( A , B ) Murine cardiac endothelial cells ( MCEC ) were treated with different concentrations of D - β - hydroxybutyrate ( βOHB ) or acetoacetate ( AcAc ) for 24 hours . Relative BrdU absorbance was quantified compared to control treatment ( H2O for βOHB and EtOH for AcAc ) . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

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(A, B) Murine cardiac endothelial cells (MCEC) were treated with different concentrations of D-β-hydroxybutyrate (βOHB) or acetoacetate (AcAc) for 24 hours. Relative BrdU absorbance was quantified compared to control treatment (H2O for βOHB and EtOH for AcAc). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( I - M ) Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid - based sprouting assay . Spheroids were treated with media containing 30 mM D - β - hydroxybutyrate ( βOHB ) , 30 mM acetoacetate ( AcAc ) , 10 mM acetate , 1 mM octanoate , 1 mM butyrate , 30 mM L - β - hydroxybutyrate ( L - βOHB ) , 1 mM niacin or the respective controls ( final concentration of reagents is diluted to approximately 10 % ) and analyzed after 48 hours . The ( I , L ) average number of sprouts per spheroid , the ( J , M ) accumulated total sprout length and ( K ) average sprout length were quantified . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I-M) Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc), 10 mM acetate, 1 mM octanoate, 1 mM butyrate, 30 mM L-β-hydroxybutyrate (L- βOHB), 1 mM niacin or the respective controls (final concentration of reagents is diluted to approximately 10 %) and analyzed after 48 hours. The (I, L) average number of sprouts per spheroid, the (J, M) accumulated total sprout length and (K) average sprout length were quantified. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( B , Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid - based sprouting assay . Spheroids were treated with media containing 30 mM D - β - hydroxybutyrate ( βOHB ) , 30 mM acetoacetate ( AcAc ) or the respective controls ( final concentration of reagents is diluted to approximately 10 % ) for 48 hours . The average number of sprouts per spheroid was quantified . Data are presented as mean ± SD . n = 4 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; unpaired Student ' s t - test .

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(B, Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. The average number of sprouts per spheroid was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test.


( C , D ) Heatmaps showing expression levels of genes involved in KEGG pathways ( C ) fatty acid degradation and ( D ) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl / 6J mice that were fed a ketogenic diet or control diet for two weeks .

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(C, D) Heatmaps showing expression levels of genes involved in KEGG pathways (C) fatty acid degradation and (D) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl/6J mice that were fed a ketogenic diet or control diet for two weeks.


( A , B ) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU , double positive cells are indicated by arrowheads , and quantification of double positive cells in heart sections per high power field ( HPF ) . Scale bar : 50 μm . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU, double positive cells are indicated by arrowheads, and quantification of double positive cells in heart sections per high power field (HPF). Scale bar: 50 μm. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( F ) Quantification of Ki67 + endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks ( gastrocnemius and soleus muscle ) or two weeks ( brain , lungs , subcutaneous adipose tissue , liver ) . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

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(F) Quantification of Ki67+ endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks (gastrocnemius and soleus muscle) or two weeks (brain, lungs, subcutaneous adipose tissue, liver). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( B ) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG . Scale bar : 50 µm .

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(B) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG. Scale bar: 50 µm.


( A - C ) Tissue lysates from ( A ) spleen , ( B ) liver and ( C ) kidney from 6 - to 9 - day old indicated mice were immunoblotted to determine cleavage of caspase - 1 . Each lane represents a lysate from a different mouse . Positive control lanes ( + ) are BMDMs stimulated with LPS / ATP ; negative control lanes ( - ) are respective organ homogenates from caspase - 1 - / - mice .

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(A-C) Tissue lysates from (A) spleen, (B) liver and (C) kidney from 6- to 9-day old indicated mice were immunoblotted to determine cleavage of caspase-1. Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from caspase-1-/- mice.


( D - G ) Tissue lysates from ( D , F ) spleen and ( E , G ) liver from 6 - to 9 - day old indicated mice were immunoblotted to determine pro - IL - 1β cleavage to IL - 1β ( D - E ) or GSDMD cleavage to its p30 and p20 fragments ( F - G ) . Each lane represents a lysate from a different mouse . Positive control lanes ( + ) are BMDMs stimulated with LPS / ATP ; negative control lanes ( - ) are respective organ homogenates from ( D - E ) caspase - 1 - / - or ( F - G ) Gsdmd - / - mice .

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

(D-G) Tissue lysates from (D, F) spleen and (E, G) liver from 6- to 9-day old indicated mice were immunoblotted to determine pro-IL-1β cleavage to IL-1β (D-E) or GSDMD cleavage to its p30 and p20 fragments (F-G). Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from (D-E) caspase-1-/- or (F-G) Gsdmd-/- mice.


Coomassie - stained proteins from bovine retina that were co - immunoprecipitated by the anti - RIBEYE antibody 2D9 ( lane 3 ) . Lane 2 , control precipitation by irrelevant mouse IgGs ; lane 1 , input ( 10 % of the total loaded in each lane ) Western blot of the immunoprecipitated proteins ( as in A , 1 % of the total protein was loaded per lane ) probed with anti - RIBEYE ( Ba ) , anti - CASPR1 ( Bb ) and anti - CNTN1 ( Bc ) . The 2D9 antibody immunoprecipitated RIBEYE ( Ba , lane 3 ) , CASPR1 ( Bb , lane 3 ) and CNTN1 ( Bc , lane 3 ) ; these proteins were not detectable in the control immunoprecipitations . Bd ) As a loading control , heavy chains of the precipitating antibody ( IgG hc ) were visualized by the goat anti - mouse IgG antibody conjugated to

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

Coomassie- stained proteins from bovine retina that were co-immunoprecipitated by the anti-RIBEYE antibody 2D9 (lane 3). Lane 2, control precipitation by irrelevant mouse IgGs; lane 1, input (10% of the total loaded in each lane) Western blot of the immunoprecipitated proteins (as in A, 1% of the total protein was loaded per lane) probed with anti-RIBEYE (Ba), anti-CASPR1 (Bb) and anti-CNTN1 (Bc). The 2D9 antibody immunoprecipitated RIBEYE (Ba, lane 3), CASPR1 (Bb, lane 3) and CNTN1 (Bc, lane 3); these proteins were not detectable in the control immunoprecipitations. Bd) As a loading control, heavy chains of the precipitating antibody (IgG hc) were visualized by the goat anti-mouse IgG antibody conjugated to peroxidase


Localization of CASPR in semi - thin sections of retinas from either MOG / CFA - injected mice ( EAE mice ) or CFA - injected control mice ( 9 days after injection ) . Semi - thin sections were double - immunolabelled with antibodies against CASPR1 and RIBEYE ( A - B OPL , outer plexiform layer ; INL , nuclear layer ; IPL , inner plexiform layer . Scale bars :

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Localization of CASPR in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelled with antibodies against CASPR1 and RIBEYE (A-B OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm


Localization o visual - arrestin ( v - arrestin ) in semi - thin sections of retinas from either MOG / CFA - injected mice ( EAE mice ) or CFA - injected control mice ( 9 days after injection ) . Semi - thin sections were double - immunolabelle with antibodies against v - arrestin and RIBEYE ( C - D ) V - arrestin , visual arrestin ; OPL , outer plexiform layer ; INL , nuclear layer ; IPL , inner plexiform layer . Scale bars :

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Localization o visual-arrestin (v-arrestin) in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelle with antibodies against v-arrestin and RIBEYE (C-D) V-arrestin, visual arrestin; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm


Localization of CNTN1 in semi - thin ( 0 . 5μm - thin ) sections of retinas from either MOG / CFA - injected mice ( EAE mice ) or CFA - injected control mice ( 9 days after injection ) . ( A , B ) Sections were double - immunolabelled with antibodies against CNTN1 and RIBEYE proteins in the OPL and IPL . ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , nuclear layer ; IPL , inner plexiform

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

Localization of CNTN1 in semi-thin (0.5μm-thin) sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). (A,B) Sections were double-immunolabelled with antibodies against CNTN1 and RIBEYE proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer


Quantification of the immunosignals of the indicated proteins in the OPL and IPL . ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , nuclear layer ; IPL , inner plexiform layer ; n . s . , non - significant ; * * * , p < 0 . 001 ( Mann - Whitney U test , actual p - values are given in C ) ( N = 6 independent set of experiments ; n = 295 images ( CFA ) ; n = 274 images ( MOG / CFA ) for quantification of RIBEYE immunosignals ; n = 95 ( CFA ) ; n = 92 ( MOG / CFA ) for quantification of CASPR1 immunosignals ; n = 91 ( CFA ) , n = 82 ( MOG / CFA ) for CNTN1 quantification ; n = 109 ( CFA ) ; n = 100 ( MOG / CFA ) for quantification of v - arrestin . Scale bars :

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

Quantification of the immunosignals of the indicated proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer; n.s., non-significant; ***, p<0.001 (Mann-Whitney U test, actual p-values are given in C) (N=6 independent set of experiments; n=295 images (CFA); n=274 images (MOG/CFA) for quantification of RIBEYE immunosignals; n=95 (CFA); n=92 (MOG/CFA) for quantification of CASPR1 immunosignals; n=91 (CFA), n=82 (MOG/CFA) for CNTN1 quantification; n=109 (CFA); n=100 (MOG/CFA) for quantification of v-arrestin. Scale bars: 20μm


MOG / CFA - but not CFA - injected mice contain reactive antibodies for retinal proteins in blood and show altered spatial visual behavior . ( A ) 7 - 9 days after injection , blood from MOG / CFA - injected ( red ) but not from CFA - injected mice ( black ) , elicited strong antibody responses in ELISA . Antibodies for RIBEYE and SV2 were used as positive controls ( green ) , secondary antibody only as a negative control ( white ) . Normalized data are given as mean ± S . E . M . ( N = 4 ( CFA ) ; N = 5 ( MOG / CFA ) . * * p < 0 . 01 ( significance was assessed by Mann - Whitney test , precise p - values given in the figure )

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MOG/CFA- but not CFA-injected mice contain reactive antibodies for retinal proteins in blood and show altered spatial visual behavior. (A) 7-9 days after injection, blood from MOG/CFA-injected (red) but not from CFA-injected mice (black), elicited strong antibody responses in ELISA. Antibodies for RIBEYE and SV2 were used as positive controls (green), secondary antibody only as a negative control (white). Normalized data are given as mean ± S.E.M. (N=4 (CFA); N=5 (MOG/CFA). ** p<0.01 (significance was assessed by Mann-Whitney test, precise p-values given in the figure)


Control Western blot of HEK293 protein lysates obtained from cells transfected with mCherry ( lane 1 ) or CASPR1 - mCherry cDNA ( lane 2 ) and probed with anti - mCherry

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Control Western blot of HEK293 protein lysates obtained from cells transfected with mCherry (lane 1) or CASPR1-mCherry cDNA (lane 2) and probed with anti-mCherry antibody


( C , D ) Western blot of protein lysates as in B ) probed by blood samples from mice before and 7 to 9 days after MOG / CFA injection . Only blood samples obtained from MOG / CFA - injected mice revealed immunoreactivity against CASPR1 ( lanes 15 , 16 ; top panels ) whereas samples obtained before immunization ( lanes 7 , 8 ; top panels ) did not . The same filters were re - probed with anti - actin antibodies as loading

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

(C,D) Western blot of protein lysates as in B) probed by blood samples from mice before and 7 to 9 days after MOG/CFA injection. Only blood samples obtained from MOG/CFA-injected mice revealed immunoreactivity against CASPR1 (lanes 15,16; top panels) whereas samples obtained before immunization (lanes 7,8; top panels) did not. The same filters were re-probed with anti-actin antibodies as loading control


( C , D ) Western blot of protein lysates as in B ) probed by blood samples from mice before and 7 to 9 days after MOG / CFA injection . Only blood samples obtained from MOG / CFA - injected mice revealed immunoreactivity against CASPR1 ( lanes 15 , 16 ; top panels ) whereas samples obtained before immunization ( lanes 7 , 8 ; top panels ) did not . The same filters were re - probed with anti - actin antibodies as loading control . ( E ) Summary of data from ( C , D ) shown as mean ± S . E . M . ( N = 4 , before injection ; N = 5 , MOG / CFA - injected ) . * * * , p < 0 . 001 ; n . s . , non - significant ( unpaired two - tailed Student ' s t - test , precise p - values given in the figure )

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(C,D) Western blot of protein lysates as in B) probed by blood samples from mice before and 7 to 9 days after MOG/CFA injection. Only blood samples obtained from MOG/CFA-injected mice revealed immunoreactivity against CASPR1 (lanes 15,16; top panels) whereas samples obtained before immunization (lanes 7,8; top panels) did not. The same filters were re-probed with anti-actin antibodies as loading control. (E) Summary of data from (C,D) shown as mean ± S.E.M. (N=4, before injection; N=5, MOG/CFA-injected). ***, p<0.001; n.s., non-significant (unpaired two-tailed Student's t-test, precise p-values given in the figure)


Spatially guided visual behavior , measured as frequency threshold with a virtual optokinetic system 9 days after immunization , is significantly decreased in MOG / CFA - injected mice compared to CFA - injected control litters . Data are shown as median ; boxes and whiskers illustrate 25 - 75 and 5 - 95 percentiles of values , respectively . Points below and above whiskers are drawn as individual points . At each trial , mice were measured three times . Home - made and commercial MOG / CFA suspensions were compared as well as PLP - injected mice with control - injected mice . Statistical analysis was done by ANOVA ( 0 , 05 significance level ) , mean comparison probabilities were Bonferroni corrected . N = 32 , n = 96 ( MOG / CFA ) ; N = 24 , n = 72 ( CFA ) . * * * , p < 0 . 001 ( Mann - Whitney test ) . Results from mice injected with home - made and commercial MOG / CFA suspensions as well as results obtained from PLP / CFA - injected mice ( and respective control - injected mice ) are

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

Spatially guided visual behavior, measured as frequency threshold with a virtual optokinetic system 9 days after immunization, is significantly decreased in MOG/CFA-injected mice compared to CFA-injected control litters. Data are shown as median; boxes and whiskers illustrate 25-75 and 5-95 percentiles of values, respectively. Points below and above whiskers are drawn as individual points. At each trial, mice were measured three times. Home-made and commercial MOG/CFA suspensions were compared as well as PLP-injected mice with control-injected mice. Statistical analysis was done by ANOVA (0,05 significance level), mean comparison probabilities were Bonferroni corrected. N=32, n=96 (MOG/CFA); N=24, n=72 (CFA). ***, p<0.001 (Mann-Whitney test). Results from mice injected with home-made and commercial MOG/CFA suspensions as well as results obtained from PLP/CFA-injected mice (and respective control-injected mice) are displayed


Optic nerve parameters are unaltered in early EAE ( 9 days after injection ) . ( A , B ) Cryostat sections of the optic nerve from MOG / CFA - injected mice ( own lab - made suspension ; for comparison with commercial MOG / CFA suspensions , see Appendix Fig . S12 ) and CFA - injected control mice were stained with anti - Nav antibody ( 1 : 50 dilution ) to label the node of Ranvier and with anti - CASPR1 antibody ( 1 : 500 dilution ) to stain the paranodal region . Length of the node of Ranvier and length of the paranodal region were quantified in C ) D )

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Optic nerve parameters are unaltered in early EAE (9 days after injection). (A,B) Cryostat sections of the optic nerve from MOG/CFA-injected mice (own lab-made suspension; for comparison with commercial MOG/CFA suspensions, see Appendix Fig. S12) and CFA-injected control mice were stained with anti-Nav antibody (1:50 dilution) to label the node of Ranvier and with anti-CASPR1 antibody (1:500 dilution) to stain the paranodal region. Length of the node of Ranvier and length of the paranodal region were quantified in C) D)


( E1 , E2 ) Richardson - Blue stained semi - thin sections from the indicated optic nerve samples . ( E2 , E3 , F2 , F3 ) Transmission electron micrographs of the optic nerves from MOG / CFA - and CFA - injected animals . Abbreviations : ax , axon ; my , myelin sheath . Axon number and myelin thickness were quantified in G ) and H ) . Scale bars : 1μm ( A , B , E , F )

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(E1, E2) Richardson-Blue stained semi-thin sections from the indicated optic nerve samples. (E2, E3, F2, F3) Transmission electron micrographs of the optic nerves from MOG/CFA- and CFA-injected animals. Abbreviations: ax, axon; my, myelin sheath. Axon number and myelin thickness were quantified in G) and H). Scale bars: 1μm (A,B,E,F)


Retinal slices from MOG / CFA - injected and CFA - injected SypHy mice were stimulated by applying a 25mM K + - containing depolarization solution for 1min ( Fig . 6A ) . In response to depolarization , we observed a strong increase in fluorescence ( Fig . 6A ) that can be best fitted by a double - exponential curve ( B ) . Amplitudes of fast and slow release ( together with the respective time constants ) of the first depolarization response are plotted in C )

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Retinal slices from MOG/CFA-injected and CFA-injected SypHy mice were stimulated by applying a 25mM K+-containing depolarization solution for 1min (Fig. 6A). In response to depolarization, we observed a strong increase in fluorescence (Fig. 6A) that can be best fitted by a double-exponential curve (B). Amplitudes of fast and slow release (together with the respective time constants) of the first depolarization response are plotted in C)


The amount of CASPR1 protein in the synapse is also altered in the PLP mouse model of multiple sclerosis . Semi - thin sections of PLP / CFA and CFA - injected mice were probed with the indicated antibodies ( A , B ; see also Appendix Fig . S13 )

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The amount of CASPR1 protein in the synapse is also altered in the PLP mouse model of multiple sclerosis. Semi-thin sections of PLP/CFA and CFA-injected mice were probed with the indicated antibodies (A, B; see also Appendix Fig. S13)


The synaptic amount of CASPR1 is strongly reduced both in the OPL and IPL . RIBEYE is only reduced in the OPL but not significantly in the IPL . CNTN1 is strongly reduced in both OPL and IPL ( C , see also Appendix Fig . S13 ) . N = 4 PLP / CFA - injected mice ; N = 4 CFA - injected mice ; n = 140 slices ( CFA , RIBEYE ) ; n = 143 slices ( PLP / CFA , RIBEYE ) ; n = 47 slices ( CFA , CASPR1 ) ; n = 53 slices ( PLP / CFA , CASPR1 ) ; n = 49 slices ( CFA , CNTN1 ) ; n = 46 slices ( PLP / CFA , CNTN1 ) ; n = 44 slices ( CFA , v - arrestin ) ; n = 44 slices ( PLP / CFA , v - arrestin )

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The synaptic amount of CASPR1 is strongly reduced both in the OPL and IPL. RIBEYE is only reduced in the OPL but not significantly in the IPL. CNTN1 is strongly reduced in both OPL and IPL (C, see also Appendix Fig. S13). N=4 PLP/CFA-injected mice; N=4 CFA-injected mice; n=140 slices (CFA, RIBEYE); n=143 slices (PLP/CFA, RIBEYE); n=47 slices (CFA, CASPR1); n=53 slices (PLP/CFA, CASPR1); n=49 slices (CFA, CNTN1); n=46 slices (PLP/CFA, CNTN1); n=44 slices (CFA, v-arrestin); n=44 slices (PLP/CFA, v-arrestin)


( D , E ) Cryostat sections of the optic nerve from PLP and control mice were stained with anti - Nav - channel antibody to label the node of Ranvier and with anti - CASPR1 antibody to stain the paranodal region . Length of the node of Ranvier and length of the paranodal region were quantified in G ) H ) . ( F ) transmission electron micrographs of the optic nerves from PLP / CFA - and CFA - injected animals ( F1 - F4 ) . Axon numbers and myelin thickness were quantified in I ) J )

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(D,E) Cryostat sections of the optic nerve from PLP and control mice were stained with anti-Nav-channel antibody to label the node of Ranvier and with anti-CASPR1 antibody to stain the paranodal region. Length of the node of Ranvier and length of the paranodal region were quantified in G) H). (F) transmission electron micrographs of the optic nerves from PLP/CFA- and CFA-injected animals (F1-F4). Axon numbers and myelin thickness were quantified in I) J)


Confocal micrographs show CK7 ( green ) and vimentin ( red ) in patient - derived HGSC cells cultured in 3D collagen for 7 d . Scale bars : 50 μm .

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Confocal micrographs show CK7 (green) and vimentin (red) in patient-derived HGSC cells cultured in 3D collagen for 7 d. Scale bars: 50 μm.


Confocal micrographs of EphA2 ( red ) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin ( 5 μM for OCKI _ p01 ) . The intensity of EphA2 is comparable only between mock and treatment conditions for each patient . Scale bars : 20 μm .

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Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm.


Chart illustrates EphA2 fold change after treatment . Mock is set to one . N = 3 .

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Chart illustrates EphA2 fold change after treatment. Mock is set to one. N = 3.


EphA2 ( total and phosphorylated at S897 or Y588 ) in OVCAR3 , OVCAR4 and OVCAR8 after treatment with 0 - 20 μM cisplatin for 72 h was assessed by immunoblotting ( A ) and quantified for pS897 / pY588 ratio ( B ) . N = 4 .

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EphA2 (total and phosphorylated at S897 or Y588) in OVCAR3, OVCAR4 and OVCAR8 after treatment with 0-20 μM cisplatin for 72 h was assessed by immunoblotting (A) and quantified for pS897/pY588 ratio (B). N = 4.


EphA2 ( total and phosphorylated ) in corresponding untreated cells .

EphA2 (total and phosphorylated) in corresponding untreated cells.


Cytotoxicity assay results after cell treatment with 0 - 20 μM cisplatin for 72 h . N = 6 .

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Cytotoxicity assay results after cell treatment with 0-20 μM cisplatin for 72 h. N = 6.


Quantitative assessment of EphA2 ( total and phosphorylated ) and pS897 / pY588 ratio in early passage patient - derived HGSC cultures treated with 0 - 20 μM cisplatin for 72 h ( see immunoblots in Appendix Fig S2A ) . N = 6 patients , pooled .

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Quantitative assessment of EphA2 (total and phosphorylated) and pS897/pY588 ratio in early passage patient-derived HGSC cultures treated with 0-20 μM cisplatin for 72 h (see immunoblots in Appendix Fig S2A). N = 6 patients, pooled.


Corresponding EphA2 pS897 / pY588 ratios for individual patient cells .

Corresponding EphA2 pS897/pY588 ratios for individual patient cells.


EphA2 ( total and phosphorylated ) in OVCAR4 and OVCAR8 after treatment with 0 - 80 μM carboplatin for 72 h ( G ) along with pS897 / pY588 quantification ( H ) . N = 3 .

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EphA2 (total and phosphorylated) in OVCAR4 and OVCAR8 after treatment with 0-80 μM carboplatin for 72 h (G) along with pS897/pY588 quantification (H). N = 3.


Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors ( day 53 ; after 13 d i . p . treatment ) . Color scale unit : p / sec / cm2 / sr . Charts illustrate the tumor growth ( B ) and ascites volumes at the end of the follow - up ( C ) . Carboplatin treatment started on day 35 . Bioluminescent unit : p / sec / cm2 / sr . N = 5 mice / group .

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Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors (day 53; after 13 d i.p. treatment). Color scale unit: p/sec/cm2/sr. Charts illustrate the tumor growth (B) and ascites volumes at the end of the follow-up (C). Carboplatin treatment started on day 35. Bioluminescent unit: p/sec/cm2/sr. N = 5 mice/group.


Representative micrographs of hematoxylin - eosin staining in the xenografts tumors . Scale bars : 100

Representative micrographs of hematoxylin-eosin staining in the xenografts tumors. Scale bars: 100 μm


EphA2 - pS897 immunofluorescence in the xenografts tumors . Scale bars : 10

EphA2-pS897 immunofluorescence in the xenografts tumors. Scale bars: 10 μm


Quantitative assessment of tumor EphA2 ( F ) and EphA2 - pS897 ( G ) . N = 5 mice / group .

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Quantitative assessment of tumor EphA2 (F) and EphA2-pS897 (G). N = 5 mice/group.


Confocal micrographs of tumor EphA2 - pS897 ( red ) and cleaved caspase - 3 ( H ; clCasp3 , green ) and corresponding clCasp3 quantification ( I ) . Scale bars : 20 μm . N = 5 mice / group .

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Confocal micrographs of tumor EphA2-pS897 (red) and cleaved caspase-3 (H; clCasp3, green) and corresponding clCasp3 quantification (I). Scale bars: 20 μm. N = 5 mice/group.


Immunoblot images show EphA2 ( A ; total , pS897 and pY588 ) and corresponding pS897 / pY588 ratios ( B ) of TYK - nu and TYK - nu . R after treatment with 0 - 10 μM cisplatin for 72 h . N = 5 .

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Immunoblot images show EphA2 (A; total, pS897 and pY588) and corresponding pS897/pY588 ratios (B) of TYK-nu and TYK-nu.R after treatment with 0-10 μM cisplatin for 72 h . N = 5.


EphA2 ( total and phosphorylated ) in the cells treated with 0 - 80 μM carboplatin for 72 h .

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EphA2 (total and phosphorylated) in the cells treated with 0-80 μM carboplatin for 72 h.


EphA2 ( total and phosphorylated ) in the

EphA2 (total and phosphorylated) in the cells


EphA2 ( total and pS897 ) in TYK - nu after treatment with inhibitors against MEK ( UO126 , 10 μM ) , PKA ( H89 . 2HCl , 10 μM ) , PI3K ( LY294002 , 10 μM ) or its alpha subunit ( BYL - 719 , 5 μM ) and RSK ( BI - D1870 , 10 μM ) in combination with 0 - 10 μM cisplatin for 72 h ( E ) . Corresponding EphA2 - pS897 quantification for BI - D1870 treatment ( F ) N = 5 .

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EphA2 (total and pS897) in TYK-nu after treatment with inhibitors against MEK (UO126, 10 μM), PKA (H89.2HCl, 10 μM), PI3K (LY294002, 10 μM) or its alpha subunit (BYL-719, 5 μM) and RSK (BI-D1870, 10 μM) in combination with 0-10 μM cisplatin for 72 h (E). Corresponding EphA2-pS897 quantification for BI-D1870 treatment (F) N = 5.


EphA2 ( total and pS897 ) in TYK - nu . R treated with 0 - 20 μM cisplatin without or with 10 μM BI - D1870 for 72 h .

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EphA2 (total and pS897) in TYK-nu.R treated with 0-20 μM cisplatin without or with 10 μM BI-D1870 for 72 h.


EphA2 , RSK and ERK1 / 2 ( total and phosphorylated ) in TYK - nu ( H ) and OVCAR4 ( I ) treated with 0 - 30 μM carboplatin without or with 50 μM LJH685 for 72 h .

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EphA2, RSK and ERK1/2 (total and phosphorylated) in TYK-nu (H) and OVCAR4 (I) treated with 0-30 μM carboplatin without or with 50 μM LJH685 for 72 h.


Charts show OC cell viability after 72 h treatment with 0 - 20 μM cisplatin and 10 μM BI -

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Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 10 μM BI-D1870


Charts show OC cell viability after 72 h treatment with 0 - 20 μM cisplatin and 50 μM

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Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 50 μM LJH685


RSK and EphA2 ( total and phosphorylated ) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h .

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RSK and EphA2 (total and phosphorylated) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h.


Charts illustrate the corresponding quantified inhibition of EphA2 - pS897 and increase of EphA2 - pY588 shown in ( C ) .

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Charts illustrate the corresponding quantified inhibition of EphA2-pS897 and increase of EphA2-pY588 shown in (C).


Immunoblot images ( A ) and quantification ( B ) show GPRC5A 46 / 41 kDa ratios in OC cells treated with 0 - 10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h . The ratio in mock cells was set to 1 . N = 4 .

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Immunoblot images (A) and quantification (B) show GPRC5A 46/41 kDa ratios in OC cells treated with 0-10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h. The ratio in mock cells was set to 1. N = 4.


Confocal micrographs of EphA2 ( red ) and GPRC5A ( green ) in TYK - nu and TYK - nu . R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h . Arrows point co - localization of the receptors . Scale bars : 20 μm .

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Confocal micrographs of EphA2 (red) and GPRC5A (green) in TYK-nu and TYK-nu.R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h. Arrows point co-localization of the receptors. Scale bars: 20 μm.


EphA2 ( total and phosphorylated ) , RSK1 / 2 and GPRC5A in TYK - nu were assessed by immunoblotting after RSK1 / 2 knockdown for 2 d and following treatment with 0 - 5 μM cisplatin for 72 h . Asterisks indicate unspecific bands .

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EphA2 (total and phosphorylated), RSK1/2 and GPRC5A in TYK-nu were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.


EphA2 ( total and phosphorylated ) , RSK1 / 2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK - nu . R were assessed by immunoblotting after RSK1 / 2 knockdown for 2 d and following treatment with 0 - 5 μM cisplatin for 72 h . Asterisks indicate unspecific bands .

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EphA2 (total and phosphorylated), RSK1/2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK-nu.R were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.


Chart illustrates the viability of the RSK1 / 2 depleted TYK - nu . R after cisplatin treatment . N = 3 .

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Chart illustrates the viability of the RSK1/2 depleted TYK-nu.R after cisplatin treatment. N = 3.


Confocal micrographs show cleaved caspase - 3 ( clCasp3 , green ) , mRFP ( orange ) and phalloidin ( F - actin , red ; only shown for CAF ) in 3D OVCAR8 - RFP and OCKI _ p22 CAF mono - and co - cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h . Scale bars : 100 μm .

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Confocal micrographs show cleaved caspase-3 (clCasp3, green), mRFP (orange) and phalloidin (F-actin, red; only shown for CAF) in 3D OVCAR8-RFP and OCKI_p22 CAF mono- and co-cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 100 μm.


Quantitative assessment of clCasp3 + cells in the 3D cultures . N = 4 .

Quantitative assessment of clCasp3+ cells in the 3D cultures. N = 4.


Confocal micrographs show cytokeratin 7 ( CK7 , green ) and phalloidin ( F - actin , red ) in 3D patient OCKI _ p13 mono - and co - culture with OCKI _ p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h . Scale bars : 50 μm . Quantitative assessment of clCasp3 + OCKI _ p13 cells in 3D co - cultures of patient - derived cells . N = 4 .

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Confocal micrographs show cytokeratin 7 (CK7, green) and phalloidin (F-actin, red) in 3D patient OCKI_p13 mono- and co-culture with OCKI_p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 50 μm. Quantitative assessment of clCasp3+ OCKI_p13 cells in 3D co-cultures of patient-derived cells. N = 4.


Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock ( N = 4 ) , carboplatin ( N = 4 ) and carboplatin + BI - D1870 ( N = 5 ) treated mice . Scale bars : 100 μm .

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Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.


Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock ( N = 4 ) , carboplatin ( N = 4 ) and carboplatin + BI - D1870 ( N = 5 ) treated mice . Scale bars : 100 μm .

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Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.


Representative confocal micrographs show EphA2 ( red ) , RSK ( green : top ) and GPRC5A ( green : bottom ) in frozen sections of HGSC patient tumors . S indicates the stroma . Scale bars : 50 μm .

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Representative confocal micrographs show EphA2 (red), RSK (green: top) and GPRC5A (green: bottom) in frozen sections of HGSC patient tumors. S indicates the stroma. Scale bars: 50 μm.


Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient - derived HGSC cells and CAFs .

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Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient-derived HGSC cells and CAFs.


GPRC5A in earlier ( top ) and later passage ( bottom ) HGSC cells . See normalized GPRC5A relative to OCKI _ p01 below the immunoblot images .

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GPRC5A in earlier (top) and later passage (bottom) HGSC cells. See normalized GPRC5A relative to OCKI_p01 below the immunoblot images.


Charts illustrate EphA2 - pS897 inhibition ( circles ) and EphA2 - pY588 increase ( squares ) by 10 µM BI - D1870 alone ( unbroken line ) or in combination with 0 - 10 µM cisplatin ( dotted line ) in the GPRC5Ahigh HGSC cells . See Appendix S7B for immunoblots .

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Charts illustrate EphA2-pS897 inhibition (circles) and EphA2-pY588 increase (squares) by 10 µM BI-D1870 alone (unbroken line) or in combination with 0-10 µM cisplatin (dotted line) in the GPRC5Ahigh HGSC cells. See Appendix S7B for immunoblots.


Cell viability of the GPRC5Ahigh OCKI _ p02 and OCKI _ p06 after treatment with a combination of 0 - 20 µM cisplatin and 10 µM BI - D1870 for 72 h . N = 3 .

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Cell viability of the GPRC5Ahigh OCKI_p02 and OCKI_p06 after treatment with a combination of 0-20 µM cisplatin and 10 µM BI-D1870 for 72 h. N = 3.


Cell viability upon 72 h combinatorial treatment with 0 - 20 µM cisplatin and 25 µM LJH685 in patient - derived

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Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 25 µM LJH685 in patient-derived CAFs


Cell viability upon 72 h combinatorial treatment with 0 - 20 µM cisplatin and 10 µM BI - D1870 in mesothelial cells ( G ; N = 3 ) .

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Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 10 µM BI-D1870 in mesothelial cells (G; N = 3).


Chart illustrates the changes of EphA2 - pS897 and EphA2 - pY588 by BI - D1870 alone and with cisplatin in mesothelial cells . Upon combination with 10 µM cisplatin for 72 h , 10 µM BI - D1870 decreased EphA2 - pS897 and EphA2 - pY588 . See Appendix Fig S7J for immunoblot .

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Chart illustrates the changes of EphA2-pS897 and EphA2-pY588 by BI-D1870 alone and with cisplatin in mesothelial cells. Upon combination with 10 µM cisplatin for 72 h, 10 µM BI-D1870 decreased EphA2-pS897 and EphA2-pY588. See Appendix Fig S7J for immunoblot.


Confocal micrographs of EphA2 ( red ) and GPRC5A ( green ) in primary OCKI _ p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin . Scale bars : 20 μm .

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Confocal micrographs of EphA2 (red) and GPRC5A (green) in primary OCKI_p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin. Scale bars: 20 μm.


Representative immunostainings for GPRC5A scoring in HGSC primary and metastatic tumors . For maximum intensity scores an optical 4 - point scale ( 0 : negative , 1 : mild , 2 : moderate , 3 : high ) was used . For survival analyses a 2 - point scale ( low : scores 0 - 2 , high : score 3 ) was used . Scale bars : 200 μm .

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Representative immunostainings for GPRC5A scoring in HGSC primary and metastatic tumors. For maximum intensity scores an optical 4-point scale (0: negative, 1: mild, 2: moderate, 3: high) was used. For survival analyses a 2-point scale (low: scores 0-2, high: score 3) was used. Scale bars: 200 μm.


Kaplan Meier survival curves illustrate the overall ( OS ) and progression - free ( PFS ) survival of patients with high or low GPRC5A . In primary tumors , mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow ( C ) . In patients with metastatic tumors , mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow . Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow ( D ) . No significant difference in mean PFS in primary tumor .

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Kaplan Meier survival curves illustrate the overall (OS) and progression-free (PFS) survival of patients with high or low GPRC5A. In primary tumors, mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow (C). In patients with metastatic tumors, mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow. Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow (D). No significant difference in mean PFS in primary tumor.


Kaplan Meier survival estimates for the PFS of patients with high or low ( top 25 % vs bottom 25 % ) GPRC5A ( F ) , EPHA2 ( G ) , GPRC5A + EPHA2 ( H ) and GPRC5A + EPHA2 + RSK1 ( RPS6KA1 ) + RSK2 ( RPS6KA3 ) combined ( I ) mRNA expression . Mean PFS for GPRC5A + EPHA2high was 15 months vs 26 months for GPRC5A + EPHA2low ( H ) . Mean PFS for GPRC5A + EPHA2 + RPS6KA1 / 3 high was 14 months vs 31 months for GPRC5A + EPHA2 + RPS6KA1 / 3low ( I ) .

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Kaplan Meier survival estimates for the PFS of patients with high or low (top 25% vs bottom 25%) GPRC5A (F), EPHA2 (G), GPRC5A+EPHA2 (H) and GPRC5A+EPHA2+RSK1(RPS6KA1)+RSK2(RPS6KA3) combined (I) mRNA expression. Mean PFS for GPRC5A+EPHA2high was 15 months vs 26 months for GPRC5A+EPHA2low (H). Mean PFS for GPRC5A+EPHA2+RPS6KA1/3 high was 14 months vs 31 months for GPRC5A+EPHA2+RPS6KA1/3low (I).


Assays were performed in HEK293 cells for A ) F508del - CFTR For the F508del - CFTR measurements since this mutant does not respond well to Fsk alone ( Fsk + VX - 770 potentiator is required to obtain a detectable signal ) we compared the CFTR inh172 ( inhibitor ) response ( F1 / ' F - CFTRinh172 ' ; where F1 represents peak RFU immediately before inhibitor addition and ' F - CFTRinh172 ' is the minimum RFU reached at the end of the CFTR inh172 measurement cycle ) . For each of F508del - CFTR data for three proteins not detected as interactors ( first three bars following CFTR only ) are shown as negative controls . Fluorescence signals were normalized to the CFTR - only control and the statistical significance of differences assed by unpaired t - test ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Samples run in n = 3 replicates , and all values are mean ± s . d .

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Assays were performed in HEK293 cells for A) F508del-CFTR For the F508del-CFTR measurements since this mutant does not respond well to Fsk alone (Fsk + VX-770 potentiator is required to obtain a detectable signal) we compared the CFTR inh172 (inhibitor) response (F1/'F-CFTRinh172'; where F1 represents peak RFU immediately before inhibitor addition and 'F-CFTRinh172' is the minimum RFU reached at the end of the CFTR inh172 measurement cycle). For each of F508del-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.


Assays were performed in HEK293 cells for B ) wt - CFTR . For the wildtype measurements , we compared the maximum Fsk response ( Maximum F / F0 ; where F0 represents baseline RFU right before the stimulation ) . For each of wt - CFTR data for three proteins not detected as interactors ( first three bars following CFTR only ) are shown as negative controls . Fluorescence signals were normalized to the CFTR - only control and the statistical significance of differences assed by unpaired t - test ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Samples run in n = 3 replicates , and all values are mean ± s . d .

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Assays were performed in HEK293 cells for B) wt-CFTR. For the wildtype measurements, we compared the maximum Fsk response (Maximum F /F0; where F0 represents baseline RFU right before the stimulation). For each of wt-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.


A ) Blot images showing bands B and C as well as the calnexin internal control .

A) Blot images showing bands B and C as well as the calnexin internal control.


B ) Blot quantification showing relative fold change of band B and band C versus treatment with a non - targeting siRNA ( Neg1 ) . One - way Anova followed by Dunnett ' s post hoc test was performed to determine significance versus baseline ( siNeg1 + DMSO treatment , * p < 0 . 05 ) and 5 µM VX - 661 ( # p < 0 . 05 ) . Measurements were obtained from biological triplicates ( n = 3 ) and all values are mean ± s . d .

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B) Blot quantification showing relative fold change of band B and band C versus treatment with a non-targeting siRNA (Neg1). One-way Anova followed by Dunnett's post hoc test was performed to determine significance versus baseline (siNeg1 + DMSO treatment, *p<0.05) and 5 µM VX-661 (#p<0.05). Measurements were obtained from biological triplicates (n=3) and all values are mean ± s.d.


B ) qPCR Validation of lentivirus - mediated shRNA knockdown of FGL2 and CFTR in intestinal organoids . Significance of sample differences was calculated using the one - tailed student t - test ( n = 2 biological replicates ) . All values are mean ± s . d . EV = empty vector .

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B) qPCR Validation of lentivirus-mediated shRNA knockdown of FGL2 and CFTR in intestinal organoids. Significance of sample differences was calculated using the one-tailed student t-test (n=2 biological replicates). All values are mean ± s.d. EV = empty vector.


C ) Representative confocal microscopy images of calcein green labelled FIS organoids of a healthy individual and a CF patient , before and 60 min after stimulation with Fsk ( 5 uM ) . Scale bar , 200 μm .

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C) Representative confocal microscopy images of calcein green labelled FIS organoids of a healthy individual and a CF patient, before and 60 min after stimulation with Fsk (5 uM). Scale bar, 200 μm.


D ) Quantification of FIS response in wt - CFTR organoids ( ORG - 01 ( HC ) ) shown in ( C ) as area under the curve at 60 min . Significance of sample differences was calculated using two - way ANOVA ( n = 2 biological replicates ) . All values are mean ± s . d . E ) Quantification of FIS response in F508del - CFTR organoids ( ORG - BX - 002 ( dF / dF ) ) shown in ( C ) as area under the curve at 60 min . Significance of sample differences was calculated using two - way ANOVA ( n = 2 biological replicates ) . All values are mean ± s . d .

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D) Quantification of FIS response in wt-CFTR organoids (ORG-01(HC)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d. E) Quantification of FIS response in F508del-CFTR organoids (ORG-BX-002 (dF/dF)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d. F


Wild type ( WT ) and TRIM21 knockout ( KO ) mice were infected with 10 ^ 5 FFU LCMV clone 13 ( 6 mice per group ) and 2 mice were uninfected ( UI ) . Weights were compared over 15 days post infection ( pi ) .

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Wild type (WT) and TRIM21 knockout (KO) mice were infected with 10^5 FFU LCMV clone 13 (6 mice per group) and 2 mice were uninfected (UI). Weights were compared over 15 days post infection (pi).


Wild type ( WT ) and TRIM21 knockout ( KO ) mice were infected with 10 ^ 5 FFU LCMV clone 13 ( 6 mice per group ) and 2 mice were uninfected ( UI ) . Kaplan - Meier survival were compared over 15 days post infection ( pi ) .

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Wild type (WT) and TRIM21 knockout (KO) mice were infected with 10^5 FFU LCMV clone 13 (6 mice per group) and 2 mice were uninfected (UI). Kaplan-Meier survival were compared over 15 days post infection (pi).


C ) Sera from WT mice was analysed for N protein ( N ) and glycoprotein ( GP ) - specific antibodies by ELISA , each data point corresponds to one mouse from the same experiment .

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C) Sera from WT mice was analysed for N protein (N) and glycoprotein (GP)-specific antibodies by ELISA, each data point corresponds to one mouse from the same experiment.


D ) N - specific antibody ELISA was used to compare antibody responses in sera of WT and KO mice .

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D) N-specific antibody ELISA was used to compare antibody responses in sera of WT and KO mice.


E - F ) In vitro neutralisation experiments were performed with sera collected day 8pi from WT , KO and UI mice . E ) Sera was pre - incubated with LCMV , then the virus - serum mix was added to MEF cells and LCMV infection titre after 16 hours was measured by FFA . F ) Sera was electroporated into MEFs , then cells were plated in triplicate and LCMV was added 4 hours later . LCMV infection titre after 16 hours was measured by FFA ( three replicates ) .

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E-F) In vitro neutralisation experiments were performed with sera collected day 8pi from WT, KO and UI mice. E) Sera was pre-incubated with LCMV, then the virus-serum mix was added to MEF cells and LCMV infection titre after 16 hours was measured by FFA. F) Sera was electroporated into MEFs, then cells were plated in triplicate and LCMV was added 4 hours later. LCMV infection titre after 16 hours was measured by FFA (three replicates).


G ) Anti - N mAb KL53 was electroporated into WT and KO MEFs and subsequent LCMV infection titres were measured by FFA .

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G) Anti-N mAb KL53 was electroporated into WT and KO MEFs and subsequent LCMV infection titres were measured by FFA.


H ) Anti - N mAb KL53 was co - electroporated with recombinant N protein into WT and KO MEFs , and immunoblotting for N was performed after 3 hours . Electroporation of recombinant KL53 expressing the TRIM21 non - binding mutation H433A was unable to mediate N protein degradation .

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H) Anti-N mAb KL53 was co-electroporated with recombinant N protein into WT and KO MEFs, and immunoblotting for N was performed after 3 hours. Electroporation of recombinant KL53 expressing the TRIM21 non-binding mutation H433A was unable to mediate N protein degradation.


Wild type ( WT ) and TRIM21 knockout ( KO ) mice were infected with 0 . 5 x 10 ^ 5 FFU LCMV clone 13 ( 6 mice per group ) , and either received anti - N mAb KL53 ( + ) or control ( - ) intraperitoneally on days 1 and 3pi . Two mice were uninfected ( UI ) . A ) Weights were monitored throughout infection , with final day 8 weights of individual mice presented separately .

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Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). A) Weights were monitored throughout infection, with final day 8 weights of individual mice presented separately.


Wild type ( WT ) and TRIM21 knockout ( KO ) mice were infected with 0 . 5 x 10 ^ 5 FFU LCMV clone 13 ( 6 mice per group ) , and either received anti - N mAb KL53 ( + ) or control ( - ) intraperitoneally on days 1 and 3pi . Two mice were uninfected ( UI ) . Viral titres in the spleen , liver , lung and kidney of all mice were determined by FFA day 8pi . Each data point represents one mouse , with results from two repeat experiments combined .

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Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). Viral titres in the spleen, liver, lung and kidney of all mice were determined by FFA day 8pi. Each data point represents one mouse, with results from two repeat experiments combined.


A ) CD8 T cells in WT mice were depleted by administration of anti - CD8 mAb 1 day prior to infection with 10 ^ 5 FFU LCMV . Anti - N mAb KL53 or PBS control was passively transferred IP on days 1 and 3pi . Viral titres in the spleen , lung , liver and kidney of all mice were determined by FFA day 8pi .

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A) CD8 T cells in WT mice were depleted by administration of anti-CD8 mAb 1 day prior to infection with 10^5 FFU LCMV. Anti-N mAb KL53 or PBS control was passively transferred IP on days 1 and 3pi. Viral titres in the spleen, lung, liver and kidney of all mice were determined by FFA day 8pi.


B ) Spleens from WT and KO mice + / - mAb KL53 day 8pi with 0 . 5 x 10 ^ 5 FFU LCMV were analysed for the presence of LCMV N - specific CTLs by staining with the class I N396 - 404 tetramer .

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B) Spleens from WT and KO mice +/- mAb KL53 day 8pi with 0.5 x 10^5 FFU LCMV were analysed for the presence of LCMV N-specific CTLs by staining with the class I N396-404 tetramer.


C ) Spleens from naïve WT and KO mice day 10pi with 0 . 5 x 10 ^ 5 FFU LCMV were stained with N tetramer .

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C) Spleens from naïve WT and KO mice day 10pi with 0.5 x 10^5 FFU LCMV were stained with N tetramer.


D ) Macrophages were depleted in WT mice by administration of clodronate liposomes 1 day prior to infection with 0 . 5 x 10 ^ 5 FFU LCMV . KL53 was administered on days 1 and 3pi , and N396 - specific CTLs in the spleen were measured day 8pi by tetramer staining .

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D) Macrophages were depleted in WT mice by administration of clodronate liposomes 1 day prior to infection with 0.5 x 10^5 FFU LCMV. KL53 was administered on days 1 and 3pi, and N396-specific CTLs in the spleen were measured day 8pi by tetramer staining.


A ) Splenocytes from uninfected CD45 . 1 mice , either pulsed with 3 concentrations of N peptide and cell trace violet ( CTV ) or unlabelled control cells , were transfused intravenously into WT and KO mice ( CD45 . 2 ) that had been infected with 0 . 5 x 10 ^ 5 FFU LCMV 8 days earlier . After 3 hours , spleens from recipient mice were harvested and the proportion of CTV - labelled CD45 . 1 cells was analysed by flow cytometry . Histograms from single representative uninfected ( UI ) , WT and KO mice are presented , showing the proportion of CD45 . 1 cells remaining for each of the labelled fractions normalised to mode . Summary data from all individual mice in the same experiment is presented in associated scatter plot , showing the mean ± standard error . B ) Labelled splenocytes as for ( A ) were transfused into WT and KO mice that had been infected with LCMV 8 days earlier , and received mAb KL53 on days 1 and 3pi . Flow cytometry histograms from single representative mice of each genotype . Summary data from all mice in the experiment is presented , showing the mean ± standard error .

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A) Splenocytes from uninfected CD45.1 mice, either pulsed with 3 concentrations of N peptide and cell trace violet (CTV) or unlabelled control cells, were transfused intravenously into WT and KO mice (CD45.2) that had been infected with 0.5 x 10^5 FFU LCMV 8 days earlier. After 3 hours, spleens from recipient mice were harvested and the proportion of CTV-labelled CD45.1 cells was analysed by flow cytometry. Histograms from single representative uninfected (UI), WT and KO mice are presented, showing the proportion of CD45.1 cells remaining for each of the labelled fractions normalised to mode. Summary data from all individual mice in the same experiment is presented in associated scatter plot, showing the mean ± standard error. B) Labelled splenocytes as for (A) were transfused into WT and KO mice that had been infected with LCMV 8 days earlier, and received mAb KL53 on days 1 and 3pi. Flow cytometry histograms from single representative mice of each genotype. Summary data from all mice in the experiment is presented, showing the mean ± standard error.


Genes with higher dominant PAS usage ( DPU ) ( A ) or with lower APA Shannon index ( B ) express at higher levels ( liver shown as example ) . The number of genes in each group is indicated on top . Mann - Whitney U test significance levels are indicated for neighboring groups : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . The boxplots show the Q1 to Q3 quartile values ( the box limits ) , the median ( the horizontal green lines ) and values within the 1 . 5 * IQR ( the whiskers ) .

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Genes with higher dominant PAS usage (DPU) (A) or with lower APA Shannon index (B) express at higher levels (liver shown as example). The number of genes in each group is indicated on top. Mann-Whitney U test significance levels are indicated for neighboring groups: *P < 0.05; **P < 0.01; ***P < 0.001. The boxplots show the Q1 to Q3 quartile values (the box limits), the median (the horizontal green lines) and values within the 1.5 * IQR (the whiskers).


Spearman correlations between gene expression level and dominant PAS usage ( DPU ) in each tissue for all genes ( blue ) , genes with DPU equal to or above 90 % and genes with DPU less than 90 % . Genes with DPU less than 90 % exhibit weaker Spearman correlations between gene expression level and dominant PAS usage in each tissue . An empty square indicates that the P value of Spearman ' s rho is above 0 . 01 . ( ESC : embryonic stem cells , CER : cerebellum , COR : cerebral cortex , HEA : heart , KID : kidney , LIV : liver , LUN : lung , MUS : muscle , SPL : spleen . )

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

Spearman correlations between gene expression level and dominant PAS usage (DPU) in each tissue for all genes (blue), genes with DPU equal to or above 90% and genes with DPU less than 90%. Genes with DPU less than 90% exhibit weaker Spearman correlations between gene expression level and dominant PAS usage in each tissue. An empty square indicates that the P value of Spearman's rho is above 0.01. (ESC: embryonic stem cells, CER: cerebellum, COR: cerebral cortex, HEA: heart, KID: kidney, LIV: liver, LUN: lung, MUS: muscle, SPL: spleen.)


Fractions of orthologous genes in different groups exhibiting different subcellular localizations of mRNA isoforms in rat neurons . Same : isoforms enriched in same cellular location . Diff : isoforms enriched in different cellular locations , i . e . one isoform in neuropil and another one in soma . ( Fisher exact test : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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

Fractions of orthologous genes in different groups exhibiting different subcellular localizations of mRNA isoforms in rat neurons. Same: isoforms enriched in same cellular location. Diff: isoforms enriched in different cellular locations, i.e. one isoform in neuropil and another one in soma. (Fisher exact test: *P < 0.05; **P < 0.01; ***P < 0.001).


2D clustering of Pearson correlation coefficients between all pairwise sample combinations of C57BL / 6J ( BL6 ) or SPRET / EiJ ( SPR ) allelic PAS usages from 5 , 264 genes ( see Methods ) . 2D clustering of Pearson correlation coefficients between all pairwise combinations of BL6 or SPR allelic gene expression levels from 5 , 264 genes . All clustering was performed hierarchically using Pearson correlations between samples .

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

2D clustering of Pearson correlation coefficients between all pairwise sample combinations of C57BL/6J (BL6) or SPRET/EiJ (SPR) allelic PAS usages from 5,264 genes (see Methods). 2D clustering of Pearson correlation coefficients between all pairwise combinations of BL6 or SPR allelic gene expression levels from 5,264 genes. All clustering was performed hierarchically using Pearson correlations between samples.


B - K . Transmission electron microscopy ( TEM ) images of 5 week old adult retinas showing the overall structure of groups of ommatidia in ( B ) wild type ; ( C ) ADAM17 - / - mutant ; ( D ) ADAM17 - / Deficiency ; ( E , F ) RNAi against ADAM17 and control ( lacZ ) , expressed throughout the retina ; ( G , H ) RNAi against ADAM17 and control ( lacZ ) , expressed in the neurons ; ( I , J ) RNAi against ADAM17 and control ( lacZ ) , expressed in the pigmented glial cells ; and ( K ) a kuz - / - mutant .

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

B-K. Transmission electron microscopy (TEM) images of 5 week old adult retinas showing the overall structure of groups of ommatidia in (B) wild type; (C) ADAM17-/- mutant; (D) ADAM17-/Deficiency; (E, F) RNAi against ADAM17 and control (lacZ), expressed throughout the retina; (G, H) RNAi against ADAM17 and control (lacZ), expressed in the neurons; (I,J) RNAi against ADAM17 and control (lacZ), expressed in the pigmented glial cells; and (K) a kuz-/- mutant.


L . Quantitation of the percentage of normal , abnormal and missing rhabdomeres from TEM images for the genotypes above , expressed as a stacked bar chart ; n = 180 ommatidia from 3 different fly retinas for each .

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

L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from TEM images for the genotypes above, expressed as a stacked bar chart; n=180 ommatidia from 3 different fly retinas for each.


M . Whole - mount retina stained with anti - ADAM17 ( green ) and phalloidin to mark the photoreceptor membranes ( red ) .

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M. Whole-mount retina stained with anti-ADAM17 (green) and phalloidin to mark the photoreceptor membranes (red).


N . Line intensity profiles delineating expression of ADAM17 and phalloidin across individual ommatidia , n = 10 fly retinas . Scale bars : 10μm .

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N. Line intensity profiles delineating expression of ADAM17 and phalloidin across individual ommatidia, n=10 fly retinas. Scale bars: 10μm.


A - C . TEM images of 1day old adult retinas showing the overall structure of clusters of ommatidia ( A1 , B1 , C1 ) , or a single ommatidium ( A2 , B2 , C2 ) in wild type ( A1 , A2 ) , ADAM17 - / - mutant ( B1 , B2 ) or ADAM17 - / Df ( C1 , C2 ) . Red arrows show lipid droplets in PGCs , green shading highlights PGCs . Scale bars for A2 , B2 and C2 : 2μm and 10μm for all other panels .

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

A-C. TEM images of 1day old adult retinas showing the overall structure of clusters of ommatidia (A1, B1, C1), or a single ommatidium (A2, B2, C2) in wild type (A1, A2), ADAM17-/- mutant (B1, B2) or ADAM17-/Df (C1, C2). Red arrows show lipid droplets in PGCs, green shading highlights PGCs. Scale bars for A2, B2 and C2: 2μm and 10μm for all other panels.


D - J . Fluorescent images of 1 day old fly retinas stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and the photoreceptor membranes respectively ; ( D ) wild type ; ( E ) ADAM17 - / - mutant ; ( F ) ADAM17 - / Deficiency ; ( G ) knockdown of ADAM17 throughout the retina ; ( H ) knockdown in glial cells ; ( I ) knockdown in neurons ; and ( J ) kuz - / - ; n = 10 fly retinas . Scale bars

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

D-J. Fluorescent images of 1 day old fly retinas stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and the photoreceptor membranes respectively; (D) wild type; (E) ADAM17-/- mutant; (F) ADAM17-/ Deficiency; (G) knockdown of ADAM17 throughout the retina; (H) knockdown in glial cells; (I) knockdown in neurons; and (J) kuz-/-; n=10 fly retinas. Scale bars 10μm


Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 , * * p < 0 . 01 .

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

Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.


Quantitation of the BODIPY signal shown as normalised LD size of lipid droplets for the genotypes The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 , * * p < 0 . 01 .

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

Quantitation of the BODIPY signal shown as normalised LD size of lipid droplets for the genotypes The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.


M - N . Q - PCR analysis of mRNA transcripts of lipogenic genes - Acetyl CoA carboxylase ( ACC ) and Fatty Acid Synthase ( FASN ) from heads of wild type and ADAM17 - / - mutants ; n = 4 and n = 3 biological replicates for M and N respectively , with 3 technical replicates for each genotype . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

M-N. Q-PCR analysis of mRNA transcripts of lipogenic genes -Acetyl CoA carboxylase (ACC) and Fatty Acid Synthase (FASN) from heads of wild type and ADAM17-/- mutants; n=4 and n=3 biological replicates for M and N respectively, with 3 technical replicates for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


A - D . Fluorescent images of 1 day old retinas , stained with BODIPY ( green ) and FM - dye ( red ) to stain lipid droplets and the photoreceptor membranes respectively ; ( A ) wild type ; ( B ) ADAM17 - / - mutant ; ( C ) overexpression of lipase in glial cells of ADAM17 - / - mutant ; ( D ) overexpression of lipase in neurons of ADAM17 - / - mutant . Scale bars : 10μm .

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

A-D. Fluorescent images of 1 day old retinas, stained with BODIPY (green) and FM-dye (red) to stain lipid droplets and the photoreceptor membranes respectively; (A) wild type; (B) ADAM17-/- mutant; (C) overexpression of lipase in glial cells of ADAM17-/- mutant; (D) overexpression of lipase in neurons of ADAM17-/- mutant. Scale bars: 10μm.


Quantitation of the BODIPY signal shown as ( E ) LD numbers ; ( F ) normalised LD size for the genotypes n = 10 for each genotype . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean and the diamond an outlier . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

Quantitation of the BODIPY signal shown as (E) LD numbers; (F) normalised LD size for the genotypes n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


Quantitation of the BODIPY signal shown as ( G ) integrated total lipid , for the genotypes n = 10 for each genotype . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean and the diamond an outlier . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

Quantitation of the BODIPY signal shown as (G) integrated total lipid, for the genotypes n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


H - K . TEM images of 5 week old adult retinas corresponding to ( H ) wildtype ; ( I ) ADAM17 - / - mutant ; ( J ) glial overexpression of lipase in wild - type glial cells ; ( K ) neuronal overexpression of lipase in the ADAM17 - / - mutant . Scale bars : 10μm .

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H-K. TEM images of 5 week old adult retinas corresponding to (H) wildtype; (I) ADAM17-/- mutant; (J) glial overexpression of lipase in wild-type glial cells; (K) neuronal overexpression of lipase in the ADAM17-/-mutant. Scale bars: 10μm.


L . Quantitation of the percentage of normal , abnormal and missing rhabdomeres observed in the TEM images for the genotypes mentioned above , presented as a stacked graph plot ; n = 180 ommatidia from 3 different fly retinas for each . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres observed in the TEM images for the genotypes mentioned above, presented as a stacked graph plot; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


A . Alkaline phosphatase ( AP ) - shedding assays performed for Eiger in S2R + cells expressing either GFP , Drosophila ADAM17 , or activity dead mutants of Drosophila ADAM17 , in the presence of either PMA , or PMA and TAPI - 1 ( DMSO is used as control ; see methods ) ; n = 5 . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

A. Alkaline phosphatase (AP)-shedding assays performed for Eiger in S2R+ cells expressing either GFP, Drosophila ADAM17, or activity dead mutants of Drosophila ADAM17, in the presence of either PMA, or PMA and TAPI-1 (DMSO is used as control; see methods); n=5. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


B - C . Fluorescent images of retinas from 1 day old flies grown on either DMSO or TAPI - 1 , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively . Scale bars : 10μm .

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B-C. Fluorescent images of retinas from 1 day old flies grown on either DMSO or TAPI-1, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively. Scale bars: 10μm.


D . Integrated total lipid counts from retinas corresponding to flies reared either on DMSO or TAPI - 1 starting from larval stages . n = 10 fly retinas . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

D. Integrated total lipid counts from retinas corresponding to flies reared either on DMSO or TAPI-1 starting from larval stages. n=10 fly retinas. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


E - H . Fluorescent images of 1week old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( E ) wild type ; ( F ) eiger - / - ; ( G ) grindelwald - / - ; ( H ) wengen - / - . Scale bars : 10μm .

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E-H. Fluorescent images of 1week old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (E) wild type; (F) eiger-/-; (G) grindelwald-/-; (H) wengen-/-. Scale bars: 10μm.


I . Quantitation of the BODIPY signal shown as integrated total lipid measurements for the genotypes mentioned above . n = 10 fly retinas . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean and the diamond an outlier . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

I. Quantitation of the BODIPY signal shown as integrated total lipid measurements for the genotypes mentioned above. n=10 fly retinas. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


J - K . mRNA level transcripts of ACC and FASN measured from head lysates of one week old wild type , eiger - / - and grindelwald - / - . n = 3 biological replicates , with 3 technical replicates for each genotype . The box end points represent the maximum and minimum values respectively , the central band is the median and the square is the mean Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

J-K. mRNA level transcripts of ACC and FASN measured from head lysates of one week old wild type, eiger-/- and grindelwald-/- . n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


L - O . TEM images of two week old retinas corresponding to ( L ) control ; ( M ) eiger - / - ; ( N ) grindelwald - / - ; ( O ) wengen - / - . n = 180 ommatidia from 3 different fly retinas for each . Scale bars : 10μm .

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L-O. TEM images of two week old retinas corresponding to (L) control; (M) eiger-/-; (N) grindelwald-/-; (O) wengen-/-. n=180 ommatidia from 3 different fly retinas for each. Scale bars: 10μm.


P . Quantitation of the percentage of normal , abnormal and missing rhabdomeres from 2 week old retinas observed in TEM , corresponding to the genotypes mentioned above ; n = 180 ommatidia from 3 different fly retinas for each . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

P. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from 2 week old retinas observed in TEM, corresponding to the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


A - D . Fluorescent images of 1 day old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( A , B ) wild type ; ( C , D ) ADAM17 - / - mutants , reared either on DMSO or AD4 . Scale bars : 10μm .

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

A-D. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A, B) wild type; (C, D) ADAM17-/- mutants, reared either on DMSO or AD4. Scale bars: 10μm.


E . Integrated total lipid counts from retinas corresponding to the genotypes and treatments mentioned above ; n = 10 for each . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 .

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

E. Integrated total lipid counts from retinas corresponding to the genotypes and treatments mentioned above; n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.


F - I . TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17 - / - mutants grown on either DMSO or AD4 . Scale bars : 10μm .

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

F-I. TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17-/- mutants grown on either DMSO or AD4. Scale bars: 10μm.