnified images of ( a ) and ( d ) ) , 200 nm ( magnified images of ( b ) and ( c ) ] .

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G U2OS cells expressing GFP-CPAP (green) and treated with RBM14 siRNA were analyzed using CLEM. Separate z-plane images of the cell with GFP-CPAP foci and the threefold magnified images are aligned from bottom to top along z-axis (left LM panels). White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. Corresponding regions of EM images are shown on the right. White squares (a-d) represent the region around GFP-CPAP foci. Note that other than pre-existing centrioles (a, 1-4), ectopic small CPAP foci are recognizable as amorphous electron-dense structures (b-d). One of pre-existing centrioles (1) is elongated because of exogenous GFP-CPAP expression. Scale bar, 10 μm (LM), 2 μm (EM, low magnified image), 500 nm [magnified images of (a) and (d)), 200 nm (magnified images of (b) and (c)].


B - E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold - treated for 30 min , followed by 30 - 60 min incubation at 37 ° C and stained with antibodies against centrin ( magenta in ( B ) and ( D ) ) or γ - tubulin ( magenta in ( E ) ) as well as α - tubulin ( green ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point . Values are mean percentages ± standard error of mean ( SEM ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 ( one - tailed t - test ) .

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B-E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30-60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).


A U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin - 2 ( green ) as well as HsSAS - 6 ( magenta ) . DNA is shown in blue . Insets show approximately twofold magnified views of fluorescent foci around the centrosome . Scale bar , 5 μm . Histograms represent the frequency of mitotic cells with the indicated phenotype . Values are mean percentages ± SEM from three independent experiments ( n = 60 for each condition ) .

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A U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin-2 (green) as well as HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the frequency of mitotic cells with the indicated phenotype. Values are mean percentages ± SEM from three independent experiments (n = 60 for each condition).


B Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were stained with antibodies against HsSAS - 6 as well as centrin - 2 . Immunofluorescence micrographs are shown in Supplementary Fig S6D . When we counted HsSAS - 6 foci in this paper , we only counted cells before metaphase / anaphase transition considering the disappearance of HsSAS - 6 from anaphase in mitosis . Histograms represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) .

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B Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were stained with antibodies against HsSAS-6 as well as centrin-2. Immunofluorescence micrographs are shown in Supplementary Fig S6D. When we counted HsSAS-6 foci in this paper, we only counted cells before metaphase/anaphase transition considering the disappearance of HsSAS-6 from anaphase in mitosis. Histograms represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


C U2OS cells treated with the indicated siRNAs were stained with anti - α - tubulin antibodies and Hoechst 33258 . Histograms represent frequency of mitotic cells with the multipolar spindle in the indicated conditions . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) .

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C U2OS cells treated with the indicated siRNAs were stained with anti-α-tubulin antibodies and Hoechst 33258. Histograms represent frequency of mitotic cells with the multipolar spindle in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


D RBM14 - depleted HeLa cells expressing GFP - centrin ( green ) were stained with antibodies against CP110 ( magenta ) and HsSAS - 6 ( blue ) . Scale bar , 1 μm . A representative stained procentriole - like structure as well as endogenous centrioles is shown together with a schematic . Histograms represent length between the centers of the foci of the indicated proteins . Values are mean percentages ± SEM from seven individual cells .

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D RBM14-depleted HeLa cells expressing GFP-centrin (green) were stained with antibodies against CP110 (magenta) and HsSAS-6 (blue). Scale bar, 1 μm. A representative stained procentriole-like structure as well as endogenous centrioles is shown together with a schematic. Histograms represent length between the centers of the foci of the indicated proteins. Values are mean percentages ± SEM from seven individual cells.


E , F Ectopic DsRed - centrin - 2 foci in RBM14 - depleted cells recruit HsSAS - 6 - GFP . ( E ) Live imaging of RBM14 - depleted U2OS cells expressing HsSAS - 6 - GFP ( green ) and DsRed - centrin - 2 ( magenta ) . Time is denoted in hh : min , and scale bar is 10 μm . Time zero corresponds to the onset of recording . Pre - existing HsSAS - 6 - GFP foci ( white arrowheads ) and ectopic HsSAS - 6 - GFP focus ( red arrowhead ) were traced throughout the time - lapse recording . White broken lines represent cell shapes , and blue broken lines represent nuclear shapes . ( F ) Histograms represent the percentages of ectopic DsRed - centrin - 2 foci with HsSAS - 6 - GFP recruitment ( n = 69 foci for control , and n = 67 foci for RBM14 siRNA ) .

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E, F Ectopic DsRed-centrin-2 foci in RBM14-depleted cells recruit HsSAS-6-GFP. (E) Live imaging of RBM14-depleted U2OS cells expressing HsSAS-6-GFP (green) and DsRed-centrin-2 (magenta). Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of recording. Pre-existing HsSAS-6-GFP foci (white arrowheads) and ectopic HsSAS-6-GFP focus (red arrowhead) were traced throughout the time-lapse recording. White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. (F) Histograms represent the percentages of ectopic DsRed-centrin-2 foci with HsSAS-6-GFP recruitment (n = 69 foci for control, and n = 67 foci for RBM14 siRNA).


G Histograms represent frequency of mitotic cells with excess foci of HsSAS - 6 in the indicated conditions . In this experiment , cells were treated with double amount of RBM14 siRNA ( 20 pmol ) compared to that in the other experiments . Values are mean percentages ± SEM from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - tailed t - test ) .

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G Histograms represent frequency of mitotic cells with excess foci of HsSAS-6 in the indicated conditions. In this experiment, cells were treated with double amount of RBM14 siRNA (20 pmol) compared to that in the other experiments. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


H HeLa cells expressing GFP - centrin ( green ) and RFP - H2B ( magenta ) and treated with RBM14 siRNA were analyzed using Live CLEM . Pre - existing GFP - centrin foci ( white arrowheads ) and ectopic GFP - centrin foci ( red arrowheads ) were traced throughout the time - lapse recording . Z - stacked confocal images spanning the entire height of the cells ( < 30 μm ) are shown ( upper panels ) . Time is denoted in hh : min . Time zero corresponds to the onset of ectopic formation of GFP - centrin foci . The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images ( lower panels ) . Red square ( ectopically formed GFP - centrin foci ) represents the region corresponding to that of the EM images acquired from serial sections . Note that ectopic GFP - centrin foci that form a spindle pole include not only electron - dense structures ( 1 , 3 ) but also a morphologically recognizable centriole - like structure ( 2 ) . Scale bars , 10 μm ( live imaging ) and 500 nm ( EM ) .

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H HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA were analyzed using Live CLEM. Pre-existing GFP-centrin foci (white arrowheads) and ectopic GFP-centrin foci (red arrowheads) were traced throughout the time-lapse recording. Z-stacked confocal images spanning the entire height of the cells (< 30 μm) are shown (upper panels). Time is denoted in hh:min. Time zero corresponds to the onset of ectopic formation of GFP-centrin foci. The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images (lower panels). Red square (ectopically formed GFP-centrin foci) represents the region corresponding to that of the EM images acquired from serial sections. Note that ectopic GFP-centrin foci that form a spindle pole include not only electron-dense structures (1, 3) but also a morphologically recognizable centriole-like structure (2). Scale bars, 10 μm (live imaging) and 500 nm (EM).


( C - I ) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h , prior to stimulation with nigericin for 45 min or ATP for 1 h . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , and ASC ( the arrow indicates ASC in the whole cell lysate ( WCL ) ; activated caspase - 1 ( p20 ) and cleaved IL - 1β ( p17 ) in the culture supernatants ( SN ) of BMDMs ( C , G ) . Caspase - 1 activity ( D , H ) , IL - 1β secretion ( E , I ) , and LDH release ( F , J ) in the SN were measured . Coomassie Blue staining was used as the supernatant loading control , while lamin B was used as the lysate loading control .

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(C-I) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC (the arrow indicates ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (C, G). Caspase-1 activity (D, H), IL-1β secretion (E, I), and LDH release (F, J) in the SN were measured. Coomassie Blue staining was used as the supernatant loading control, while lamin B was used as the lysate loading control.


( A - C ) BMDMs were primed with LPS and then treated with LicoB ( 20 μΜ ) for 1 h , followed by stimulation with nigericin , ATP , poly ( I : C ) , or MSU . Pam3CSK4 - primed BMDMs were treated with LicoB ( 20 μM ) and then transfected with LPS . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , and ASC in the whole cell lysate ( WCL ) ; and activated caspase - 1 ( p20 ) and cleaved IL - 1β ( p17 ) in the culture supernatants ( SN ) of BMDMs . ( A ) . Caspase - 1 activity ( B ) and IL - 1β secretion ( C ) in the SN were measured .

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(A-C) BMDMs were primed with LPS and then treated with LicoB (20 μΜ) for 1 h, followed by stimulation with nigericin, ATP, poly (I:C), or MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then transfected with LPS. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs. (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured.


( D - F ) LPS - primed BMDMs were treated with LicoB ( 20 μM ) for 1 h and then stimulated with nigericin for 45 min , or poly ( dA : dT ) / Salmonella for 6 h . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , and ASC in the whole cell lysate ( WCL ) ; and activated caspase - 1 ( p20 ) and cleaved IL - 1β ( p17 ) in the culture supernatants ( SN ) of BMDMs ( D ) . Caspase - 1 activity ( E ) and IL - 1β secretion ( F ) in the SN were measured .

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(D-F) LPS-primed BMDMs were treated with LicoB (20 μM) for 1 h and then stimulated with nigericin for 45 min, or poly(dA:dT)/Salmonella for 6 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (D). Caspase-1 activity (E) and IL-1β secretion (F) in the SN were measured.


( A - C ) LPS - primed BMDMs were pre - treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h . Western blot analysis was used to detect cross - linked ASC in the Triton X - insoluble pellet , the arrow indicates ASC ( A ) . Caspase - 1 activity ( B ) and IL - 1β secretion ( C ) in the SN were measured again to verify NLRP3 activation .

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(A-C) LPS-primed BMDMs were pre-treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h. Western blot analysis was used to detect cross-linked ASC in the Triton X-insoluble pellet, the arrow indicates ASC (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured again to verify NLRP3 activation.


( E ) Western blot analysis of cross - linked ASC in the Triton X - insoluble pellet from LPS - primed BMDMs pre - treated with LicoB ( 20 μM ) or vehicle and then stimulated with nigericin , poly ( dA : dT ) , or Salmonella .

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(E) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, poly (dA:dT), or Salmonella.


( A ) Western blot analysis of protein in whole cell lysates ( WCL ) from BMDMs stimulated with LPS for 3 h and then treated with LicoB for 1 h ( LicoB after LPS ) , or BMDMs first treated with LicoB for 1 h and then stimulated with LPS for 3 h ( LicoB before LPS ) .

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(A) Western blot analysis of protein in whole cell lysates (WCL) from BMDMs stimulated with LPS for 3 h and then treated with LicoB for 1 h (LicoB after LPS), or BMDMs first treated with LicoB for 1 h and then stimulated with LPS for 3 h (LicoB before LPS).


( I ) Cell lysates of LPS - primed BMDMs treated with / without nigericin were incubated with sepharose or LicoB - sepharose . The pull - down samples and input were analysed using Western blot analysis . ( J ) Cell lysates of LPS - primed BMDMs were incubated with sepharose or LicoB - sepharose in the presence of different concentrations of free LicoB ( 0 . 5 mM and 1 mM ) . The pull - down samples and input were analysed using Western blot .

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(I) Cell lysates of LPS-primed BMDMs treated with/without nigericin were incubated with sepharose or LicoB-sepharose. The pull-down samples and input were analysed using Western blot analysis. (J) Cell lysates of LPS-primed BMDMs were incubated with sepharose or LicoB-sepharose in the presence of different concentrations of free LicoB (0.5 mM and 1 mM). The pull-down samples and input were analysed using Western blot.


( K ) HEK - 293T cells were transfected with Flag - NLRP3 or Flag - vector and then treated with LicoB ( 40 μM ) . Immunoprecipitation was performed with anti - DYKDDDDK ( Flag ) affinity gel agarose beads ; Western blot analysis has been shown .

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(K) HEK-293T cells were transfected with Flag-NLRP3 or Flag-vector and then treated with LicoB (40 μM). Immunoprecipitation was performed with anti-DYKDDDDK (Flag) affinity gel agarose beads; Western blot analysis has been shown.


( A ) Survival of WT mice pre - treated with vehicle , LicoB ( 20 mg / kg ) , LicoB ( 40 mg / kg ) , MCC950 ( 40 mg / kg ) , or LicoB ( 40 mg / kg ) + MCC950 ( 40 mg / kg ) , followed by i . p . injection with LPS ( 20 mg / kg ) ; mice survival was monitored for 72 h ( n = 10 mice per group ) .

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(A) Survival of WT mice pre-treated with vehicle, LicoB (20 mg/kg), LicoB (40 mg/kg), MCC950 (40 mg/kg), or LicoB (40 mg/kg) + MCC950 (40 mg/kg), followed by i.p. injection with LPS (20 mg/kg); mice survival was monitored for 72 h (n=10 mice per group).


C57BL / 6 mice received vehicle or LicoB ( 40 mg / kg ) for 17 consecutive doses , once every two days for 34 days . Following that treatment , the serum levels of ALT ( H ) ( n = 8 mice per group ) .

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

C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of ALT (H) (n=8 mice per group).


C57BL / 6 mice received vehicle or LicoB ( 40 mg / kg ) for 17 consecutive doses , once every two days for 34 days . Following that treatment , the serum levels of AST ( I ) , and CRE ( J ) were measured , ( n = 8 mice per group ) .

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 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

C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of AST (I), and CRE (J) were measured, (n=8 mice per group).


( A - D ) Mice were pre - treated with LicoB or MCC950 for 1 h , then i . p . injected with MSU ( 50 mg / kg ) and treated for 6 h ( n = 6 mice per group ) . The levels of IL - 1β in the peritoneal lavage fluid ( A ) and serum ( B ) were measured using ELISA . Quantification of peritoneal exudate cells ( PECs ) ( C ) and neutrophils ( Ly6G and CD11b ) ( D ) using flow cytometry .

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

(A-D) Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with MSU (50 mg/kg) and treated for 6 h (n=6 mice per group). The levels of IL-1β in the peritoneal lavage fluid (A) and serum (B) were measured using ELISA. Quantification of peritoneal exudate cells (PECs) (C) and neutrophils (Ly6G and CD11b) (D) using flow cytometry.


Under the same growth conditions ( permitted water , free food , and a 12 h / 12 h dark / light cycle at 20 ± 2 ° C ) , eight - week - old male C57BL / 6 mice were continuously fed with methionine - and choline - supplemented ( MCS ) or methionine - and choline - deficient ( MCD ) diets for 6 weeks , and at the same time , gavaged with LicoB , MCC950 , or a combination of LicoB and MCC950 ( n = 8 mice per group ( B , C ( A ) Representative liver section images stained with H & E , Sirius Red , and Masson ' s trichrome stains . Scale bar : 0 . 5 cm ( top row ) and 200 μm ( 3 bottom ows ) . ( B , C ) The serum levels of ALT and AST ( n = 8 mice per group ) were measured using ELISA .

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

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950, or a combination of LicoB and MCC950 (n=8 mice per group (B, C (A) Representative liver section images stained with H&E, Sirius Red, and Masson's trichrome stains. Scale bar: 0.5 cm (top row) and 200 μm (3 bottom ows). (B, C) The serum levels of ALT and AST (n=8 mice per group) were measured using ELISA.