® Retinal arteriolar αSMA coverage in vehicle or AngII - treated mice . Green arrowheads denote stenosis , blue arrowheads label αSMA - negative gap . ( S ) αSMA - negative gaps in arterioles per retina ( Jag1 + / + ctrl n = 4 , Jag1 + / + AngII n = 4 , Jag1Ndr / Ndr ctrl n = 3 - 5 , Jag1Ndr / Ndr AngII n = 5 , two - way ANOVA , with Sidak ' s multiple comparison . Interaction P = 0 . 1054 , treatment P = 0 . 0815 ; Genotype P = 0 . 0747 . Ší ' ák ' s multiple comparisons test : AngII treated Jag1 + / + vs AngII treated Jag1Ndr / Ndr * P = 0 . 0315 ) . ) .

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 B-CONTROLLED_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 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

® Retinal arteriolar αSMA coverage in vehicle or AngII-treated mice. Green arrowheads denote stenosis, blue arrowheads label αSMA-negative gap. (S) αSMA-negative gaps in arterioles per retina (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=3-5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P = 0.1054, treatment P=0.0815; Genotype P=0.0747. Ší'ák's multiple comparisons test: AngII treated Jag1+/+ vs AngII treated Jag1Ndr/Ndr *P= 0.0315).).


A - D ( A ) P30 retina , three CD31 + capillary layers pseudo - colored for superficial , intermediate and deep layers . Scale bar 50 µm . ( B ) P30 SCP branching point number ( n = 6 - 8 per group , unpaired t test , ns , P = 0 . 5346 ) . ( C ) P30 ICP vascular length ( n = 6 per group , unpaired t test , * P = 0 . 0124 ) . ( D ) P30 ICP branching point number ( n = 6 per group , unpaired t test , * * P = 0 . 0057 ) . E - H ( E ) 3 - 6 months old retina , three pseudo - colored CD31 + capillary layers . Scale bar 50 µm . ( F ) Adult SCP branching point number ( n = 6 per group , unpaired t test , ns , P = 0 . 5365 ) . ( G ) Adult ICP vascular length ( n = 6 - 7 per group , unpaired t test , * * P = 0 . 0078 ) . ( H ) Adult ICP branching point number ( n = 6 - 7 per group , unpaired t test , * * P = 0 .

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

A-D (A) P30 retina, three CD31+ capillary layers pseudo-colored for superficial, intermediate and deep layers. Scale bar 50 µm. (B) P30 SCP branching point number (n=6-8 per group, unpaired t test, ns, P=0.5346). (C) P30 ICP vascular length (n=6 per group, unpaired t test, *P=0.0124). (D) P30 ICP branching point number (n=6 per group, unpaired t test, **P=0.0057). E-H (E) 3-6 months old retina, three pseudo-colored CD31+ capillary layers. Scale bar 50 µm. (F) Adult SCP branching point number (n=6 per group, unpaired t test, ns, P=0.5365). (G) Adult ICP vascular length (n=6-7 per group, unpaired t test, **P=0.0078). (H) Adult ICP branching point number (n=6-7 per group, unpaired t test, **P=0.001


I - J ( I ) ICP COLIV and CD31 ( arrowhead labels COLIV + CD31 - capillary ) . Scale bar 50 µm . ( J ) Empty COLIV sleeve length in ICP per field ( n = 6 per group , unpaired t test , ns , P = 0 . 1583 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (I) ICP COLIV and CD31 (arrowhead labels COLIV+ CD31- capillary). Scale bar 50 µm. (J) Empty COLIV sleeve length in ICP per field (n=6 per group, unpaired t test, ns, P=0.1583).


( K ) Retinal capillary plexuses in 3 - 6 months old ( top panels ) and one - year - old mice ( bottom panels ) . White arrowheads label discontinuous ICP . Blue arrowhead labels SCP , green ICP and magenta DCP .

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

(K) Retinal capillary plexuses in 3-6 months old (top panels) and one-year-old mice (bottom panels). White arrowheads label discontinuous ICP. Blue arrowhead labels SCP, green ICP and magenta DCP.


( L - M ) Vertical sprout number between SCP ( S ) and ICP ( I ) and ICP ( I ) and DCP ( D ) in ( L ) adult ( n = 6 per group , two - way ANOVA , genotype * * * P = 0 . 0004 , Sidak ' s multiple comparisons test S - I ns , P = 0 . 0592 , I - D * * * * P < 0 . 0001 ) and ( M ) one - year - old mice ( n = 8 per group , two - way ANOVA , genotype ns , P = 0 . 4507 , Sidak ' s multiple comparisons test S - I ns , P = 0 . 9483 , I - D ns , P = 0 . 6046 ) .

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

(L-M) Vertical sprout number between SCP (S) and ICP (I) and ICP (I) and DCP (D) in (L) adult (n=6 per group, two-way ANOVA, genotype ***P=0.0004, Sidak's multiple comparisons test S-I ns, P=0.0592, I-D ****P<0.0001) and (M) one-year-old mice (n=8 per group, two-way ANOVA, genotype ns, P=0.4507, Sidak's multiple comparisons test S-I ns, P=0.9483, I-D ns, P=0.6046).


N - P ( N ) P10 and ( O ) P40 neurofilament ( NF ) and CD31 staining . Scale bar 50 µm . ( P ) % area positive for NF in P10 and P40 mice ( n = 6 per group , two - way ANOVA : Interaction * * * P = 0 . 0001 ; Age * P = 0 . 0428 ; Genotype * * * P = 0 . 0004 . Sidak ' s multiple comparisons test : P10 Jag1 + / + vs P10 Jag1Ndr / Ndr P = 0 . 9246 ; P40 Jag1 + / + vs P40 Jag1Ndr / Ndr * * * * P = < 0 . 0001 ) .

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

N-P (N) P10 and (O) P40 neurofilament (NF) and CD31 staining. Scale bar 50 µm. (P) % area positive for NF in P10 and P40 mice (n=6 per group, two-way ANOVA: Interaction ***P= 0.0001; Age *P=0.0428; Genotype ***P=0.0004. Sidak's multiple comparisons test: P10 Jag1+/+ vs P10 Jag1Ndr/Ndr P= 0.9246; P40 Jag1+/+ vs P40 Jag1Ndr/Ndr ****P= <0.0001).


A Representative retinographs from a healthy adult , patient with CADASIL , healthy pediatric individual , patient with ALGS and patient with BA . Boxed regions and black arrowheads magnify arteriovenous crossing .

O O O O O O O O O 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 Representative retinographs from a healthy adult, patient with CADASIL, healthy pediatric individual, patient with ALGS and patient with BA. Boxed regions and black arrowheads magnify arteriovenous crossing.


B - F ( B ) Arteriovenous crossings per retina ( n = 14 per group , unpaired t test , ns , P = 0 . 3854 . ( C ) Major arterioles ( n = 14 per group , unpaired t test , ns , P = 0 . 2902 ) and ( D ) venules ( n = 14 per group , unpaired t test , ns , P = 0 . 1536 ) . ( E ) Arterial ( n = 14 per group , unpaired t test , ns , P = 0 . 0759 ) and ( F ) venous tortuosity ( n = 14 per group , unpaired t test , ns , P = 0 . 1050 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F (B) Arteriovenous crossings per retina (n=14 per group, unpaired t test, ns, P=0.3854. (C) Major arterioles (n=14 per group, unpaired t test, ns, P=0.2902) and (D) venules (n=14 per group, unpaired t test, ns, P=0.1536). (E) Arterial (n=14 per group, unpaired t test, ns, P=0.0759) and (F) venous tortuosity (n=14 per group, unpaired t test, ns, P=0.1050).


G - K ( G ) Arteriovenous crossings per retina ( CTRL n = 5 , ALGS n = 3 , BA n = 4 , one - way ANOVA , ns , P = 0 . 0608 , Sidak ' s multiple comparisons ALGS vs . CTRL * P = 0 . 0492 , ALGS vs . BA ns , P = 0 . 0999 ) . ( H ) Major arterioles ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * P = 0 . 0026 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0039 , ALGS vs . BA ns , P = 0 . 9799 ) and ( I ) venules ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * * P = 0 . 0008 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0011 , ALGS vs . BA ns , P > 0 . 9999 ) . ( J ) Arterial ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * P = 0 . 0486 , Sidak ' s multiple comparisons ALGS vs . CTRL * P = 0 . 0392 , ALGS vs . BA ns , P = 0 . 1204 ) and ( K ) venous tortuosity ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * P = 0 . 009 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0076 , ALGS vs . BA * P = 0 . 0327 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K (G) Arteriovenous crossings per retina (CTRL n=5, ALGS n=3, BA n=4, one-way ANOVA, ns, P=0.0608, Sidak's multiple comparisons ALGS vs. CTRL *P=0.0492, ALGS vs. BA ns, P=0.0999). (H) Major arterioles (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, **P=0.0026, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0039, ALGS vs. BA ns, P=0.9799) and (I) venules (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, ***P=0.0008, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0011, ALGS vs. BA ns, P>0.9999). (J) Arterial (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, *P=0.0486, Sidak's multiple comparisons ALGS vs. CTRL *P=0.0392, ALGS vs. BA ns, P=0.1204) and (K) venous tortuosity (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, **P=0.009, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0076, ALGS vs. BA *P=0.0327).


( A ) Upper panels - Immunofluorescence imaging of PRR7 with Shank ( synaptic ) and MAP2 ( dendritic ) markers in DIV21 hippocampal neurons . Lower panels - Single plane apotome images of nuclei ( DAPI ) show PRR7 can be present ( arrow ) or absent ( arrowhead ) from nuclei .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upper panels- Immunofluorescence imaging of PRR7 with Shank (synaptic) and MAP2 (dendritic) markers in DIV21 hippocampal neurons. Lower panels- Single plane apotome images of nuclei (DAPI) show PRR7 can be present (arrow) or absent (arrowhead) from nuclei.


( B ) Synaptic and dendritic distribution of PRR7 and pre ( Bassoon ) and postsynaptic ( PSD95 ) markers ( arrowheads - triple colocalization ) .

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(B) Synaptic and dendritic distribution of PRR7 and pre (Bassoon) and postsynaptic (PSD95) markers (arrowheads-triple colocalization).


( C ) PRR7 - GFP expressed in 17DIV hippocampal neurons is present in dendritic spines ( arrows ) . Scale bar is 15 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

(C) PRR7-GFP expressed in 17DIV hippocampal neurons is present in dendritic spines (arrows). Scale bar is 15 µm.


( D ) PRR7 distribution across subcellular fractions ( TOT = whole lysate , Syn = synaptosomes , Nuc = nuclei ) isolated from rat hippocampal tissue . Fib ( Fibrillarin ) , SNAP25 and PSD95 were used as nuclear , presynaptic and postsynaptic markers , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O 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) PRR7 distribution across subcellular fractions (TOT = whole lysate, Syn = synaptosomes, Nuc = nuclei) isolated from rat hippocampal tissue. Fib (Fibrillarin), SNAP25 and PSD95 were used as nuclear, presynaptic and postsynaptic markers, respectively.


( A ) Immunocytochemistry analyses show that glutamate stimulation ( Glut , 100 µM 5 min + 25 min washout ) increased PRR7 in the nucleus . Cycloheximide ( 100 µM ) added 30 min prior to glutamate stimulation did not affect this increase ( Cont = 100 ± 12 . 5 % , Glut = 202 . 1 ± 15 . 2 % ; + cycloheximide Cont = 104 . 9 ± 3 . 6 , Glut = 231 . 4 ± 10 . 9 ) ( n = ~ 30 - 50 cells ) .

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

(A) Immunocytochemistry analyses show that glutamate stimulation (Glut, 100 µM 5 min + 25 min washout) increased PRR7 in the nucleus. Cycloheximide (100 µM) added 30 min prior to glutamate stimulation did not affect this increase (Cont=100±12.5%, Glut=202.1±15.2%; +cycloheximide Cont=104.9±3.6, Glut=231.4±10.9) (n=~30-50 cells).


( B ) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons . Epoxomicin ( 50 nM ) added 20 min prior to stimulation did not affect this decrease ( Cont = 100 ± 3 . 6 % , Glut = 32 . 3 ± 1 . 3 % ; + epoxomicin Cont = 99 . 8 ± 4 . 6 , Glut = 42 . 9 ± 1 . 1 ) . ( n = 14 - 17 dendrites , > 1200 puncta for each group ) . Statistical analyses for ( A ) and ( B ) were performed using the Mann Whitney test .

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(B) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont=100±3.6%, Glut=32.3±1.3%; +epoxomicin Cont=99.8±4.6, Glut=42.9±1.1). (n=14-17 dendrites, >1200 puncta for each group). Statistical analyses for (A) and (B) were performed using the Mann Whitney test.


( C ) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons . Treatments ( APV [ 50 µM , APV - ( 2R ) - amino - 5 - phosphonovaleric acid ] , CNQX [ 40 µM , 6 - cyano - 7 nitroquinoxaline - 2 , 3 - dione ] , TTX [ 1 µM , tetrodotoxin ] , NMDA [ 50 µM , N - Methyl - D - Aspartate ] ) as indicated . Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 ( 108 . 1 ± 19 % ) . Statistical significance was determined using one - way ANOVA in conjugation with Sidak post - hoc test .

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

(C) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons. Treatments (APV [50 µM, APV-(2R)-amino-5-phosphonovaleric acid], CNQX [40 µM, 6-cyano-7 nitroquinoxaline-2,3-dione], TTX [1 µM, tetrodotoxin], NMDA [50 µM, N-Methyl-D-Aspartate]) as indicated. Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 (108.1 ± 19%). Statistical significance was determined using one-way ANOVA in conjugation with Sidak post-hoc test.


( D ) PRR7 - GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment . Depicted are representative averaged confocal images ( 30x30 µm2 ) from optical sections ( 300 nm step size ) of PRR7 - GFP expressing neurons ( DIV17 ) before and after bleaching . Prior to stimulation there was substantial PRR7 - GFP in the nucleus due to basal neuronal activity ( upper panel ) . This was reduced by incubation overnight with MK801 ( 10 µM ) and TTX ( 1 µM ) ( lower panel ) . NMDA ( 50 µM ) stimulation , but not MK - 801 , led to recovery of nuclear PRR7 - GFP fluorescence following bleaching of nuclear ROIs at t0 ` ( blue arrow in E ) . ( E ) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0 ` and corrected for bleaching . No significant differences in PRR7 - GFPFRAP were found in control ( MK801 ) conditions . GFP intensities are displayed via look - up - table ( LUT ) . Data are represented as mean ± SEM ; n = 9 cells ( NMDA ) and n = 5 cells ( MK801 ) . Analysis was done using one - way ANOVA using Bonferroni post - hoc correction .

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(D) PRR7-GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment. Depicted are representative averaged confocal images (30x30 µm2) from optical sections (300 nm step size) of PRR7-GFP expressing neurons (DIV17) before and after bleaching. Prior to stimulation there was substantial PRR7-GFP in the nucleus due to basal neuronal activity (upper panel). This was reduced by incubation overnight with MK801 (10 µM) and TTX (1 µM) (lower panel). NMDA (50 µM) stimulation, but not MK-801, led to recovery of nuclear PRR7-GFP fluorescence following bleaching of nuclear ROIs at t0`(blue arrow in E).(E) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0` and corrected for bleaching. No significant differences in PRR7-GFPFRAP were found in control (MK801) conditions. GFP intensities are displayed via look-up-table (LUT). Data are represented as mean±SEM; n=9 cells (NMDA) and n=5 cells (MK801). Analysis was done using one-way ANOVA using Bonferroni post-hoc correction.


( F ) PRR7 - mEos translocates from distal dendrites to the nucleus upon NMDA treatment . ( Left panels ) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7 - mEos before and after photoconversion of distal dendrites ( ROIs ) through the image z - stack from from green ( 488nm ) to red ( 568nm ) using a UV laser ( 405 nm ) . ( Right panels ) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7 - mEos ( 568nm ) in the nucleus following NMDA stimulation ( 50 µM - 30min ) . ( G ) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7 - mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment . ( Average increase ; NMDA = 617 . 8 ± 47 . 7 % , n = 11 cells , p < 0 . 0005 ; MK801 = 206 . 1 ± 13 . 6 % , n = 5 cells , n . s . ) . Statistical significance was determined by two - way ANOVA using Sidak ' s correction for multiple comparisons .

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(F) PRR7-mEos translocates from distal dendrites to the nucleus upon NMDA treatment. (Left panels) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7-mEos before and after photoconversion of distal dendrites (ROIs) through the image z-stack from from green (488nm) to red (568nm) using a UV laser (405 nm). (Right panels) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7-mEos (568nm) in the nucleus following NMDA stimulation (50 µM- 30min).(G) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7-mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment. (Average increase; NMDA=617.8±47.7%, n=11 cells, p<0.0005; MK801=206.1±13.6%, n=5 cells, n.s.). Statistical significance was determined by two-way ANOVA using Sidak's correction for multiple comparisons.


( A ) Co - Immunoprecipitations ( Co - IPs ) of PRR7 with GluN1 , PSD95 and GluN2B from rat brain lysates . FT = flowthrough , IgG = normal IgGs .

(A) Co-Immunoprecipitations (Co-IPs) of PRR7 with GluN1, PSD95 and GluN2B from rat brain lysates. FT = flowthrough, IgG = normal IgGs.


( B ) Co - IPs from HEK293 cells of c - terminal myc - tagged PRR7 with GluN1 in the absence of other NMDAR subunits or PSD95 .

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(B) Co-IPs from HEK293 cells of c-terminal myc-tagged PRR7 with GluN1 in the absence of other NMDAR subunits or PSD95.


( C ) Co - IPs from HEK293 cells transfected with tagged NMDA or AMPA receptor subunits and PRR7 ( myc - tagged GluN2B and GluA2 and flag - tagged PRR7 ) .

(C) Co-IPs from HEK293 cells transfected with tagged NMDA or AMPA receptor subunits and PRR7 (myc-tagged GluN2B and GluA2 and flag-tagged PRR7).


( D ) ( top ) Schematics of the different PRR7 constructs used for co - IP ( FL = full length ) . ( bottom ) PRR7 deletion constructs and NMDAR subunits were expressed in HEK293 cells and Co - IPs performed as indicated .

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(D) (top) Schematics of the different PRR7 constructs used for co-IP (FL= full length). (bottom) PRR7 deletion constructs and NMDAR subunits were expressed in HEK293 cells and Co-IPs performed as indicated.


( E ) Co - IPs of PRR7 with GluN1 from high - density primary neuronal cultures following treatment with NMDA ( 100µM , 30 min , representative of n = 4 ) .

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(E) Co-IPs of PRR7 with GluN1 from high-density primary neuronal cultures following treatment with NMDA (100µM, 30 min, representative of n=4).


( A ) Western blots of lysates of primary neurons transduced with control lentiviruses ( NT ) or lentiviruses to knockdown ( KD ) or overexpress ( OE ) PRR7 . PRR7 knockdown reduced ( 75 . 6 ± 6 . 9 % ) while PRR7 overexpression ( OE ) increased ( 157 . 6 ± 22 . 8 % ) c - Jun levels compared to controls . No change was detected in c - Fos levels . One - way ANOVA test was used to analyze results with Dunett ' s post hoc test ( n = 16 gels for c - Jun and 4 gels for c - Fos ) .

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(A) Western blots of lysates of primary neurons transduced with control lentiviruses (NT) or lentiviruses to knockdown (KD) or overexpress (OE) PRR7. PRR7 knockdown reduced (75.6 ± 6.9%) while PRR7 overexpression (OE) increased (157.6 ± 22.8%) c-Jun levels compared to controls. No change was detected in c-Fos levels. One-way ANOVA test was used to analyze results with Dunett's post hoc test (n=16 gels for c-Jun and 4 gels for c-Fos).


( B ) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c - Jun protein levels were measured by Western blotting . PRR7 expression significantly stabilized c - Jun levels at 120 min ( 110 . 0 ± 7 . 5 % vs control 68 . 7 ± 7 . 9 % ) and 240 minutes ( 83 . 6 ± 5 . 2 % vs control 43 . 7 ± 3 . 4 % ) . Values for c - Jun were normalized to their respective 0 min time point ( n = 4 gels for each group performed blind ) . T - tests between similar time points were used to analyze results .

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(B) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c-Jun protein levels were measured by Western blotting. PRR7 expression significantly stabilized c-Jun levels at 120 min (110.0 ± 7.5% vs control 68.7 ± 7.9%) and 240 minutes (83.6 ± 5.2% vs control 43.7 ± 3.4%). Values for c-Jun were normalized to their respective 0 min time point (n=4 gels for each group performed blind). T-tests between similar time points were used to analyze results.


( C ) PRR7 , FBW7 and / or a control protein ( PSD95 ) were transfected into HEK293 cells and c - Jun ubiquitination was measured by immunoprecipitating c - Jun and blotting for ubiquitin . Ubiquitination of c - Jun in the presence of FBW7 ( 178 . 3 ± 26 . 9 % ) was significantly reduced in the presence of PRR7 ( 103 . 7 ± 20 . 6 % ) but not a control protein ( PSD95 ) ( n = 5 gels for all groups ) .

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(C) PRR7, FBW7 and/or a control protein (PSD95) were transfected into HEK293 cells and c-Jun ubiquitination was measured by immunoprecipitating c-Jun and blotting for ubiquitin. Ubiquitination of c-Jun in the presence of FBW7 (178.3 ± 26.9%) was significantly reduced in the presence of PRR7 (103.7 ± 20.6%) but not a control protein (PSD95) (n=5 gels for all groups).


( D ) Western blots of c - Junubiquitination in hippocampal primary neuronal cultures . PRR7 knockdown ( KD ) elevated ( 149 ± 10 . 62 % , n = 6 gels ) c - Junubiquitination level in comparison to control neurons ( NT ) . Statistical significance was determined by t - test .

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(D) Western blots of c-Junubiquitination in hippocampal primary neuronal cultures. PRR7 knockdown (KD) elevated (149±10.62%, n=6 gels) c-Junubiquitination level in comparison to control neurons (NT). Statistical significance was determined by t-test.


( E ) Western blots showing knockdown ( KD ) or overexpression ( OE ) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc .

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(E) Western blots showing knockdown (KD) or overexpression (OE) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc.


( A ) Co - IPs of PRR7 with c - Jun from lysates of HEK293 cells overexpressing PRR7 .

(A) Co-IPs of PRR7 with c-Jun from lysates of HEK293 cells overexpressing PRR7.


( B ) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c - Jun .

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(B) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c-Jun.


( C ) Co - IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7 .

(C) Co-IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7.


( D ) Pulldown assays in vitro using purified PRR7 and FBW7 .

(D) Pulldown assays in vitro using purified PRR7 and FBW7.


( E ) Co - IPs of endogenous PRR7 and FBW7 from lysates of ratbrain .

(E) Co-IPs of endogenous PRR7 and FBW7 from lysates of ratbrain.


( F ) Co - IPs of PRR7 with c - Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7 . FBW7 expression increased the amount of c - Jun pulled down with PRR7 ( n = 4 gels for each panel , representative blots ) . Lower bar graphs - quantitation for Western blots show c - Jun / FBW7 binding is 41 . 4 ± 13 . 9 % greater in the presence of PRR7 ( normalized for FBW7 expression ) . Similarly , c - Jun / PRR7 binding is 97 . 4 ± 38 . 1 % greater in the presence of FBW7 ( normalized for PRR7 expression ) . Statistical significance was calculated using unpaired t - tests .

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(F) Co-IPs of PRR7 with c-Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. FBW7 expression increased the amount of c-Jun pulled down with PRR7 (n=4 gels for each panel, representative blots). Lower bar graphs- quantitation for Western blots show c-Jun/FBW7 binding is 41.4±13.9% greater in the presence of PRR7 (normalized for FBW7 expression). Similarly, c-Jun/PRR7 binding is 97.4±38.1% greater in the presence of FBW7 (normalized for PRR7 expression). Statistical significance was calculated using unpaired t-tests.


( A ) Western blots of phospho - c - Jun in primary neuronal cultures . Quantification of phospho - c - Jun represents a ratio of phospho - c - Jun to total c - Jun and all ratios were normalized to NT . PRR7 knockdown ( KD ) decreased , while PRR7 overexpression ( OE ) increased phospho - c - Jun levels ( black bars ) . Glutamate stimulation ( gray bars ) increased phospho - c - Jun / c - Jun ratio in NT but not in KD or OE neurons ( n = 7 gels for all groups ) . Significance was determined using two - way ANOVA with Sidak post - hoc test .

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(A) Western blots of phospho-c-Jun in primary neuronal cultures. Quantification of phospho-c-Jun represents a ratio of phospho-c-Jun to total c-Jun and all ratios were normalized to NT. PRR7 knockdown (KD) decreased, while PRR7 overexpression (OE) increased phospho-c-Jun levels (black bars). Glutamate stimulation (gray bars) increased phospho-c-Jun/c-Jun ratio in NT but not in KD or OE neurons (n=7 gels for all groups). Significance was determined using two-way ANOVA with Sidak post-hoc test.


( B ) AP - 1 driven luciferase luminescence assays . PRR7 expression in HEK293 cells increased AP - 1 activity ( 190 . 6 ± 13 . 9 % , n = 12 ; RLU = relative light units ) . Statistical significance was determined using t - tests .

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(B) AP-1 driven luciferase luminescence assays. PRR7 expression in HEK293 cells increased AP-1 activity (190.6 ± 13.9%, n=12; RLU= relative light units). Statistical significance was determined using t-tests.


( D ) Quantitation of cell death using 30 minutes of PI in - situ staining following 48h of WT - PRR7 - GFP or mNLS - PRR7 - GFP overexpression in primary hippocampal neuronal cultures ( n = 24 cells per group ) . Images represent a max projection of five z - stack sections across the cell body .

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(D) Quantitation of cell death using 30 minutes of PI in-situ staining following 48h of WT-PRR7-GFP or mNLS-PRR7-GFP overexpression in primary hippocampal neuronal cultures (n=24 cells per group). Images represent a max projection of five z-stack sections across the cell body.


( E ) NMDA treatment ( 25 min , 100 µM followed by 14h chase ) caused 83 . 6 ± 3 . 5 % cell death in control neurons compared to 60 . 3 ± 4 . 6 % cell death in neurons with PRR7 expression knocked down . A c - Jun inhibitory peptide reduced NMDA - mediated cell death to 66 . 2 ± 6 . 0 % in control neurons and to 55 . 6 ± 5 . 3 % in neurons knocked down for PRR7 , but this difference was not statistically significant ( n = 5 independent experiments ) . Two - way ANOVA was used in conjugation with Tukey post - hoc test . ( Left ) Representative images showing transduced neurons ( green - GFP ) and PI staining ( red ) .

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(E) NMDA treatment (25 min, 100 µM followed by 14h chase) caused 83.6±3.5% cell death in control neurons compared to 60.3±4.6% cell death in neurons with PRR7 expression knocked down. A c-Jun inhibitory peptide reduced NMDA-mediated cell death to 66.2±6.0% in control neurons and to 55.6±5.3% in neurons knocked down for PRR7, but this difference was not statistically significant (n=5 independent experiments). Two-way ANOVA was used in conjugation with Tukey post-hoc test. (Left) Representative images showing transduced neurons (green- GFP) and PI staining (red).


( F ) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c - Jun or a dominant negative c - Jun4A ( 4A ) . Overexpression of WT c - Jun caused extensive neuronal cell death ( 84 . 8 ± 2 . 4 % ) that was reduced in neurons knocked down for PRR7 ( 43 . 8 ± 9 . 2 ) . Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity ( 50 . 8 ± 7 . 9 % ) but was not additive with PRR7 knockdown ( 42 . 3 ± 8 . 1 % ) ( n = 3 independent experiments ) . Significance was determined using two - way ANOVA in conjugation with Tukey post - hoc test . Images are representative 10X widefield micrographs showing transduced neurons ( green - GFP ) and PI staining ( red ) . Data is presented as mean ± sem . All % values represent comparisons to control .

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(F) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c-Jun or a dominant negative c-Jun4A (4A). Overexpression of WT c-Jun caused extensive neuronal cell death (84.8±2.4%) that was reduced in neurons knocked down for PRR7 (43.8±9.2). Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity (50.8±7.9%) but was not additive with PRR7 knockdown (42.3±8.1%) (n=3 independent experiments). Significance was determined using two-way ANOVA in conjugation with Tukey post-hoc test. Images are representative 10X widefield micrographs showing transduced neurons (green- GFP) and PI staining (red). Data is presented as mean±sem. All % values represent comparisons to control.


C - D . ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination ( B ) , whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination ( C ) . Protein complexes were assembled in vitro on ice in the presence of dsDNA ( 32 bp ) and their in vitro ubiquitination at 30o C was subsequently monitored in a time - course : at indicative time - points , aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies ( Top ) . For each protein complex , data - points from three replicate experiments ( three technical replicates ) were used in fitting to a one - phase association model ( Bottom ) .

C-D. ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination (B), whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination (C). Protein complexes were assembled in vitro on ice in the presence of dsDNA (32 bp) and their in vitro ubiquitination at 30o C was subsequently monitored in a time-course: at indicative time-points, aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies (Top). For each protein complex, data-points from three replicate experiments (three technical replicates) were used in fitting to a one-phase association model (Bottom).


A . USP1 - UAF1 - mediated deubiquitination of V5 - FANCI and FANCD2 was assessed in the absence or presence of DNA ( 51 bp ) , when ubiquitinated versions of these proteins were in isolation ( IUb and D2Ub ) or within singly / doubly ubiquitinated ID2 complexes ( IUbD2 , IUbD2Ub and ID2Ub ) . At indicated time - points , aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies .

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A. USP1-UAF1-mediated deubiquitination of V5-FANCI and FANCD2 was assessed in the absence or presence of DNA (51 bp), when ubiquitinated versions of these proteins were in isolation (IUb and D2Ub) or within singly/doubly ubiquitinated ID2 complexes (IUbD2, IUbD2Ub and ID2Ub). At indicated time-points, aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies.


C . Deletion of N - terminus ( ∆ N ) of USP1 ( residues 1 - 54 ) results in greatly reduced FANCD2 deubiquitination . Assays were performed in triplicate ( technical replicates ) as in A , but all reactions contained DNA . Left : Western blotting of reaction products at zero and 30 minutes using FANCD2 and V5 antibodies . Right : Residual FANCI and FANCD2 ubiquitination following USP1 - UAF1 treatment for 30 min . Replicate residual ubiquitination values and statistically significant changes ( one - way ANOVA test with Bonferroni correction ) are shown . * * * p < 0 . 001 .

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C. Deletion of N-terminus (∆N) of USP1 (residues 1-54) results in greatly reduced FANCD2 deubiquitination. Assays were performed in triplicate (technical replicates) as in A, but all reactions contained DNA. Left: Western blotting of reaction products at zero and 30 minutes using FANCD2 and V5 antibodies. Right: Residual FANCI and FANCD2 ubiquitination following USP1-UAF1 treatment for 30 min. Replicate residual ubiquitination values and statistically significant changes (one-way ANOVA test with Bonferroni correction) are shown. ***p<0.001.


Alanine mutagenesis of key FANCD2 ' s residues ( H209 , V243 and P244 ) participating in the extended FANCD2 - ubiquitin interface ( IUbD2 - DNA and IUbD2Ub - DNA structures ) , results in enhanced FANCI deubiquitination by USP1 - UAF1 . C . FANCI deubiquitination by USP1 - UAF1 ( incubation with 50 nM USP1 - UAF1 for 30 minutes at room temperature ) in the presence of DNA and , in the absence or presence of wild - type ( D2WT ) or mutant ( D2H209A , VP243AA ) FANCD2 . Replicate residual ubiquitination values ( three technical replicates ) and statistically significant changes ( t - test and one - way ANOVA test with Bonferroni correction ) are shown .

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Alanine mutagenesis of key FANCD2's residues (H209, V243 and P244) participating in the extended FANCD2-ubiquitin interface (IUbD2-DNA and IUbD2Ub-DNA structures), results in enhanced FANCI deubiquitination by USP1-UAF1. C. FANCI deubiquitination by USP1-UAF1 (incubation with 50 nM USP1-UAF1 for 30 minutes at room temperature) in the presence of DNA and, in the absence or presence of wild-type (D2WT) or mutant (D2H209A, VP243AA) FANCD2. Replicate residual ubiquitination values (three technical replicates) and statistically significant changes (t-test and one-way ANOVA test with Bonferroni correction) are shown.


D . FANCI was ubiquitinated with DyLight - 680 labelled ubiquitin ( IUb - DL680 ) and its deubiquitination by USP1 - UAF1 ( incubation with 100 nM USP1 - UAF1 for 20 minutes at room temperature ) , was assessed at increasing concentrations ( 20 , 200 or 200 nM ) of , either wild - type ( D2WT ) , or mutant ( D2H209A , VP243AA ) FANCD2 . Experiment was repeated three times ( technical replicates ) , and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb - DL680 at 20 nM FANCD2 ( average from six values , for both D2WT and D2H209A , VP243AA ) . Mean ( with range ) fold - increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration ( t - test and two - way ANOVA test with Bonferroni correction ) are shown .

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D. FANCI was ubiquitinated with DyLight-680 labelled ubiquitin (IUb-DL680) and its deubiquitination by USP1-UAF1 (incubation with 100 nM USP1-UAF1 for 20 minutes at room temperature), was assessed at increasing concentrations (20, 200 or 200 nM) of, either wild-type (D2WT), or mutant (D2H209A, VP243AA) FANCD2. Experiment was repeated three times (technical replicates), and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb-DL680 at 20 nM FANCD2 (average from six values, for both D2WT and D2H209A, VP243AA). Mean (with range) fold-increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration (t-test and two-way ANOVA test with Bonferroni correction) are shown.


( B ) Wild type mice received strain GFP - K79 via the tail vein and 1 hr later , animals were perfused and the meninges , cortex and choroid plexus were obtained for demonstration of intravenously injected bacteria . The arrows represent bacteria co - localized with capillaries ( e . g . , within capillaries ) and arrowheads represent bacteria outside the capillaries ( e . g . , exited from the capillaries ) . A few bacteria were successfully passed through the meningeal and cortex capillaries , but no bacteria were demonstrated in the choroid plexus at this time .

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(B) Wild type mice received strain GFP-K79 via the tail vein and 1 hr later, animals were perfused and the meninges, cortex and choroid plexus were obtained for demonstration of intravenously injected bacteria. The arrows represent bacteria co-localized with capillaries (e.g., within capillaries) and arrowheads represent bacteria outside the capillaries (e.g., exited from the capillaries). A few bacteria were successfully passed through the meningeal and cortex capillaries, but no bacteria were demonstrated in the choroid plexus at this time.


( C ) Sulfo - NHS - biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood - brain barrier permeability , i . e . , extravasation is indicative of leakage from the intravascular lumen . Sulfo - NHS - Biotin was confined to the capillaries of the meninges and cortex ( as shown with arrows ) , and there was no evidence of extravasation . Scale bar = 100 μm .

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(C) Sulfo-NHS-biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood-brain barrier permeability, i.e., extravasation is indicative of leakage from the intravascular lumen. Sulfo-NHS-Biotin was confined to the capillaries of the meninges and cortex (as shown with arrows), and there was no evidence of extravasation. Scale bar = 100 μm.


( D ) Relative invasion frequency of GBS strain K79 in HBMEC with or without EGFR inhibitor ( gefitinib ) .

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(D) Relative invasion frequency of GBS strain K79 in HBMEC with or without EGFR inhibitor (gefitinib).


( E ) Bacterial counts recovered from the blood and brain in wild type mice receiving vehicle control ( n = 5 ) or gefitinib ( 10 mg / kg ) ( n = 6 ) , infected with strain K79 for 1 h .

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(E) Bacterial counts recovered from the blood and brain in wild type mice receiving vehicle control (n = 5) or gefitinib (10 mg/kg) (n = 6), infected with strain K79 for 1 h.


( F ) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS strain K79 infection at 15 min and 60 min post infection .

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(F) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS strain K79 infection at 15 min and 60 min post infection.


( G ) EGFR protein expression in EGFR knockout HBMEC using CRISPR / Cas9 ( left panel ) and relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout and control HBMEC ( right panel ) . From left to right , p value of K79 is 0 . 0059 , p value of K160 is 0 . 00024 , p value of K161 is 0 . 00080 , p value of K181 is 0 . 00042 , p value of K226 is 0 . 00011 , p value of K237 is 0 . 00072 , p value of K238 is 0 . 0093 , p value of P539 is 0 . 00013 ( right panel ) .

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(G) EGFR protein expression in EGFR knockout HBMEC using CRISPR/Cas9 (left panel) and relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout and control HBMEC (right panel). From left to right, p value of K79 is 0.0059, p value of K160 is 0.00024, p value of K161 is 0.00080, p value of K181 is 0.00042, p value of K226 is 0.00011, p value of K237 is 0.00072, p value of K238 is 0.0093, p value of P539 is 0.00013 (right panel).


( H ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in EGFR knockout HBMEC compare to control HBMEC .

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(H) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in EGFR knockout HBMEC compare to control HBMEC.


( I ) Bacterial counts recovered from the blood and brain of EGFR conditional knockout ( n = 5 ) and control mice ( n = 5 ) 1 h after intravenous inoculation with strain K79 .

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(I) Bacterial counts recovered from the blood and brain of EGFR conditional knockout (n = 5) and control mice (n = 5) 1 h after intravenous inoculation with strain K79.


( A ) Tyrosine phosphorylation of EGFR in HBMEC pre - treated with 20 μM S1P2 antagonist ( JTE - 013 ) or vehicle control and infected with GBS strain K79 infection for 60 min . EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79 .

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(A) Tyrosine phosphorylation of EGFR in HBMEC pre-treated with 20 μM S1P2 antagonist (JTE-013) or vehicle control and infected with GBS strain K79 infection for 60 min. EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79.


( B ) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors . All the inhibitors were used at 10 μM . From left to right , p value of DJB - V - 39 is 0 . 0047 , p value of SKI is 0 . 0062 , p value of FTY720 is 0 . 0022 , and p value of HB56 is 0 . 0045 .

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(B) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors. All the inhibitors were used at 10 μM. From left to right, p value of DJB-V-39 is 0.0047, p value of SKI is 0.0062, p value of FTY720 is 0.0022, and p value of HB56 is 0.0045.


( C ) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR / Cas9 ( left panel ) . Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC . From left to right , p value of K79 is 0 . 0018 , p value of K160 is 0 . 0091 , p value of K161 is 0 . 0054 , p value of K181 is 0 . 00040 , p value of K226 is 0 . 00032 , p value of K237 is 0 . 040 , p value of K238 is 0 . 041 and p value of P539 is 0 . 0025 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR/Cas9 (left panel). Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC. From left to right, p value of K79 is 0.0018, p value of K160 is 0.0091, p value of K161 is 0.0054, p value of K181 is 0.00040, p value of K226 is 0.00032, p value of K237 is 0.040, p value of K238 is 0.041 and p value of P539 is 0.0025.


( D ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control .

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

(D) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control.


( E ) Bacterial counts recovered from the blood and brain of wild type ( n = 5 ) and S1P2 - / - mice ( n = 5 ) infected with strain K79 for 1h .

(E) Bacterial counts recovered from the blood and brain of wild type (n = 5) and S1P2-/- mice (n = 5) infected with strain K79 for 1h.


( A ) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC .

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

(A) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC.


( B ) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control , EGFR knockout and S1P2 knockout HBMEC .

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

(B) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control, EGFR knockout and S1P2 knockout HBMEC.


( C ) Bacterial counts recovered from the blood and brain in wild type mice ( n = 5 ) and CysLT2 - / - mice ( n = 5 ) infected with strain K79 for 1h .

O O O O O O O O O O O O O O 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) Bacterial counts recovered from the blood and brain in wild type mice (n = 5) and CysLT2-/- mice (n = 5) infected with strain K79 for 1h.


( D ) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA ( left panel ) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC . t - test . From left to right , p value of K79 is 0 . 0039 , p value of K160 is 0 . 0082 , p value of K161 is 0 . 00091 , p value of K181 is 0 . 0087 , p value of K226 is 0 . 00020 , p value of K237 is 0 . 0039 , p value of K238 is 0 . 00066 , p value of P539 is 1 . 76E - 05 ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA (left panel) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC. t-test. From left to right, p value of K79 is 0.0039, p value of K160 is 0.0082, p value of K161 is 0.00091, p value of K181 is 0.0087, p value of K226 is 0.00020, p value of K237 is 0.0039, p value of K238 is 0.00066, p value of P539 is 1.76E-05 (right panel).


( E ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control .

(E) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control.


( F ) Bacterial counts recovered from the blood and brain in wild type mice ( n = 7 ) and CysLT1 - / - mice ( n = 7 ) infected with strain K79 for 1h .

(F) Bacterial counts recovered from the blood and brain in wild type mice (n = 7) and CysLT1-/- mice (n = 7) infected with strain K79 for 1h.


( G ) Ezrin protein expression in ezrin knockdown HBMEC using shRNA ( left panel ) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC ( right panel ) .

O O O O O O O O O O O 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) Ezrin protein expression in ezrin knockdown HBMEC using shRNA (left panel) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC (right panel).


( H Ezrin phosphorylation in HBMEC pre - treated with CysLT1 antagonist ( montelukast ) and infected with GBS strain K79 for 60 min ( left panel )

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

(H Ezrin phosphorylation in HBMEC pre-treated with CysLT1 antagonist (montelukast) and infected with GBS strain K79 for 60 min (left panel)


I ) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1 - / - and control mice infected with GBS strain K79 for 1h ( right panel ) .

O O O O O O 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) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1-/- and control mice infected with GBS strain K79 for 1h (right panel).


( J ) Intracellular K79 co - localization with EGFR and ezrin in control and S1P2 knockout HBMEC ( as shown by arrows , cyan ) . Percentage of co - localization was calculated by counting numbers of all GBS and co - localized intracellular GBS from at least three representative fields , and expressing as numbers of co - localized GBS / all GBS x 100 .

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

(J) Intracellular K79 co-localization with EGFR and ezrin in control and S1P2 knockout HBMEC (as shown by arrows, cyan). Percentage of co-localization was calculated by counting numbers of all GBS and co-localized intracellular GBS from at least three representative fields, and expressing as numbers of co-localized GBS/all GBS x 100.


( A ) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79 . Data are presented as a Kaplan - Maier plot with a log rank test used to compare percentage of survival between the groups , CFX group n = 18 , and CFX + MONT group n = 10 * p = 0 . 015 . ( CFX : ceftriaxone ; MONT : montelukast ) .

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

(A) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79. Data are presented as a Kaplan-Maier plot with a log rank test used to compare percentage of survival between the groups, CFX group n = 18, and CFX + MONT group n = 10*p = 0.015. (CFX: ceftriaxone; MONT: montelukast).


( B ) Nissl staining of the hippocampus CA1 , CA2 & CA3 regions ( low magnification ) and cortex region ( higher magnification ) ( left ) and histograms showing the number of Nissl - stained bodies ( right ) Scale bar , 50 μm . Data represent the means ± SEM from three independent experiments , with statistical analysis by One - way ANOVA , p value between control group and Ceftriaxone group is 0 . 00021 , p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0 . 013 .

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

(B) Nissl staining of the hippocampus CA1, CA2 & CA3 regions (low magnification) and cortex region (higher magnification) (left) and histograms showing the number of Nissl-stained bodies (right) Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value between control group and Ceftriaxone group is 0.00021, p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0.013.


( C - D ) Representative immunofluorescence images ( left ) and quantification ( right ) of TUNEL bodies , astrocyte ( GFAP ) , microglia ( Iba1 ) in cortex of wild type mice after 7 days of therapy . Scale bar , 50 μm . Data represent the means ± SEM from three independent experiments , with statistical analysis by One - way ANOVA , p value of TUNEL bodies between CFX group and MONT + CFX group is 0 . 0012 , p value of astrocyte between CFX group and MONT + CFX group is 0 . 0068 , and p value of microglia cells between CFX group and MONT + CFX group is 0 . 0029 . ( E ) Y - maze test for spatial learning and memory of different group of wild type mice . The total duration of time spent at novel arm ( left ) and the percentage of entries to novel arm of Y - maze ( right ) . Data represent the means ± SEM was statistically analyzed by One - way ANOVA , p value of total duration of time spent at novel arm between control group ( n = 6 ) and CFX group ( n = 5 ) is 0 . 00012 , p value between CFX group ( n = 5 ) and MONT + CFX group ( n = 6 ) is 0 . 00017 ; p value of total duration of % of entry to novel arm between control group ( n = 6 ) and CFX group ( n = 5 ) is 0 . 0102 , and p value between CFX group ( n = 5 ) and MONT + CFX group ( n = 6 ) is 0 . 0216 .

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

(C-D) Representative immunofluorescence images (left) and quantification (right) of TUNEL bodies, astrocyte (GFAP), microglia (Iba1) in cortex of wild type mice after 7 days of therapy. Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value of TUNEL bodies between CFX group and MONT + CFX group is 0.0012, p value of astrocyte between CFX group and MONT + CFX group is 0.0068, and p value of microglia cells between CFX group and MONT + CFX group is 0.0029. (E) Y-maze test for spatial learning and memory of different group of wild type mice. The total duration of time spent at novel arm (left) and the percentage of entries to novel arm of Y-maze (right). Data represent the means ± SEM was statistically analyzed by One-way ANOVA, p value of total duration of time spent at novel arm between control group (n = 6) and CFX group (n = 5) is 0.00012, p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.00017; p value of total duration of % of entry to novel arm between control group (n = 6) and CFX group (n = 5) is 0.0102, and p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.0216.


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , Xnr1 ( 50 pg )

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), Xnr1 (50 pg)


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , BMP4 ( 100 pg ) .

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), BMP4 (100 pg).


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng )

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng)


( D wt Wip1 , but not Wip1 ( D277A ) , down - regulates the activity of Activin - or BMP - responsive promoters in HEK293T cells . FAST1 , were co - transfected as indicated for efficient activation of the promoters . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test .

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

(D wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. FAST1, were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


E ) wt Wip1 , but not Wip1 ( D277A ) , down - regulates the activity of Activin - or BMP - responsive promoters in HEK293T cells . Smad1 and Smad4 were co - transfected as indicated for efficient activation of the promoters . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test .

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

E) wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. Smad1 and Smad4 were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


( F - Q ) Dorsal ( F - N ) or ventral ( O - Q ) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10 . 25 as analyzed by in situ hybridization . Embryos are shown in vegetal views with dorsal to the top . Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos . Control , an embryo injected with LacZ only . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) and LacZ ( 100 pg ) . Scale bar , 150 μm .

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

(F-Q) Dorsal (F-N) or ventral (O-Q) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10.25 as analyzed by in situ hybridization. Embryos are shown in vegetal views with dorsal to the top. Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos. Control, an embryo injected with LacZ only. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng) and LacZ (100 pg). Scale bar, 150 μm.


( A , Nodal - induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT - PCR . Animal caps ere treated with - ) , no injection of Co MO , Wip1 MO and / or Wip1 mRNA . The dose of reagent injected : Xnr1 ( 5 pg ) , Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) and Wip1 ( 1 ng ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps ere treated with -), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Xnr1 (5 pg), Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).


B ) Activin / Nodal - induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT - PCR . Animal caps in ( B ) were treated with Activin protein ( 5 ng / ml ) . ( - ) , no injection of Co MO , Wip1 MO and / or Wip1 mRNA . The dose of reagent injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) and Wip1 ( 1 ng ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activin/Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps in (B) were treated with Activin protein (5 ng/ml). (-), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).


( C ) Knockdown of Wip1 up - regulates the activity of Activin / Nodal - responsive promoter . HEK293T cells transfected with Co siRNA ( 50 nM ) or Wip1 siRNA ( 50 nM ) were stimulated with Activin protein ( 10 ng / ml ) for 10 h and then subjected to luciferase assays . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * * * P < 0 . 001 by unpaired Student ' s t - test .

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

(C) Knockdown of Wip1 up-regulates the activity of Activin/Nodal-responsive promoter. HEK293T cells transfected with Co siRNA (50 nM) or Wip1 siRNA (50 nM) were stimulated with Activin protein (10 ng/ml) for 10 h and then subjected to luciferase assays. Data are expressed as the mean ± SEM (n=3 biological replicates). ***P<0.001 by unpaired Student's t-test.


Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate . ( - ) , no injection of Noggin mRNA . The amount of MO and mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G and J ; 30 ng for F and I ) and Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm .

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

Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. (-), no injection of Noggin mRNA. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.


Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate . Embryos in ( E - J ) are shown in dorsal views with anterior to the top . The amount of MO and mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G and J ; 30 ng for F and I ) and Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm .

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

Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. Embryos in (E-J) are shown in dorsal views with anterior to the top. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.


( K , Activin induction of p21 is up - regulated upon Wip1 knockdown . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection , were stimulated with Activin ( 10 ng / ml ) for 3 to 12 h as indicated t = 0 in ( K ) is 48 h after siRNA transfection . P - Smad2 serves as an indicator of active Activin / Nodal signaling . Arrows ndicate non - specific bands .

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

(K, Activin induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with Activin (10 ng/ml) for 3 to 12 h as indicated t=0 in (K) is 48 h after siRNA transfection. P-Smad2 serves as an indicator of active Activin/Nodal signaling. Arrows ndicate non-specific bands.


L ) TGF - β induction of p21 is up - regulated upon Wip1 knockdown . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection , were stimulated with increasing concentration of TGF - β1 ( 10 , 20 ng / ml ) for 3 h .

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

L) TGF-β induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with increasing concentration of TGF-β1 (10, 20 ng/ml) for 3 h.


( A , Effects of the gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T cells were transfected or not with wt Wip1 , Wip1 ( D277A ) , as indicated and subjected to CCK - 8 assays before ( 0 h ) and after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - test . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with wt Wip1, Wip1(D277A), as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.


B ) Effects of the gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK - 8 assays before ( 0 h ) and after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - test . n . s . , not significant .

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B) Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.


Depletion of Wip1 augments TGF - β - induced migration and invasion of MDA - MB231 breast cancer cells . MDA - MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20 - μL pipette tip and then incubated in the absence or presence of TGF - β1 for the indicated times ( C , 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

Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20-μL pipette tip and then incubated in the absence or presence of TGF-β1 for the indicated times (C, D)


Depletion of Wip1 augments TGF - β - induced migration and invasion of MDA - MB231 breast cancer cells . MDA - MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF - β1 for 20 h ( E , F ) .

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

Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF-β1 for 20 h (E, F).


( A - D ) Knockdown of Wip1 expands the expression of dorsal mesodermal markers . Embryos injected dorsally with Co MO ( 40 ng ) or Wip1 MO ( 40 ng ) were subjected to in situ hybridization at stage 10 . 25 . Embryos are shown in dorso - vegetal views with dorsal to the top . Scale bar , 150 μm .

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(A-D) Knockdown of Wip1 expands the expression of dorsal mesodermal markers. Embryos injected dorsally with Co MO (40 ng) or Wip1 MO (40 ng) were subjected to in situ hybridization at stage 10.25. Embryos are shown in dorso-vegetal views with dorsal to the top. Scale bar, 150 μm.


( E - I ) Morphological phenotypes of Wip1 morphants . Embryos were injected radially in the marginal zone with Co MO ( 80 ng ) , Wip1 MO ( 80 ng ) , wt Wip1 ( 1 ng ) and Wip1 ( D277A ) mRNA as indicated and cultured to tadpole stages . Embryos are shown in lateral views with anterior to the left . Un Co , uninjected control . Scale bar , 1 mm . ( J ) Quantification of the phenotypes shown in ( E - I ) . Severe defects include microcephaly , shortened and kinked body axis , and no eye . Mild defects indicate normal body axis with malformed eyes .

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(E-I) Morphological phenotypes of Wip1 morphants. Embryos were injected radially in the marginal zone with Co MO (80 ng), Wip1 MO (80 ng), wt Wip1 (1 ng) and Wip1(D277A) mRNA as indicated and cultured to tadpole stages. Embryos are shown in lateral views with anterior to the left. Un Co, uninjected control. Scale bar, 1 mm. (J) Quantification of the phenotypes shown in (E-I). Severe defects include microcephaly, shortened and kinked body axis, and no eye. Mild defects indicate normal body axis with malformed eyes.


( A Wip1 and Smad4 form an endogenous protein complex . Cell lysates of HEK293T cells , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) ( - ) , no treatment with protein ligand . IgG , control IgG . An arrow in ( A ) indicates non - specific bands .

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(A Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) (-), no treatment with protein ligand. IgG, control IgG. An arrow in (A) indicates non-specific bands.


B ) Wip1 and Smad4 form an endogenous protein complex . Cell lysates of HEK293T cells , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) and vice versa ( B ) . ( - ) , no treatment with protein ligand . IgG , control IgG .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) and vice versa (B). (-), no treatment with protein ligand. IgG, control IgG.


( C ) Upon ligand stimulation , Wip1 forms a complex with Smad4 in the nucleus . HeLa cells were treated or not with TGF - β1 ( 40 ng / ml ) for 2 h , fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4 . Blue : DAPI ; Red : PLA signal . Scale bar , 20 μm . ( D ) Quantitative analysis of Wip1 / Smad4 interactions shown in ( C ) . Data are represented as the mean ± SEM ( n = 3 biological replicates ) . Twenty cells were analyzed per sample . * * * P < 0 . 001 by Student ' s t - test .

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(C) Upon ligand stimulation, Wip1 forms a complex with Smad4 in the nucleus. HeLa cells were treated or not with TGF-β1 (40 ng/ml) for 2 h, fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4. Blue: DAPI; Red: PLA signal. Scale bar, 20 μm. (D) Quantitative analysis of Wip1/Smad4 interactions shown in (C). Data are represented as the mean ± SEM (n=3 biological replicates). Twenty cells were analyzed per sample. ***P<0.001 by Student's t-test.


( E ) Wip1 interferes with the ability of Smad4 to bind to Smad2 . HEK293T cells co - transfected with the indicated constructs were treated or not with TGF - β1 for 1 h and then harvested . An arrow denotes Myc - tagged Wip1 protein .

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(E) Wip1 interferes with the ability of Smad4 to bind to Smad2. HEK293T cells co-transfected with the indicated constructs were treated or not with TGF-β1 for 1 h and then harvested. An arrow denotes Myc-tagged Wip1 protein.


( F ) Wip1 has a negative effect on nuclear accumulation of Smad4 . HEK293T cells were transfected with Co siRNA or Wip1 siRNA , treated with TGF - β1 ( 40 ng / ml , 1 h ) 48 h after siRNA transfection and subjected to nuclear / cytoplasmic fractionation and western blotting analysis . β - tubulin and lamin A / C serve as cytoplasmic and nuclear markers , respectively . C , cytoplasm ; N , nucleus . The experiment was repeated two times with similar results . ( G ) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in ( F ) .

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(F) Wip1 has a negative effect on nuclear accumulation of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA, treated with TGF-β1 (40 ng/ml, 1 h) 48 h after siRNA transfection and subjected to nuclear/cytoplasmic fractionation and western blotting analysis. β-tubulin and lamin A/C serve as cytoplasmic and nuclear markers, respectively. C, cytoplasm; N, nucleus. The experiment was repeated two times with similar results. (G) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in (F).


( A ) Overexpression of Wip1 down - regulates Smad4 phosphorylation at Thr277 . MOCK - or Flag - Wip1 - transfected HEK293T cells were treated with 1 h pulse of FGF2 ( 10 ng / ml ) , followed by addition of U0126 ( 40 μM ) and harvesting for western blotting at the indicated times . - FGF2 , no treatment with FGF2 .

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

(A) Overexpression of Wip1 down-regulates Smad4 phosphorylation at Thr277. MOCK- or Flag-Wip1-transfected HEK293T cells were treated with 1 h pulse of FGF2 (10 ng/ml), followed by addition of U0126 (40 μM) and harvesting for western blotting at the indicated times. - FGF2, no treatment with FGF2.


( B ) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1 - depleted HEK293T cells , showing that knockdown of Wip1 up - regulates Smad4 phosphorylation at the MAPK site . A bracket denotes phospho - Smad4 .

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(B) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1-depleted HEK293T cells, showing that knockdown of Wip1 up-regulates Smad4 phosphorylation at the MAPK site. A bracket denotes phospho-Smad4.


( C ) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay . Myc - tagged Smad4 was expressed in HEK293T cells , which were then treated or not with FGF2 ( 40 ng / ml , 2 h ) , and the Myc - Smad4 was immunoprecipitated using anti - Myc antibody and subsequently incubated with or without recombinant human Wip1 protein ( 0 . 5 μg ) in the absence or presence of Mg2 + as indicated .

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

(C) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay. Myc-tagged Smad4 was expressed in HEK293T cells, which were then treated or not with FGF2 (40 ng/ml, 2 h), and the Myc-Smad4 was immunoprecipitated using anti-Myc antibody and subsequently incubated with or without recombinant human Wip1 protein (0.5 μg) in the absence or presence of Mg2+ as indicated.


( D ) FGF - induced destabilization of Smad4 is enhanced further in Wip1 - depleted animal cap cells as analyzed by western blotting . The amount of reagent injected : Myc - Smad4 ( 400 pg ) , FGF8 ( 100 pg ) , Wnt8 ( 400 pg ) , Co MO ( 60 ng ) and Wip1 MO ( 60 ng ) .

(D) FGF-induced destabilization of Smad4 is enhanced further in Wip1-depleted animal cap cells as analyzed by western blotting. The amount of reagent injected: Myc-Smad4 (400 pg), FGF8 (100 pg), Wnt8 (400 pg), Co MO (60 ng) and Wip1 MO (60 ng).


( E ) Polyubiquitination of wt Smad4 is promoted in Wip1 - silenced cells . HEK293T cells were transfected with the indicated combinations of Flag - ubiquitin , Co siRNA , Wip1 siRNA , Myc - Smad4 and Myc - Smad4 ( T277A ) and then treated with FGF2 ( 10 ng / ml , 4 h ) in the presence of the proteasome inhibitor MG132 , and Myc - Smad4 and its phosphorylation - resistant mutant were immunoprecipitated with anti - Myc antibody . Ubiquitin - conjugated Smad4 ( Smad4 - Ubn ) was detected by western blotting with anti - Flag antibody .

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

(E) Polyubiquitination of wt Smad4 is promoted in Wip1-silenced cells. HEK293T cells were transfected with the indicated combinations of Flag-ubiquitin, Co siRNA, Wip1 siRNA, Myc-Smad4 and Myc-Smad4(T277A) and then treated with FGF2 (10 ng/ml, 4 h) in the presence of the proteasome inhibitor MG132, and Myc-Smad4 and its phosphorylation-resistant mutant were immunoprecipitated with anti-Myc antibody. Ubiquitin-conjugated Smad4 (Smad4-Ubn) was detected by western blotting with anti-Flag antibody.


( F ) Wip1 prolongs the half - life of Smad4 . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide ( CHX , 100 ng / ml ) for the indicated times and harvested for western blotting . An arrow indicates non - specific bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wip1 prolongs the half-life of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide (CHX, 100 ng/ml) for the indicated times and harvested for western blotting. An arrow indicates non-specific bands.


( A - J ) Embryos injected dorsally with wt Wip1 ( 1 ng ) , wt Smad4 ( 1 ng ) , Smad4 ( T277A ) ( 1 ng ) and Smad4 ( T277E ) ( 1 ng ) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10 . 25 . Control , control embryo injected with LacZ mRNA only . Scale bar , 150 μm . ( K ) Quantification of the results from in situ hybridization analysis in ( A - J ) .

(A-J) Embryos injected dorsally with wt Wip1 (1 ng), wt Smad4 (1 ng), Smad4(T277A) (1 ng) and Smad4 (T277E) (1 ng) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10.25. Control, control embryo injected with LacZ mRNA only. Scale bar, 150 μm. (K) Quantification of the results from in situ hybridization analysis in (A-J).


( L ) Wip1 - mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho - mimetic amino acid at the MAPK site . ( - ) , no co - injection of wt or mutated Smad4 .

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(L) Wip1-mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho-mimetic amino acid at the MAPK site. (-), no co-injection of wt or mutated Smad4.


( M ) MDA - MB231 and MDA - MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4 ( T277A ) and then subjected to CCK - 8 assays to measure cell growth . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test .

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(M) MDA-MB231 and MDA-MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4(T277A) and then subjected to CCK-8 assays to measure cell growth. Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


( N ) Anchorage - independent colony - formation assays were performed in MDA - MB231 and MDA - MB468 cells transfected with Wip1 or negative control constructs . Scale bar , 100 μm . ( O ) Quantification of the assays shown in ( N ) . The number of colonies larger than 50 μm in diameter was scored . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test .

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(N) Anchorage-independent colony-formation assays were performed in MDA-MB231 and MDA-MB468 cells transfected with Wip1 or negative control constructs. Scale bar, 100 μm. (O) Quantification of the assays shown in (N). The number of colonies larger than 50 μm in diameter was scored. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


a - e , Scanning electron microscopy ( SEM ) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . a , Normal eyes in DTS7 flies reared at 22 ° C . b , Rough eyes in DTS7 flies reared at 28 ° C . c , d , Degeneration was suppressed by expression of Drosophila HDAC6 ( dHDAC6 ; c ) or human HDAC6 ( hHDAC6 ; d ) , but not a catalytically dead mutant of hHDAC6 ( cat . mut . ; e ) .

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a-e, Scanning electron microscopy (SEM) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. a, Normal eyes in DTS7 flies reared at 22 °C. b, Rough eyes in DTS7 flies reared at 28 °C. c, d, Degeneration was suppressed by expression of Drosophila HDAC6 (dHDAC6; c) or human HDAC6 (hHDAC6; d), but not a catalytically dead mutant of hHDAC6 (cat. mut.; e).


f - j , SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . f , Normal eyes in AR52 flies reared without ( - ) DHT . g , Rough eyes in AR52 flies reared with ( + ) DHT . Degeneration was suppressed by expression of Drosophila HDAC6 ( h ) or human HDAC6 ( i ) , but not a catalytically dead mutant of human HDAC6 ( j ) .

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f-j, SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. f, Normal eyes in AR52 flies reared without (-) DHT. g, Rough eyes in AR52 flies reared with (+) DHT. Degeneration was suppressed by expression of Drosophila HDAC6 (h) or human HDAC6 (i), but not a catalytically dead mutant of human HDAC6 (j).


k - p , Detection of UPS reporter in imaginal eye discs from third - instar larvae by confocal microscopy . High level fluorescence was found in flies expressing GFP ( k , positive control ) , but fluorescence was barely detectable in control flies expressing CL1 - GFP ( l , negative control ) . CL1 - GFP accumulates in DTS7flies with temperature - dependent proteasome impairment ( compare m to n ) and in AR52flies with ligand - dependent degeneration ( compare o to p ) . The retinal phenotypes of 200 to > 1 , 000 flies of each genotype were examined . Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3 , respectively . ( DHT , dihydrotestosterone . )

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

k-p, Detection of UPS reporter in imaginal eye discs from third-instar larvae by confocal microscopy. High level fluorescence was found in flies expressing GFP (k, positive control), but fluorescence was barely detectable in control flies expressing CL1-GFP (l, negative control). CL1-GFP accumulates in DTS7flies with temperature-dependent proteasome impairment (compare m to n) and in AR52flies with ligand-dependent degeneration (compare o to p). The retinal phenotypes of 200 to >1,000 flies of each genotype were examined. Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3, respectively. (DHT, dihydrotestosterone.)


a - e , Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in ( f ) . a , An autophagosome ( red arrow ) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT . b , Higher magnification of the autophagosome in a . c , Multiple autophagolysosomes ( red arrows ) containing dense , amorphous material from an AR52fly reared on DHT . d , A juxtanuclear multilamellar body ( red arrow ) from a DTS7fly reared at 28 ° C . e , A multivesicular body ( red arrow ) from a DTS7fly reared at 28 ° C . f , A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 ° C compared to those reared at 22 ° C , and in AR52 flies reared on DHT compared to those reared off DHT . Data show mean ± s . d . , n = 59 - 82 neurons in 5 sections per condition . No accumulation of autophagic figures was found in AR12 flies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f). a, An autophagosome (red arrow) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT. b, Higher magnification of the autophagosome in a. c, Multiple autophagolysosomes (red arrows) containing dense, amorphous material from an AR52fly reared on DHT. d, A juxtanuclear multilamellar body (red arrow) from a DTS7fly reared at 28 °C. e, A multivesicular body (red arrow) from a DTS7fly reared at 28 °C. f, A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 °C compared to those reared at 22 °C, and in AR52 flies reared on DHT compared to those reared off DHT. Data show mean ± s.d., n = 59-82 neurons in 5 sections per condition. No accumulation of autophagic figures was found in AR12 flies.


g - l , SEM images of fly eyes expressing the indicated transgenes . RNAi knockdown ( KD ) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 ° C ( compare h , i to g ) and AR52 flies reared on DHT ( compare k , l to j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye phenotypes are presented in Supplementary Fig . S2 . ( N , nucleus ; Rh , rhabdomere . )

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

g-l, SEM images of fly eyes expressing the indicated transgenes. RNAi knockdown (KD) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 °C (compare h, i to g) and AR52 flies reared on DHT (compare k, l to j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2. (N, nucleus; Rh, rhabdomere.)