A . - E . for β - galactosidase in 3 - months old VEGF - Ahyper ( lacZ / wt ) mouseeyes . A . Low magnification image shows β - gal + ( in blue ) in RPE ( arrow ) , retina ( blue arrow ) , ciliary ( black arrow ) , lens ( orange arrow ) . of Figures 1B to 1E are higher magnification of Figure 1A . Scale bar 200 μm . B . Strong expression of VEGF with β - gal + are observed in RPE ( short arrow ) inner layer ( INL ) ( long arrow ) , while less is observed in ganglion layer ( black arrow ) of retina . Scale bar 100 μm . C . Ciliary epithelial are β - gal + , similarly as RPE ( arrow ) . Scale bar 100 μm . D . VEGF - A is strongly expressed in secondary nuclearfiber in adult lens ( arrow ) . Scale bar 50 μm . E . No β - gal + are observed in corneas of adult VEGF - Ahyper ( lacZ / wt ) mouseeyes . arrow shows stroma of cornea black arrow shows corneal epithelium . Scale bar 100 μm .

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A.-E. Staining for β-galactosidase in 3-months old VEGF-Ahyper(lacZ/wt)mouseeyes.A. Low magnification image shows β-gal+ cells (in blue) in the RPE (white arrow), retina (blue arrow), ciliary body (black arrow), and lens (orange arrow). Images of Figures 1B to 1E are higher magnification images of Figure 1A. Scale bar 200 μm.B. Strong expression of VEGF with β-gal+ cells are observed in the RPE (short white arrow) and the inner nuclear layer (INL) (long white arrow), while less staining is observed in the ganglion cell layer (black arrow) of the retina. Scale bar 100 μm.C. Ciliary body epithelial cells are β-gal+, similarly as RPE cells (white arrow). Scale bar 100 μm.D. VEGF-A is strongly expressed in secondary nuclearfiber cells in the adult lens (white arrow). Scale bar 50 μm.E. No β-gal+ cells are observed in the corneas of adult VEGF-Ahyper(lacZ/wt)mouseeyes. White arrow shows the stroma of the cornea and the black arrow shows the corneal epithelium. Scale bar 100 μm.


G . RPE maintain strong expression of VEGF - A ( β - gal + [ arrow ] ) in retina with progressive . Immunofluorescence for β - gal in retina of a 24 - months old VEGF - Ahypermouse is shown . Ocular macrophages ( F4 / 80 + [ yellow arrow ] ) show no strong β - galactosidase expression , while some immunolabeling for β - galactosidase is detected in retinal of inner layer ( orange arrow ) of retina . RPE : retinal pigment epithelium ; ONL : outer layer ; INL : inner layer . Scale bar 50 μm .

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G. RPE cells maintain strong expression of VEGF-A (β-gal+ cells [white arrow]) in the retina with progressive age. Immunofluorescence for β-gal in the retina of a 24-months old VEGF-Ahypermouse is shown. Ocular macrophages (F4/80+ cells [yellow arrow]) show no strong β-galactosidase expression, while some immunolabeling for β-galactosidase is detected in retinal cells of the inner nuclear layer (orange arrow) of the retina. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer. Scale bar 50 μm.


H . VEGF - A continues to be highly expressed in secondary fiber in aged lens ( shown is a lens of a 24 - months old VEGF - Ahyper mouse ; β - galactosidasestaining ( arrow ) reflects VEGF - A expression ) . Cor : lenscortex ; Cap : lens capsule . DAPI labels nuclei ( blue ) . Scale bar 25 μm .

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H. VEGF-A continues to be highly expressed in secondary nuclear fiber cells in the aged lens (shown is a lens of a 24-months old VEGF-Ahyper mouse; nuclear β-galactosidasestaining (arrow) reflects cellular VEGF-A expression). Cor: lenscortex; Cap: lens capsule. DAPI labels nuclei (blue). Scale bar 25 μm.


A . Left graph : VEGF - Aprotein in lenses of VEGF - Ahypermice ( KI ) are increased ~ 3 - fold throughout life ( measured at 2 - at 8 . 5 - months of ; n = 7 mice / group , 2 independent experiments ) . Graph shows mean ± SD . Right graph : VEGF - AmRNA are similarly increased in lenses of adult VEGF - Ahypermice ( 9 - months old ; n = 7 mice / group , 3 independent experiments ) . Graph shows mean ± . * * P - value : 0 . 0095 .

A. Left graph: VEGF-Aprotein levels in lenses of VEGF-Ahypermice (KI) are increased ~3-fold throughout life (measured at 2- and at 8.5-months of age; n=7 mice/group, 2 independent experiments). Graph shows mean ± SD. Right graph: VEGF-AmRNA levels are similarly increased in lenses of adult VEGF-Ahypermice (9-months old; n=7 mice/group, 3 independent experiments). Graph shows mean ± SEM. **P-value: 0.0095.


B . VEGF - A isoforms ( mainly VEGF - A120 VEGF - A164 ) VEGF - A receptor Flk1 are expressed in adult lens . Flt1 expression was not detected in lens . N = 3 mice / group .

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B. VEGF-A isoforms (mainly VEGF-A120 and VEGF-A164) and the VEGF-A receptor Flk1 are expressed in the adult lens. Flt1 expression was not detected in the lens. N=3 mice/group.


C . Increased serum of lipid peroxidation marker 4 - HNE ( a marker of increased oxidative damage ) in aged , but not in young VEGF - Ahyper mice . * P - value : 0 . 0325 . N = 7 mice / group ( 2 independent experiments ) . Graph shows mean ± .

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C. Increased serum levels of the lipid peroxidation marker 4-HNE (a marker of increased oxidative damage) in aged, but not in young VEGF-Ahyper mice. *P-value: 0.0325. N=7 mice/group (2 independent experiments). Graph shows mean ± SEM.


A . Increased ERK1 / 2 phosphorylation is observed in lenses from VEGF - Ahyper mice ( KI ) with increased VEGF - A already prior to cataract formation ( 11 - months old mice with no cataracts ( 0 cataract ) ) , which is maintained in lenses with mature cataracts ( lens of a 26 - months old VEGF - Ahyper mouse with + 3 cataract ) . of ERK1 / 2 phosphorylation in 11 - months old lenses ( n = 3 / group ) . Graph shows mean ± . * * * P - value : 0 . 0005 .

A. Increased ERK1/2 phosphorylation is observed in lenses from VEGF-Ahyper mice (KI) with increased VEGF-A levels already prior to cataract formation (11-months old mice with no cataracts (0 cataract)), which is maintained in lenses with mature cataracts (lens of a 26-months old VEGF-Ahyper mouse with +3 cataract). Quantification of ERK1/2 phosphorylation in 11-months old lenses (n=3/group). Graph shows mean ± SEM. ***P-value: 0.0005.


B . - C . Cataracts with opacification of lens ( B . , black arrow ) extruded nuclei ( C . , arrow ) are observed with progressive in VEGF - Ahyper mice ( here a representative 30 - months old lens with a cataract is shown ) .

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B.-C. Cataracts with opacification of the lens (B., black arrow) and extruded nuclei (C., white arrow) are observed with progressive age in VEGF-Ahyper mice (here a representative 30-months old lens with a cataract is shown).


D . - E . Histology of lens from b . - c . shows extrusion of sclerotic nucleus ( arrows in C . D . ) massive degenerative changes of lens cortex with vacuolization ( black arrows in D . E . ) . Scale bar 500 μm in ( D . ) 100 μm in ( E . ) .

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D.-E. Histology of lens from b.-c. shows extrusion of sclerotic nucleus (white arrows in C. and D.) and massive degenerative changes of the lens cortex with vacuolization (black arrows in D. and E.). Scale bar 500 μm in (D.) and 100 μm in (E.).


F . - G . Representative histological of 21 - months old eyes from WT VEGF - Ahyper mice . sclerotic nucleus ( red arrow in G . ) was extruded lenscortex capsule are separated from nucleus ( arrow in G . ) . retina in aged VEGF - Ahyper mice is atrophic shows degenerative changes ( yellow arrow in G . ) , while retina ( * ) lens ( * * ) appears normal in - matched WT littermate mice . Scale bars 200 μm . DAPI ( blue , nuclei ) ; phalloidin ( green ) .

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F.-G. Representative histological images of 21-months old eyes from WT and VEGF-Ahyper mice. The sclerotic nucleus (red arrow in G.) was extruded and the lenscortex and capsule are separated from the nucleus (white arrow in G.). The retina in aged VEGF-Ahyper mice is atrophic and shows degenerative changes (yellow arrow in G.), while the retina (*) and lens (**) appears normal in age-matched WT littermate mice. Scale bars 200 μm. DAPI (blue, nuclei); phalloidin (green).


A . lens progress with in VEGF - Ahypermice . lens were graded from + 1 ( mild ) , + 2 ( moderate ) , to + 3 ( mature cataract with fully opacified lens ) . While only a small subset of > 18 months - old WT mice have mature ( + 3 ) cataracts , majority of VEGF - Ahypermice of this group has mature cataracts . Percentile ( % ) of mice with graded cataracts is indicated in each group . 0 = no cataract . Absolute mouse numbers of each group are shown in Appendix Figure S1 . B . Targeting NLRP3 inhibits normal - dependent cataract formation , delays ( but does not prevent ) VEGF - A - induced cataract formation in VEGF - Ahypermice . Percentile ( % ) of mice with graded cataracts is indicated in each group . 0 = no cataract . Absolute mouse numbers of each group are shown in Appendix Figure S1 .

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A. lens progress with age in VEGF-Ahypermice. lens were graded from +1 (mild), +2 (moderate), to +3 (mature cataract with fully opacified lens). While only a small subset of >18 months-old WT mice have mature (+3) cataracts, the majority of VEGF-Ahypermice of this age group has mature cataracts. Percentile (%) of mice with graded cataracts is indicated in each age group. 0 = no cataract. Absolute mouse numbers of each group are shown in Appendix Figure S1.B. Targeting NLRP3 inhibits normal age-dependent cataract formation, and delays (but does not prevent) VEGF-A-induced cataract formation in VEGF-Ahypermice. Percentile (%) of mice with graded cataracts is indicated in each age group. 0 = no cataract. Absolute mouse numbers of each group are shown in Appendix Figure S1.


C . Expression of IL - 1β in lenses of 21 - months old VEGF - Ahyper mice with + 3 cataracts is increased compared to lenses from - matched WT mice with + 3 cataracts , but not in lenses from 11 - months old mouse groups with no cataracts . * P - value : 0 . 0259 . N = 7 / group ( 3 independent experiments ) .

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C. Expression of IL-1β in lenses of 21-months old VEGF-Ahyper mice with +3 cataracts is increased compared to lenses from age-matched WT mice with +3 cataracts, but not in lenses from 11-months old mouse groups with no cataracts. *P-value: 0.0259. N=7/group (3 independent experiments).


D . VEGF - Ahyper mice have decreased of major antioxidant in lens , reduced glutathione ( μmol / gm lens ) , prior to cataract formation . Lenses with 0 to + 1 cataracts from 11 - months old mice were used for measurements . * * P - value : 0 . 0097 . N = 7 / group ( 2 independent experiments ) .

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D. VEGF-Ahyper mice have decreased levels of the major antioxidant in the lens, reduced glutathione (μmol/gm lens weight), prior to cataract formation. Lenses with 0 to +1 cataracts from 11-months old mice were used for measurements. ** P-value: 0.0097. N=7/group (2 independent experiments).


E - F . Overexpression of NADPH oxidase gp91phox [ * p - value : 0 . 0435 ] ( E . ) of antioxidant enzymes SOD1 [ * p - value : 0 . 0330 ] , catalase ( CAT ) [ * p - value : 0 . 0448 ] GPx - 1 [ * p - value : 0 . 0443 ] ( F . ) in 21 - months old lenses of VEGF - Ahyper mice with cataracts ( + 3 ) , but not in 11 - months old lenses with no cataracts . N = 7 / group ( 3 independent experiments ) .

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E-F. Overexpression of NADPH oxidase gp91phox [*p-value: 0.0435] (E.) and of the antioxidant enzymes SOD1 [*p-value: 0.0330], catalase (CAT) [*p-value: 0.0448] and GPx-1 [*p-value: 0.0443] (F.) in 21-months old lenses of VEGF-Ahyper mice with cataracts (+3), but not in 11-months old lenses with no cataracts. N=7/group (3 independent experiments).


A - B . Choroidalflat mount of a VEGF - Ahypermouse shows choroidalvessels ( green [ fluorescein - conjugated isolectin B4 ] ) from which neovessels ( strong red [ CD31 ] ) originate that are covered by a RPEcell layer ( [ phalloidin ] ) , which separates CNV lesion from photoreceptors . arrows demarcate CNV lesion . Yellow arrows show round autofluorescent deposits . Scale bars 100 μm .

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A-B. Choroidalflat mount staining of a white VEGF-Ahypermouse shows choroidalvessels (green [fluorescein-conjugated isolectin B4]) from which neovessels (strong red [CD31]) originate and that are covered by a RPEcell layer (white [phalloidin]), which separates the CNV lesion from the photoreceptors. White arrows demarcate CNV lesion. Yellow arrows show round autofluorescent deposits. Scale bars 100 μm.


A - B . Choroidalflat mount of a VEGF - A hypermouse shows choroidal vessels ( green [ fluorescein - conjugated isolectin B4 ] ) from which neovessels ( strong red [ CD31 ] ) originate that are covered by a RPEcell layer ( [ phalloidin ] ) , which separates CNV lesion from photoreceptors . arrows demarcate CNV lesion . Yellow arrows show round autofluorescent deposits . Scale bars 100 μm .

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A-B. Choroidalflat mount staining of a white VEGF-A hypermouse shows choroidal vessels (green [fluorescein-conjugated isolectin B4]) from which neovessels (strong red [CD31]) originate and that are covered by a RPEcell layer (white [phalloidin]), which separates the CNV lesion from the photoreceptors. White arrows demarcate CNV lesion. Yellow arrows show round autofluorescent deposits. Scale bars 100 μm.


C . Section through a CNV lesion in a VEGF - Ahyper mouse . Bruch ' s location is indictaed by yellow arrows . Choroidal neovessels ( arrows ) are covered by a layer of RPE ( black arrows ) . Chor : choroid ; ONL : outer layer ; PR : photoreceptors . Scale bar 20 μm . A . - C . 21 - months old VEGF - Ahyper mice .

C. Section through a CNV lesion in a VEGF-Ahyper mouse. Bruch's membrane location is indictaed by yellow arrows. Choroidal neovessels (white arrows) are covered by a layer of RPE cells (black arrows). Chor: choroid; ONL: outer nuclear layer; PR: photoreceptors. Scale bar 20 μm. A.-C. 21-months old VEGF-Ahyper mice.


RPE - specific overexpression of VEGF - A in VMD2Cre + / WTROSA - STOPfl / fl - VEGF - A164mice ( VEGF - ARPEmice ) results in CNV at sites of VEGF - A overexpression ( Cre + patches ( nuclei ) ; red arrow ) , while Cre - areas with normal VEGF - A expression show no CNV ( arrow ) . Neovessels are CD31 + ( red ) . VEGF - A - induced CNV lesions RPE barrier breakdown in VMD2Cre + / WT ROSA - STOPfl / fl - VEGF - A164mice resemble those seen in VEGF - Ahypermice ( B . - C . , arrows ; phalloidin in green ) . Scale bars 100 μm .

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RPE-specific overexpression of VEGF-A in VMD2Cre+/WTROSA-STOPfl/fl-VEGF-A164mice (VEGF-ARPEmice) results in CNV at sites of VEGF-A overexpression (Cre+ patches (white nuclei); red arrow), while Cre- areas with normal VEGF-A expression show no CNV (white arrow). Neovessels are CD31+ (red). VEGF-A-induced CNV lesions and RPE barrier breakdown in VMD2Cre+/WT ROSA-STOPfl/fl-VEGF-A164mice resemble those seen in VEGF-Ahypermice (B.-C., arrows; phalloidin staining in green). Scale bars 100 μm.


D . Genetic inactivation of Flk1 specifically in RPE in VEGF - Ahypermice ( VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice ) prevents RPE barrier breakdown CNV lesion formation . Choroidalflat mounts of 5 months old VEGF - Ahypermice VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice are shown . β - gal shows uniform VEGF - A expression in these mice ( green ) . Crestaining ( red ) shows co - localization of β - gal Crestaining in nuclei of RPE in VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice ( red areas of occur in CNV lesions of VEGF - Ahypermice due to labeling of anti - mouseIgG not Cre , as anti - mouseIgG secondary antibodies were used to detect mouse anti - Cre antibody ) . Phalloidinstaining ( ) reveals extensive RPE barrier breakdown CNV lesions ( arrow ) in VEGF - Ahypermice , which is not observed in VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice . Scale bar 100 μm .

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D. Genetic inactivation of Flk1 specifically in the RPE in VEGF-Ahypermice (VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice) prevents RPE barrier breakdown and CNV lesion formation. Choroidalflat mounts of 5 months old VEGF-Ahypermice and VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice are shown. β-gal shows uniform VEGF-A expression in these mice (green). Crestaining (red) shows co-localization of β-gal and Crestaining in the nuclei of RPE cells in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice (red areas of staining occur in CNV lesions of VEGF-Ahypermice due to labeling of anti-mouseIgG and not Cre, as the anti-mouseIgG secondary antibodies were used to detect the mouse anti-Cre antibody). Phalloidinstaining (white) reveals extensive RPE barrier breakdown and CNV lesions (arrow) in VEGF-Ahypermice, which is not observed in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice. Scale bar 100 μm.


A . NLRP3 is expressed in RPE at sites of CNV lesions ( arrow ; ) . B . Lack of NLRP3 in VEGF - Ahyper / NLRP3 - / - mice ( control ) . Scale bars 100 μm .

A. NLRP3 is expressed in RPE cells at sites of CNV lesions (arrow; white).B. Lack of NLRP3 staining in VEGF-Ahyper/NLRP3-/- mice (control). Scale bars 100 μm.


C . Expression of NLRP3 [ * p - value : 0 . 0478 ] IL - 1β [ * p - value : 0 . 0398 ] , but not of IL - 18 [ p - value : 0 . 3529 ] is increased in RPE / choroid lysates from VEGF - Ahyper mice when compared to matched control littermate mice . N = 7 mice / group ( 3 independent experiments ) . Graphs show mean ± .

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C. Expression of NLRP3 [*p-value: 0.0478] and IL-1β [*p-value: 0.0398], but not of IL-18 [p-value: 0.3529] is increased in RPE/choroid lysates from VEGF-Ahyper mice when compared to matched control littermate mice. N=7 mice/group (3 independent experiments). Graphs show mean ± SEM.


D . - G . Complement pathway activation ( immunolabeling for product of complement pathway activation C5b - 9 , an inducer of NLRP3 inflammasome ) is observed in CNV lesions of VEGF - Ahypermice ( arrow in E . ) but not in - matched control mice ( D . ) Co - localization of C5b - 9 in same CNV lesion is observed with complement C1qA ( initiator of classical complement pathway an inducer of NLRP3 inflammasome ) ( F . ) , with TLR2 ( G . ) ( arrows ) . Autofluorescence in red in D . E . CD31 + vessels in red in I . J . Yellow arrows show site of choroid . Scale bars 50 μm .

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D.-G. Complement pathway activation (immunolabeling for the product of complement pathway activation C5b-9, an inducer of the NLRP3 inflammasome) is observed in CNV lesions of VEGF-Ahypermice (white arrow in E.) but not in age-matched control mice (D.) Co-localization of C5b-9 in the same CNV lesion is observed with complement C1qA (the initiator of the classical complement pathway and an inducer of the NLRP3 inflammasome) (F.), and with TLR2 (G.) (white arrows). Autofluorescence in red in D. and E. CD31+vessels in red in I. and J. Yellow arrows show site of choroid. Scale bars 50 μm.


A . CNV lesion numbers per eye in 6 - week old VEGF - Ahypermice compared with VEGF - Ahypermice lacking CASP1 / CASP11 , NLRP3 , IL1R1 , IL - 18 , TLR2 , or those heterozygous for beclin - 1 or homozygous for hypomorphic ATG16L1 . CASP1 / CASP11 , NLRP3 or IL - 1R1 deficiency results in significantly fewer CNV lesions . Each value represents total CNV lesion number / eye for an mouse . Mean ± is shown . P - values are on comparison with VEGF - Ahypermouse group . Absolute numbers of mice per group are indicated in ( ) . P - values were calculated with a two - unpaired Student ' s t - . Separately , p - values are shown between VEGF - Ahyper / CASP1 - / - / CASP11 - / - VEGF - Ahyper / NLRP3 - / - or VEGF - Ahyper / IL1R1 - / - mouse groups . WT mice developed no CNV lesions .

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A. CNV lesion numbers per eye in 6-week old VEGF-Ahypermice compared with VEGF-Ahypermice lacking CASP1/CASP11, NLRP3, IL1R1, IL-18, TLR2, or those heterozygous for beclin-1 or homozygous for hypomorphic ATG16L1. CASP1/CASP11, NLRP3 or IL-1R1 deficiency results in significantly fewer CNV lesions. Each value represents the total CNV lesion number/eye for an individual mouse. Mean ± SEM is shown. P-values are based on comparison with the VEGF-Ahypermouse group. Absolute numbers of mice per group are indicated in ( ). P-values were calculated with a two-tailed unpaired Student's t-test. Separately, p-values are shown between VEGF-Ahyper/CASP1-/-/CASP11-/- and VEGF-Ahyper/NLRP3-/- or VEGF-Ahyper/IL1R1-/-mouse groups. WT mice developed no CNV lesions.


B . Average CNV lesion ( in μm2 / lesion ) in 6 - week old VEGF - Ahypermice compared with VEGF - Ahypermice lacking CASP1 / CASP11 , NLRP3 , IL1R1 , IL - 18 , TLR2 , or those heterozygous for beclin - 1 or homozygous for hypomorphic ATG16L1 . Lack of TLR2 or CASP1 / CASP11 significantly reduces CNV lesion , while reduced autophagy moderately increases CNV . Each value represents average CNV lesion / eye for an mouse . Mean ± is shown . P - values are on comparison with VEGF - Ahypermouse group . Absolute numbers of mice per group are indicated in ( ) . P - values were calculated with a two - unpaired Student ' s t - . Separately , p - values are shown between VEGF - Ahyper / CASP1 - / - / CASP11 - / - VEGF - Ahyper / NLRP3 - / - or VEGF - Ahyper / IL1R1 - / - mouse groups .

B. Average CNV lesion area (in μm2/lesion) in 6-week old VEGF-Ahypermice compared with VEGF-Ahypermice lacking CASP1/CASP11, NLRP3, IL1R1, IL-18, TLR2, or those heterozygous for beclin-1 or homozygous for hypomorphic ATG16L1. Lack of TLR2 or CASP1/CASP11 significantly reduces CNV lesion size, while reduced autophagy moderately increases CNV size. Each value represents the average CNV lesion size/eye for an individual mouse. Mean ± SEM is shown. P-values are based on comparison with the VEGF-Ahypermouse group. Absolute numbers of mice per group are indicated in ( ). P-values were calculated with a two-tailed unpaired Student's t-test. Separately, p-values are shown between VEGF-Ahyper/CASP1-/-/CASP11-/- and VEGF-Ahyper/NLRP3-/- or VEGF-Ahyper/IL1R1-/-mouse groups.


C . - E . Representative choroidal flat mount show multifocal CNV lesions in VEGF - Ahyper mice a significant reduction in CNV numbers in those VEGF - Ahyper mice that lack either CASP1 / CASP11 or IL1R1 ( therefore IL - 1β - signaling ) . for phalloidin in green for CD31 in red . Scale bars 200 μm .

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C.-E. Representative choroidal flat mount images show multifocal CNV lesions in VEGF-Ahyper mice and a significant reduction in CNV numbers in those VEGF-Ahyper mice that lack either CASP1/CASP11 or IL1R1 (and therefore IL-1β-signaling). Staining for phalloidin in green and for CD31 in red. Scale bars 200 μm.


. - induced CNV experiments demonstrate that treatment with a caspase - 1 inhibitor ( compared to DMSO treated controls ) or a neutralizing TLR2 antibody ( compared to isotype matched IgG treated controls ) inhibits CNV lesion growth . P - values are shown . N = 15 mice / group . Representative CNV lesions in choroidal flat mounts are shown . Scale bars 100 μm .

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. Laser-induced CNV experiments demonstrate that treatment with a caspase-1 inhibitor (compared to DMSO treated controls) or a neutralizing TLR2 antibody (compared to isotype matched IgG treated controls) inhibits CNV lesion growth. P-values are shown. N=15 mice/group. Representative CNV lesions in choroidal flat mounts are shown. Scale bars 100 μm.


Figure 9 : VEGF - A - induced non - exudative AMD - like pathologies can be prevented by targeting NLRP3 , IL1R1 or caspase - 1 / caspase - 11 . A . No significant abnormalities of choroid , RPE or retina are seen in control wild - type littermate mice . Representative image of a 28 - months old control eye is shown . arrow indicates normal Bruch ' s . Chor : choroid ; RPE ; PR : photoreceptor ; ONL : outer layer . B . Aged VEGF - Ahypermice develop a progressive - dependent degeneration of RPE ( red arrow ) photoreceptors ( yellow arrow ) with accumulation of thick sub - RPE basal laminar deposits ( black arrow ) . These non - exudative AMD - like pathologies occur also at sites devoid of CNV lesions . C . - E . Genetic inactivation of CASP1 / CASP11 , NLRP3 , or IL1R1 strongly inhibits manifestation of RPE photoreceptor degeneration in aged VEGF - Ahypermice . F . - G . VEGF - Ahyper / IL18 - / - mice show significant RPE photoreceptor degeneration , with shortened irregular photoreceptor outer segments ( arrows ) . Thick sub - RPE deposits that protrude into RPE plane are observed as well ( black arrows ) . H . VEGF - Ahyper / Becn1 + / - mice show significant RPE photoreceptor degeneration . I . In contrast , - matched VEGF - Ahypomice that are hypomorphic for VEGF - A express β - galactosidase from endogenous VEGF - A locus ( as do VEGF - Ahypermice ) do not show these eye pathologies . B - I . 21 - months old mice . Scale bars 20 μm .

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Figure 9: VEGF-A-induced non-exudative AMD-like pathologies can be prevented by targeting NLRP3, IL1R1 or caspase-1/caspase-11.A. No significant abnormalities of the choroid, RPE or retina are seen in control wild-type littermate mice. Representative image of a 28-months old control eye is shown. White arrow indicates normal Bruch's membrane. Chor: choroid; RPE; PR: photoreceptor; ONL: outer nuclear layer.B. Aged VEGF-Ahypermice develop a progressive age-dependent degeneration of the RPE (red arrow) and the photoreceptors (yellow arrow) with accumulation of thick sub-RPE basal laminar deposits (black arrow). These non-exudative AMD-like pathologies occur also at sites devoid of CNV lesions.C.-E. Genetic inactivation of CASP1/CASP11, NLRP3, or IL1R1 strongly inhibits the manifestation of RPE and photoreceptor degeneration in aged VEGF-Ahypermice.F.-G. VEGF-Ahyper/IL18-/-mice show significant RPE and photoreceptor degeneration, with shortened and irregular photoreceptor outer segments (white arrows). Thick sub-RPE deposits that protrude into the RPE plane are observed as well (black arrows).H. VEGF-Ahyper/Becn1+/-mice show significant RPE and photoreceptor degeneration.I. In contrast, age-matched VEGF-Ahypomice that are hypomorphic for VEGF-A and express β-galactosidase from the endogenous VEGF-A locus (as do VEGF-Ahypermice) do not show these eye pathologies.B-I. 21-months old mice. Scale bars 20 μm.


A . Graphs show means of a - b - waves of - adapted - adapted ERG responses ( ERG intensities in μV ) for both left right eyes from 21 - months old VEGF - Ahypermice , VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice , CASP1 - / - / CASP11 - / - mice , VEGF - Ahyper / IL1R1 - / - mice , IL1R1 - / - mice , VEGF - Ahyper / IL18 - / - mice , IL18 - / - mice . None of these mice had cataracts . Graphs show mean ± .

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A. Graphs show means of a- and b-waves of dark-adapted and light-adapted ERG responses (ERG intensities in μV) for both the left and the right eyes from 21-months old VEGF-Ahypermice, VEGF-Ahyper/CASP1-/-/CASP11-/-mice, CASP1-/-/CASP11-/-mice, VEGF-Ahyper/IL1R1-/-mice, IL1R1-/-mice, VEGF-Ahyper/IL18-/-mice, and IL18-/-mice. None of these mice had cataracts. Graphs show mean ± SEM.


B . Representative ERG tracings are shown for VEGF - Ahyper mice for VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice . X - axes show milliseconds ( ms ) . Y - axes show ERG intensities ( in μV ) .

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B. Representative ERG tracings are shown for VEGF-Ahyper mice and for VEGF-Ahyper/CASP1-/-/CASP11-/- mice. X-axes show milliseconds (ms). Y-axes show ERG intensities (in μV).


Figure 11 : Lack of caspase - 1 / caspase - 11 inhibits CNV lesion formation , retinal degeneration , fundus abnormalities fundus autofluorescence in aged VEGF - Ahyper mice . A . OCTs in aged VEGF - Ahypermice show retinal degeneration with a thinning of retina CNV lesions extending from choroid into subretinal space ( arrows ) . OCTs appear normal in - matched VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice CASP1 - / - / CASP11 - / - mice . B . Fundus autofluorescence imaging shows areas of autofluorescence in fundus of aged VEGF - Ahypermice , but not in - matched VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice CASP1 - / - / CASP11 - / - mice . C . Abnormal pigment loss degenerative changes are observed in fundus of aged VEGF - Ahypermice , while fundus of - matched VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice CASP1 - / - / CASP11 - / - mice does not show these abnormalities . Representative of 21 - months old mice are shown .

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Figure 11: Lack of caspase-1/caspase-11 inhibits CNV lesion formation, retinal degeneration, fundus abnormalities and fundus autofluorescence in aged VEGF-Ahyper mice.A. OCTs in aged VEGF-Ahypermice show retinal degeneration with a thinning of the retina and CNV lesions extending from the choroid into the subretinal space (arrows). OCTs appear normal in age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice.B. Fundus autofluorescence imaging shows areas of autofluorescence in the fundus of aged VEGF-Ahypermice, but not in age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice.C. Abnormal pigment loss and degenerative changes are observed in the fundus of aged VEGF-Ahypermice, while the fundus of age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice does not show these abnormalities.Representative images of 21-months old mice are shown.


A - C Viability of indicated V . parahaemolyticus ( A - B ) or E . coli ( C ) prey strains before ( 0 h ) after ( 4 h ) co - incubation with indicated V . parahaemolyticus attackers on media containing 3 % NaCl at 30 ° C . In A B , media also contain 0 . 1 % arabinose to induce expression from plasmids . In A , prey strains contain either an empty plasmid ( pEmpty ) or a plasmid for arabinose - inducible expression of VP1389 ( pVP1389 ) . In B C , prey strains contain an empty plasmid that provides a selection marker , attackers are derivatives of a ∆ hns mutant ( parental ) . In B , attackers contain an empty plasmid , or plasmids for arabinose - inducible expression of VP1388 ( pVP1388 ) or VP1390 ( pVP1390 ) . Data are shown as mean ± SD , n = 3 technical replicates . Statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above . A significant difference was considered as P < 0 . 05 . DL , detection limit . ∆ hcp1 was used as a T6SS1 - control . experiment was performed at least three times with similar results ; results of a representative experiment are shown .

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A-C Viability counts of the indicated V. parahaemolyticus (A-B) or E. coli (C) prey strains before (0 h) and after (4 h) co-incubation with the indicated V. parahaemolyticus attackers on media containing 3% NaCl at 30°C. In A and B, media also contain 0.1% arabinose to induce expression from plasmids. In A, prey strains contain either an empty plasmid (pEmpty) or a plasmid for arabinose-inducible expression of VP1389 (pVP1389). In B and C, prey strains contain an empty plasmid that provides a selection marker, and the attackers are derivatives of a ∆hns mutant (parental). In B, the attackers contain an empty plasmid, or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit. ∆hcp1 was used as a T6SS1- control strain. The experiment was performed at least three times with similar results; results of a representative experiment are shown.


A VP1388 binds VP1390 . Immunoprecipitation using α - FLAG antibodies from E . coli co - expressing indicated C - terminal FLAG - Myc - tagged from arabinose - inducible plasmids .

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A VP1388 binds VP1390. Immunoprecipitation using α-FLAG antibodies from E. coli cells co-expressing the indicated C-terminal FLAG- and Myc-tagged proteins from arabinose-inducible plasmids.


B VP1388 VgrG1 co - precipitate with VP1390 . Immunoprecipitation using α - FLAG antibodies from V . parahaemolyticus ∆ hns / ∆ hcp1 / ∆ vp1390 derivatives harboring plasmids for arabinose - inducible expression of FLAG - tagged sfGFP or VP1390 . were grown in MLB media supplemented with chloramphenicol to maintain plasmids , 0 . 1 % arabinose . Endogenous VP1388 VgrG1 were detected using α - VP1388 α - VgrG1 antibodies , respectively .

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B VP1388 and VgrG1 co-precipitate with VP1390. Immunoprecipitation using α-FLAG antibodies from V. parahaemolyticus ∆hns/∆hcp1/∆vp1390 derivatives harboring plasmids for the arabinose-inducible expression of FLAG-tagged sfGFP or VP1390. Cells were grown in MLB media supplemented with chloramphenicol to maintain the plasmids, and 0.1% arabinose. Endogenous VP1388 and VgrG1 were detected using α-VP1388 and α-VgrG1 antibodies, respectively.


C Expression ( ) secretion ( media ) of VP1388 , VP1390 , VgrG1 from indicated V . parahaemolyticus ∆ hns - derived strains harboring an empty plasmid ( pEmpty ) or plasmids for arabinose - inducible expression of VP1388 ( pVP1388 ) or VP1390 ( pVP1390 ) . Samples were grown in media containing 3 % NaCl supplemented with 0 . 1 % arabinose at 30 ° C . polymerase β ( RNAp ) was used as a non - secreted loading control . Arrows denote bands at expected of VP1388 or VP1390 , as indicated in appropriate panels .

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C Expression (cells) and secretion (media) of VP1388, VP1390, and VgrG1 from the indicated V. parahaemolyticus ∆hns-derived strains harboring an empty plasmid (pEmpty) or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Samples were grown in media containing 3% NaCl and supplemented with 0.1% arabinose at 30°C. RNA polymerase β (RNAp) was used as a non-secreted protein loading control. Arrows denote the bands at the expected size of VP1388 or VP1390, as indicated in the appropriate panels.


A Toxicity of periplasm - targeted in E . coli . E . coli strains containing plasmids for arabinose - inducible expression of sfGFP ( used as a control ) , VP1388 or VP1390 fused to an N - terminal PelB signal peptide ( perisfGFP , periVP1388 , periVP1390 , respectively ) were spotted at 10 - fold serial dilutions onto LB agar plates supplemented with kanamycin ( to maintain plasmids ) either 0 . 2 % glucose , to repress expression , or 0 . 001 % arabinose , to induce expression . Plates were incubated over - night at 30 ° C or 37 ° C , as indicated .

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A Toxicity of periplasm-targeted proteins in E. coli. E. coli strains containing plasmids for the arabinose-inducible expression of sfGFP (used as a control), VP1388 or VP1390 fused to an N-terminal PelB signal peptide (perisfGFP, periVP1388, and periVP1390, respectively) were spotted at 10-fold serial dilutions onto LB agar plates supplemented with kanamycin (to maintain plasmids) and either 0.2% glucose, to repress protein expression, or 0.001% arabinose, to induce protein expression. Plates were incubated over-night at 30°C or 37°C, as indicated.


B VP1389 interacts with VP1390 . Co - immunoprecipitation of FLAG - tagged VP1390 or BC3020 using Myc - tagged VP1389 when co - expressed in V . parahaemolyticus Δvp1389 ( input ) . Precipitated ( output ) were detected by immunobotting using α - Myc α - FLAG antibodies . Source data are available online for this figure .

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B VP1389 interacts with VP1390. Co-immunoprecipitation of FLAG-tagged VP1390 or BC3020 using Myc-tagged VP1389 when co-expressed in V. parahaemolyticus Δvp1389 (input). Precipitated proteins (output) were detected by immunobotting using α-Myc and α-FLAG antibodies. Source data are available online for this figure.


C VP1390 induces lysis in E . coli . Time - lapse microscopy of E . coli expressing periplasm - targeted sfGFP , VP1388 , or VP1390 ( perisfGFP , periVP1388 , periVP1390 , respectively ) from an arabinose - inducible vector , grown on LB agarose pads supplemented with kanamycin ( to maintain plasmid ) , 0 . 2 % arabinose ( to induce expression ) , propidium iodide ( PI ; pink ) . Merging of phase contrast PI channels are shown . Scale bar = 2 µm .

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C VP1390 induces cell lysis in E. coli. Time-lapse microscopy of E. coli cells expressing periplasm-targeted sfGFP, VP1388, or VP1390 (perisfGFP, periVP1388, and periVP1390, respectively) from an arabinose-inducible vector, grown on LB agarose pads supplemented with kanamycin (to maintain the plasmid), 0.2% arabinose (to induce expression), and propidium iodide (PI; pink). Merging of the phase contrast and PI channels are shown. Scale bar = 2 µm.


Time - lapse microscopy of competition between V . parahaemolyticus ∆ hns ( T6SS + / VP1390 + ) , ∆ hns / ∆ hcp1 ( T6SS - / VP1390 + ) , or ∆ hns / ∆ vp1390 ( T6SS + / VP1390 - ) attackers V . parahaemolyticus ∆ vp1389 prey that express GFP . Attacker prey were mixed ( 2 : 1 ) spotted on LB agarose pads supplemented with propidium iodide ( PI ; pink ) . Merging of phase contrast , GFP ( green ) , PI ( pink ) channels , as well as PI channel alone are shown . Scale bar = 5 µm . experiment was performed three times with similar results ; results of a representative experiment are shown .

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Time-lapse microscopy of competition between V. parahaemolyticus ∆hns (T6SS+/VP1390+), ∆hns/∆hcp1 (T6SS-/VP1390+), or ∆hns/∆vp1390 (T6SS+/VP1390-) attackers and V. parahaemolyticus ∆vp1389 prey that express GFP. Attacker and prey were mixed (2:1 ratio) and spotted on LB agarose pads supplemented with propidium iodide (PI; pink). Merging of the phase contrast, GFP (green), and PI (pink) channels, as well as the PI channel alone are shown. Scale bar = 5 µm. The experiment was performed three times with similar results; results of a representative experiment are shown.


( A - C ) Telophase GSCs marked by H3S10p ( gray ) expressing old ( green ) new ( red ) histone H3 show non - overlapping patterns ( A ) . Telophase GSCs expressing old ( green ) new ( red ) histone H4 show moderate of overlap ( B ) . GSCs expressing old ( green ) new ( red ) histone H2A show high of overlap ( C ) . ( D ) A control where old new tags have been switched shows non - overlapping patterns of old ( red ) versus new ( green ) H3Rev histone in mitotic telophase GSCs marked by H3S10p ( gray ) . ( E ) of log2 ratios of total old new histone inherited by each future daughter of GSC division , where a value of 0 is equal inheritance at exactly a 1 : 1 . Data gathered over 9 separate experiments for H3Rev GSCs , H2A GSCs , H4 GSCs . Data gathered over 16 separate experiments for H3 GSCs . Values are mean + 95 % CI . ( F - H ) Telophase CBs marked by H3S10p ( gray ) expressing old ( green ) new ( red ) histone H3 also show non - overlapping patterns . ( F ) Telophase CBs expressing old ( green ) new ( red ) histone H4 ( G ) H2A ( H ) , which depict more overlap like GSCs . ( I ) A control with switched old new tags shows non - overlapping old old ( red ) versus new ( green ) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p ( gray ) .

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(A-C) Telophase GSCs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 show non-overlapping patterns (A). Telophase GSCs expressing old (green) and new (red) histone H4 show moderate levels of overlap (B). GSCs expressing old (green) and new (red) histone H2A show high levels of overlap (C). (D) A control where the old and new fluorescent tags have been switched shows the non-overlapping patterns of old (red) versus new (green) H3Rev histone in mitotic telophase GSCs marked by H3S10p (gray). (E) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev GSCs, H2A GSCs, and H4 GSCs. Data gathered over 16 separate experiments for H3 GSCs. Values are mean + 95% CI. (F-H) Telophase CBs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 also show non-overlapping patterns. (F) Telophase CBs expressing old (green) and new (red) histone H4 (G) and H2A (H), which depict more overlap like GSCs. (I) A control with switched old and new fluorescent tags shows the non-overlapping old old (red) versus new (green) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p (gray).


( B - C ) Non - overlapping patterns of old ( green ) versus new ( red ) histone H3 ( B ) overlapping patterns for H2A ( C ) in mitotic prometaphase bam mutant marked by anti - H3S10p ( gray ) , similar to WT GSCs . Scale bars : 2μm .

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(B-C) Non-overlapping patterns of old (green) versus new (red) histone H3 (B) and overlapping patterns for H2A (C) in mitotic prometaphase bam mutant cells marked by anti-H3S10p (gray), similar to WT GSCs. Scale bars: 2μm.


( F - H ) Examples showing old ( green ) versus new ( red ) H3 distribution patterns of signal strength at a single genomic locus labeled with probes ( gray ) for dad gene ( F ) , bgcn gene ( G ) , ss gene ( H ) in bam mutant GSC - like at prometaphase marked by anti - H3S10p ( blue ) . Scale bars : 1μm . Zoomed - in of each probe are displayed to right of each figure panel . Scale bars : 0 . 1μm .

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(F-H) Examples showing old (green) versus new (red) H3 distribution patterns and percentage of signal strength at a single genomic locus labeled with fluorescent probes (gray) for dad gene (F), bgcn gene (G), and ss gene (H) in bam mutant GSC-like cells at prometaphase marked by anti-H3S10p (blue). Scale bars: 1μm. Zoomed-in images of each probe are displayed to the right of each figure panel. Scale bars: 0.1μm.


( A - B ) Old ( red ) versus new ( green ) histone H3 distribution patterns of signal strength with a single genomic locus labeled with probes ( cyan ) for dad ( A ) bam ( B ) in WT telophase GSCs , marked by anti - H3S10p ( gray ) . ( C ) of probe association ratios with old versus new H3 for each candidate gene in WT GSCs . Data gathered over 8 separate experiments . D

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(A-B) Old (red) versus new (green) histone H3 distribution patterns and percentage of signal strength with a single genomic locus labeled with fluorescent probes (cyan) for dad (A) and bam (B) in WT telophase female GSCs, marked by anti-H3S10p (gray). (C) Quantification of the probe association ratios with old versus new H3 for each candidate gene in WT female GSCs. Data gathered over 8 separate experiments. D


B of Uqcrh - / - mice wild - type at 1 - 2 ( n = 5 to 10 ) , during post - weaning period from 5 - 6 of ( n = 3 to 7 ) weekly from 6 to 10 ( n = 13 to 66 ) , data are shown as mean ± SD . * * * * p < 0 . 0001 , 2 - Way ANOVA , from 7 . 5wk of for Uqcrh - / - versus sex - matched controls . Data are shown as single data points median , or categorical data , pooled , since genotype - related effects were comparable for both sexes from different cohorts . * p < 0 . 05 , * * * p < 0 . 001 , Wilcoxon rank - sum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Body weight of Uqcrh -/- mice and wild-type littermates at 1-2 weeks (n = 5 to 10), during the post-weaning period from 5-6 weeks of age (n = 3 to 7) and weekly from 6 to 10 weeks (n = 13 to 66), data are shown as mean ± SD. **** p < 0.0001, 2-Way ANOVA, from 7.5wk of age for Uqcrh-/- males and females versus sex-matched controls. Data are shown as single data points and median, or categorical data, males and females pooled, since genotype-related effects were comparable for both sexes from different cohorts. * p < 0.05, *** p <0.001, Wilcoxon rank-sum test.


C , D Clinical chemistry plasma concentrations of parameters related to acid - base balance ( Lactate Ammonia ) metabolic state ( Glucose ) n = 15 Uqcrh - / - 16 WT pups 1 - 2 - week - old ( C ) ; n = 15 f , 15 m Uqcrh - / - 15 f , 15 m WT mice aged 8 - 9 ( D ) ; sex not differentiated . Data shown as scatter dot plots with at median . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , Wilcoxon rank sum .

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

C, D Clinical chemistry plasma concentrations of parameters related to acid-base balance (Lactate and Ammonia) and metabolic state (Glucose) n = 15 Uqcrh-/- and 16 WT pups 1-2-week-old (C); n = 15 f, 15 m Uqcrh-/- and 15 f,15 m WT mice aged 8-9 weeks (D); sex not differentiated. Data shown as scatter dot plots with line at median. * p < 0.05, ** p < 0.01, **** p < 0.0001, Wilcoxon rank sum test.


F Measures related to behaviour neurological function , data generated by SHIRPA ( 9 old , F ) , n = 7f , 8m Uqcrh - / - 8f , 9m WT . Prepulse inhibition of acoustic startle as a parameter associated with sensorimotor gating ( 10 , E right panel ) , n = 7 f / 5 m Uqcrh - / - 8 f / 9 m WT mice . Sex is not differentiated . Data are shown as scatter dot plots with a representing median . * * * p < 0 . 001 , * * * * p < 0 . 0001 , Wilcoxon rank - sum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Measures related to behaviour and neurological function, data generated by the SHIRPA (9 weeks old, F), n = 7f, 8m Uqcrh-/- and 8f, 9m WT animals. Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating (10 weeks, E right panel), n = 7 f/5 m Uqcrh-/- and 8 f/ 9 m WT mice. Sex is not differentiated. Data are shown as scatter dot plots with a line representing the median. *** p < 0.001, **** p < 0.0001, Wilcoxon rank-sum test.


G Parameters related to heart function generated by Echocardiography ( ECHO ) Electrocardiography ( ECG ) , n = 15 f , 15 m Uqcrh - / - 15 f , 15 m WT mice , 6 old .

O O O O O O O O O O O O O O O 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 Parameters related to heart function generated by Echocardiography (ECHO) and Electrocardiography (ECG), n = 15 f, 15 m Uqcrh-/- and 15 f, 15 m WT mice, 6 weeks old.


A Respiratory chain enzyme activities from human control ( green ) ( red ) fibroblasts . Mean activities of controls ( n = 8 ) are set to 100 % error bars represent 1 standard deviation . Data are normalised to citrate synthase ( CS ) activity . B Respiratory chain enzyme activities in heart , brain liver , of wild - type Uqcrh - / - mice . Data are normalised to citrate synthase ( CS ) activity . Values are given as mean ± SD , n = 4 WT , 5 Uqcrh - / - ( Heart ) , 3 WT , 3 Uqcrh - / - ( Brain ) , 1 WT 2 Uqcrh - / - ( Liver ) . * p < 0 . 05 , * * * * p < 0 . 0001 , 2 - way ANOVA ( Multiple comparison ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Respiratory chain enzyme activities from human control (green) and patient (red) fibroblasts. Mean activities of controls (n = 8) are set to 100% and error bars represent 1 standard deviation. Data are normalised to citrate synthase (CS) activity. B Respiratory chain enzyme activities in heart, brain and liver, of wild-type and Uqcrh-/- mice. Data are normalised to citrate synthase (CS) activity. Values are given as mean ± SD, n = 4 WT, 5 Uqcrh-/- (Heart), 3 WT, 3 Uqcrh-/- (Brain), 1 WT and 2 Uqcrh-/- (Liver). * p < 0.05, **** p < 0.0001, 2-way ANOVA (Multiple comparison).


C Western blot of OXPHOS components in ( P ) control ( n = 2 , C1 - 2 ) fibroblasts . D BN - PAGE of OXPHOS assembly in enriched mitochondria from control fibroblasts ( n = 2 , C1 - 2 ) solubilised with DDM . Immunoblotting was performed using antibodies to a subunit of each ( CI [ NDUFB8 ] , CII [ SDHA ] , CIII [ UQCRC2 ] , CIV [ MT - CO1 ] , CV [ ATP5A ] )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of OXPHOS components in the patient (P) and control (n = 2, C1-2) fibroblasts. D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts (n = 2, C1-2) solubilised with DDM. Immunoblotting was performed using antibodies to a subunit of each complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A])


F Immunohistochemical of UQCRC2 was performed on liver , heart kidney derived from wild - type Uqcrh - / - mice at 9 of . of VDAC1 / porin was also carried out G Graphs indicate score values of immunohistochemical of both VDAC1 / porin UQCRC2 . Data are given as mean ± . * p < 0 . 05 ; n = 3 , Student ' s t - ( unpaired 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 O O

F Immunohistochemical staining of UQCRC2 was performed on liver, heart and kidney derived from wild-type and Uqcrh-/- mice at 9 weeks of age. Staining of VDAC1/porin was also carried out G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1/porin and UQCRC2. Data are given as mean ± SEM. * p < 0.05; n = 3, Student's t-test (unpaired samples).


A Western blot of control ( n = 2 , C1 - 2 ) fibroblasts transduced with a lentiviral vector ( pLVX ) containing wild - type UQCRH . Expression of wild - type UQCRH was induced using various concentrations of doxycycline ( dox ) up to 20 ng / ml for 72 hours . B BN - PAGE of control ( n = 2 , C1 - 2 ) fibroblasts transduced with a lentiviral vector ( pLVX ) containing wild - type UQCRH . Transduced fibroblasts were either uninduced or induced with 20 ng / ml doxycycline ( dox ) for 72 hours .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Expression of wild-type UQCRH was induced using various concentrations of doxycycline (dox) up to 20 ng/ml for 72 hours. B BN-PAGE analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Transduced fibroblasts were either uninduced or induced with 20 ng/ml doxycycline (dox) for 72 hours.


C Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts . D Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) nuclei ( DAPI , blue ) , in fibroblasts . E Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts transduced with wild - type UQCRH . F Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) nuclei ( DAPI , blue ) , in fibroblasts transduced with wild - type ( WT ) UQCRH . G Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts transduced with pseudogene UQCRHL . H Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) nuclei ( DAPI , blue ) , in fibroblasts transduced with pseudogene UQCRHL . I Graph indicates of UQCRC2 in control ( NHDF ) fibroblasts ( green ) fibroblasts ( red ) , those respective lines transduced ( T ) with wild - type UQCRH ( diagonal lines ) those lines transduced with pseudogene UQCRHL ( vertical lines ) measured using Image J , Data are given as mean ± . One - way ANOVA ( Kruskal - Wallis ) , * p < 0 . 05 ; n = 3 macroscopic field ( 10 - 14 for macroscopic field were measured ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts. D Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts. E Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with wild-type UQCRH. F Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with wild-type (WT) UQCRH. G Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with the pseudogene UQCRHL. H Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with the pseudogene UQCRHL. I Graph indicates the staining intensity of UQCRC2 in control (NHDF) fibroblasts (green) and patient fibroblasts (red), those respective cell lines transduced (T) with wild-type UQCRH (diagonal lines) and those cell lines transduced with the pseudogene UQCRHL (vertical lines) measured using Image J, Data are given as mean ± SEM. One-way ANOVA (Kruskal-Wallis test), * p < 0.05; n = 3 macroscopic field (10-14 cells for macroscopic field were measured).


Mitochondrial membranes from fibroblasts of control ( WT ) fibroblasts ( i , immortalised ) were solubilised with digitonin separated on native gradient gels . A BN - PAGE gel stained with Coomassie showing molecular marker ( BHM ) samples from control ( fibroblasts , ( I ) = immortalised fibroblasts ) B BN - PAGE gel stained with NADH : NTB reductase activity stain showing molecular marker ( BHM ) samples from control ( fibroblasts , ( I ) = immortalised fibroblasts ) C Further complexome of BN - PAGE gels shown in A B . Each native lane was cut into even fractions from high to low molecular mass . in each slice were digested with trypsin analysed by quantitative mass spectrometry . Shown here are average IBAQ values of identified subunits from complexes III , I IV

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

Mitochondrial membranes from fibroblasts of control (WT) and patient fibroblasts (i, immortalised) were solubilised with digitonin and separated on native gradient gels. A BN-PAGE gel stained with Coomassie showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts) B BN-PAGE gel stained with NADH:NTB reductase activity stain showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts) C Further complexome analysis of the BN-PAGE gels shown in A and B. Each native lane was cut into even fractions from high to low molecular mass. Proteins in each slice were digested with trypsin and analysed by quantitative mass spectrometry. Shown here are average IBAQ values of all identified subunits from complexes III, I and IV


D Profile of III ( averaged IBAQ values ) in ( dashed ) control ( solid ) fibroblasts . E Profile of complexes III ( red ) , I ( yellow ) IV ( green ) to visualise assess supercomplex composition stability in control ( solid lines ) ( dashed lines ) fibroblasts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Profile of complex III (averaged IBAQ values) in the patient (dashed line) and control (solid line) fibroblasts. E Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in control (solid lines) and patient (dashed lines) fibroblasts.


heart tissue from wild - type ( WT ) Uqcrh - / - mice were solubilised with digitonin separated on native gradient gels . F BN - PAGE gel stained with Coomassie ( left ) NADH : NTB reductase activity stain ( right ) showing molecular marker ( BHM ) heart tissue samples from wild - type ( WT ) Uqcrh - / - mice . G BN - PAGE immunoblot using antibody to CIII subunit UQCRC2 in heart tissue samples from wild - type ( WT ) Uqcrh - / - mice . H Further complexome of BN - PAGE gels shown in F , shown here are average IBAQ values of subunits from complexes III , I IV

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

heart tissue from wild-type (WT) and Uqcrh-/- mice were solubilised with digitonin and separated on native gradient gels. F BN-PAGE gel stained with Coomassie (left) and NADH:NTB reductase activity stain (right) showing molecular weight marker (BHM) and heart tissue samples from wild-type (WT) and Uqcrh-/- mice. G BN-PAGE and immunoblot using the antibody to CIII subunit UQCRC2 in heart tissue samples from wild-type (WT) and Uqcrh-/- mice. H Further complexome analysis of the BN-PAGE gels shown in F, shown here are average IBAQ values of subunits from complexes III, I and IV


I Profile of III ( averaged IBAQ values ) in wild - type ( + / + , solid ) Uqcrh - / - ( - / - , dashed ) heart tissue . J Profile of complexes III ( red ) , I ( yellow ) IV ( green ) to visualise assess supercomplex composition stability in wild - type ( + / + , solid ) Uqcrh - / - ( - / - , dashed ) heart tissue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Profile of complex III (averaged IBAQ values) in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue. J Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue.


B . Internalization ( cytoplasm ) recycling ( plasma ) of FGF7 - FGF10 - stimulated endogenous FGFR2 ( green ) for 0 , 15 , 40 , 120 min . in T47D . TRITC - Tf is a marker of recycling ( red ) . Nuclei are stained in blue . * , with receptor recycled to plasma . Scale bar , 5 μm . C . Red green pixels overlap fraction representing co - localization of FGFR2b with recycling endosomes marker Tf ( above ) proportion of green over total pixels representing FGFR2b in cytoplasm ( below ) upon stimulation with FGF7 ( green ) or FGF10 ( burgundy ) for 15 , 40 , or 120 min . . Values represent median ± SD of at least 3 independent experiments . Representative pictures are shown in B ( cytoplasm ) . * , p value < 0 . 005 ( Student ' s t - ) . D

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Internalization (cytoplasm) and recycling (plasma membrane) of FGF7- and FGF10-stimulated endogenous FGFR2 (green) for 0, 15, 40, and 120 min. in T47D. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm. C. Red and green pixels overlap fraction representing the co-localization of FGFR2b with the recycling endosomes marker Tf (above) and the proportion of green over total pixels representing FGFR2b in the cytoplasm (below) upon stimulation with FGF7 (dark green) or FGF10 (burgundy) for 15, 40, or 120 min.. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in B (cytoplasm). *, p value<0.005 (Student's t-test). D


H . Internalization ( cytoplasm ) recycling ( plasma ) of FGF10 - stimulated HA - FGFR2b ( green ) transfected in BT549 stimulated for 0 , 40 120 min . . were depleted or not of TTP or RCP by a pool of siRNAs . TRITC - Tf is a marker of recycling ( red ) . Nuclei are stained in blue . * , with receptor recycled to plasma . Scale bar , 5 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

H. Internalization (cytoplasm) and recycling (plasma membrane) of FGF10-stimulated HA-FGFR2b (green) transfected in BT549 stimulated for 0, 40 and 120 min.. Cells were depleted or not of TTP or RCP by a pool of siRNAs. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm.


( J ) control or 40 min . FGF10 - or TGFα - stimulated T47D left untreated or depleted of TTP , followed or not by transfection with siRNA resistant Flag - TTP ( Flag - TTP * ) or depleted of RCP followed or not by transfection with siRNA resistant RCP - GFP ( RCP - GFP * ) were immunoblotted with indicated antibodies . * * , non - specific band ( J ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) control or 40 min. FGF10- or TGFα-stimulated T47D left untreated or depleted of TTP, followed or not by transfection with siRNA resistant Flag-TTP (Flag-TTP*) or depleted of RCP followed or not by transfection with siRNA resistant RCP-GFP (RCP-GFP*) were immunoblotted with the indicated antibodies. **, non-specific band (J).


F , G Lysates from T47D or BT20 treated with DMSO , PD173074 , AG1478 , or MEK inhibitors U0126 MEK162 stimulated or not with either FGF10 or TGFα ( F ) ; T47D or BT20 treated with DMSO , or p38 inhibitor BMS582949 stimulated or not with either FGF10 or TGFα ( G ) were immunoblotted with indicated antibodies .

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

F, G Lysates from T47D or BT20 treated with DMSO, PD173074, AG1478, or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα (F); T47D or BT20 treated with DMSO, or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα (G) were immunoblotted with the indicated antibodies.


Lysates from T47D stimulated or not with FGF10 , EGF for different time periods or treated with PD173074 ( C ) , MEK162 ( D ) , AG1478 ( E ) before stimulation were immunoblotted with indicated antibodies .

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

Lysates from T47D stimulated or not with FGF10, EGF for different time periods or treated with PD173074 (C), MEK162 (D), AG1478 (E) before stimulation were immunoblotted with the indicated antibodies.


G , H of EdU incorporation in T47D pre - treated or not with FGF10 for 40 min . stimulated or not with FGF10 or EGF ( G ) or incubated with MEK162 , PD173074 , or AG1478 before pre - treatment ( H ) . N = 6 . P value = < 0 . 05 * , < 0 . 01 * * , < 0 . 001 * * * ( one - way ANOVA with Tukey ) .

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

G, H Percentage of EdU incorporation in T47D pre-treated or not with FGF10 for 40 min. and stimulated or not with FGF10 or EGF (G) or incubated with MEK162, PD173074, or AG1478 before pre-treatment (H). N= 6. P value = <0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).


A . Co - localization of FGFR2 ( red ) , EGFR ( green ) , recycling marker Tf ( blue ) in T47D stimulated or not with FGF10 or TGFα for 40 min . Scale bar , 5 μm . C . Co - localization of EGFR ( green ) , T693 phosphorylated EGFR ( blue ) , recycling marker Tf ( red ) in T47D stimulated or not with FGF10 or TGFα for 40 min . Scale bar , 5 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

A. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm. C. Co-localization of EGFR (green), T693 phosphorylated EGFR (blue), and the recycling marker Tf (red) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm.


E . Co - localization of FGFR2 ( red ) , EGFR ( green ) , recycling marker Tf ( blue ) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A stimulated or not with either FGF10 or TGFα for indicated time periods . Scale bar , 5 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

E. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods. Scale bar, 5 μm.


E , F Lysates from T47D ( E ) BT20 ( F ) depleted of EGFR , transfected with wt or T693A , stimulated or not with either FGF10 or TGFα for indicated time intervals were immunoblotted with indicated antibodies .

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

E, F Lysates from T47D (E) and BT20 (F) depleted of EGFR, transfected with wt or T693A, and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies.


A - D of EdU incorporation in T47D ( A , C ) or BT20 ( B , D ) depleted of EGFR or not , transfected with wt or T693A , stimulated or not with FGF10 ( A , B ) or with TGFα ( C , D ) pre - treated ( C , D ) or not ( A , B ) with FGF10 for 40 min . Values represent median ± SD of N = 4 . P value = < 0 . 05 * , < 0 . 01 * * , < 0 . 001 * * * ( one - way ANOVA with Tukey ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentage of EdU incorporation in T47D (A, C) or BT20 (B, D) depleted of EGFR or not, transfected with wt or T693A, and stimulated or not with FGF10 (A, B) or with TGFα (C, D) and pre-treated (C, D) or not (A, B) with FGF10 for 40 min. Values represent the median ± SD of N= 4. P value =<0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).


A ) Seeding efficiency was compared between control non - neurodegenerative FTLD - TDP samples . were transfected using Lipofectamine after mitotic arrest with AraC were fixed 3 to 6 days post - transfection with SarkoSpin fractions ( Laferrière et al , 2019 ) . Surface masks generated with Imaris show DAPI ( blue ) , TDP - 43 ( NuclTDP - 43 , cyan ) neoaggregates ( NeoAgg , orange ) . are presented at same scale , 20 μm as indicated in AraC panel Day 6 . B ) Higher magnification imaging of seeded for 6 days revealed presence of large TDP - 43 - HA in seeded with FTLD - TDP - A multiple TDP - 43 - HA in seeded with FTLD - TDP - C . Surface representation generated with Imaris showing DAPI , TDP - 43 - HA ( green ) , phosphorylated TDP - 43 ( pTDP - 43 , magenta ) neoaggregates ( NeoAgg , orange ) . are presented at same scale , 5 μm as indicated in FTLD - TDP - C top 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 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 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

A) Seeding efficiency was compared between control non-neurodegenerative and FTLD-TDP patient samples. Cells were transfected using Lipofectamine after mitotic arrest with AraC and were fixed 3 to 6 days post-transfection with SarkoSpin fractions (Laferrière et al, 2019). Surface masks generated with Imaris show DAPI (blue), nuclear TDP-43 (NuclTDP-43, cyan) and neoaggregates (NeoAgg, orange). All images are presented at the same scale, 20 μm as indicated in AraC panel Day 6. B) Higher magnification imaging of cells seeded for 6 days revealed presence of large cytoplasmic TDP-43-HA aggregates in cells seeded with FTLD-TDP-A and multiple cytoplasmic TDP-43-HA aggregates in cells seeded with FTLD-TDP-C. Surface representation generated with Imaris showing DAPI , TDP-43-HA (green), phosphorylated TDP-43 (pTDP-43, magenta) and neoaggregates (NeoAgg, orange). All images are presented at the same scale, 5 μm as indicated in FTLD-TDP-C top panel.


B ) Bar graph showing seeding effect at day 5 day 6 post - seeding in untreated , denaturation or autoclaving denaturation conditions . While seeding in untreated condition showed a significant increase in neoaggregates ( One - way ANOVA , with post - hoc Fisher ' s LSD , Day 5 : F = ( 5 , 12 ) = 6 . 713 , p = 0 . 0033 , non - neurodegenerative control vs FTLD - TDP - A p = 0 . 0016 , Day 6 : F ( 5 , 12 ) = 4 . 271 , p = 0 . 0183 , non - neurodegenerative control vs FTLD - TDP - A p = 0 . 0397 ) , this effect was abolished in denaturing conditions ( N = 3 biological replicates for non - neurodegenerative control , FTLD - TDP - A FTLD - TDP - C , bar graph showing mean ± ) .

B) Bar graph showing the seeding effect at day 5 and day 6 post-seeding in untreated, temperature denaturation or autoclaving denaturation conditions. While seeding in untreated condition showed a significant increase in neoaggregates (One-way ANOVA, with post-hoc Fisher's LSD test, Day 5: F=(5,12)=6.713, p=0.0033, non-neurodegenerative control vs FTLD-TDP-A p=0.0016, Day 6: F(5,12)=4.271, p=0.0183, non-neurodegenerative control vs FTLD-TDP-A p=0.0397), this effect was abolished in denaturing conditions (N=3 biological replicates for non-neurodegenerative control, FTLD-TDP-A and FTLD-TDP-C, bar graph showing mean ± SEM).


C ) TOP : Aggregation kinetics of full ( fl ) recombinant human TDP - 43 ( rec - hTDP - 43 ) measured as an increase in absorbance . addition of TEV protease induces cleavage of MBP tag subsequent aggregation of rec - hTDP - 43 . Rec - hTDP - 43 used for seeding were collected 18 hours after addition of TEV protease . BOTTOM LEFT : rec - hTDP - 43 stained by Thioflavin T ( ThT ) . BOTTOM RIGHT : Transmission Electron Microscopy ( ) image of rec - hTDP - 43 . are presented at same scale of 1 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

C) TOP: Aggregation kinetics of full length (fl) recombinant human TDP-43 (rec-hTDP-43) measured as an increase in absorbance. The addition of TEV protease induces the cleavage of the MBP tag and subsequent aggregation of rec-hTDP-43. Rec-hTDP-43 aggregates used for seeding were collected 18 hours after the addition of TEV protease. BOTTOM LEFT: rec-hTDP-43 aggregates stained by Thioflavin T (ThT). BOTTOM RIGHT: Transmission Electron Microscopy (TEM) image of the rec-hTDP-43 aggregates. All images are presented at the same scale of 1 μm.


E ) of neoaggregates ( left y - axis ) pTDP - 43 ( right y - axis ) at day 5 day 6 post - transfection for increasing concentrations of rec - hTDP - 43 . 2 - way ANOVA repeated measure , with post - hoc Fisher ' s LSD , F ( 7 , 80 ) = 5 . 886 , p < 0 . 0001 , Day 5 : AraC vs 50 ng p = 0 . 0497 , AraC vs 500 ng p = 0 . 0002 , AraC vs 5 µg p = 0 . 0161 . 50 ng vs 100 ng p = 0 . 0208 , 50 ng vs 500 ng p = 0 . 0169 , 50 ng vs 2 . 5 µg p = 0 . 0272 , 100 ng vs 1 µg p < 0 . 0001 , 100 ng vs 5 µg p = 0 . 006 , 250 ng vs 1 μg p = 0 . 0005 , 250 ng vs 5 µg p = 0 . 034 , 500 ng vs 1 µg p < 0 . 0001 , 500 ng vs 5 µg p = 0 . 0047 , 1 µg vs 2 . 5 µg p < 0 . 0001 , 2 . 5 µg vs 5 µg p = 0 . 0081 , Day 6 : AraC vs 250 ng p = 0 . 0385 , AraC vs 1 µg p = 0 . 0006 , 50 ng vs 1 µg p = 0 . 0016 , 100 ng vs 1 µg p = 0 . 0101 , 100 ng vs 5 µg p = 0 . 0173 , 250 ng vs 5 µg p = 0 . 0006 , 500 ng vs 1 µg p = 0 . 0099 , 500 ng vs 5 µg p = 0 . 0176 , 1 µg vs 2 . 5 µg p = 0 . 0144 , 1 µg vs 5 µg p < 0 . 0001 , 2 . 5 µg vs 5 µg p = 0 . 0121 . To reduce complexity , only comparisons with AraC are marked in graph . N = 6 biological replicates for each ; bar graph showing mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of neoaggregates (left y-axis) and pTDP-43 (right y-axis) at day 5 and day 6 post-transfection for increasing concentrations of rec-hTDP-43. 2-way ANOVA repeated measure, with post-hoc Fisher's LSD, F(7,80)=5.886, p<0.0001, Day 5: AraC vs 50 ng p=0.0497, AraC vs 500 ng p=0.0002, AraC vs 5 µg p=0.0161. 50 ng vs 100 ng p=0.0208, 50 ng vs 500 ng p=0.0169, 50 ng vs 2.5 µg p=0.0272, 100 ng vs 1 µg p<0.0001, 100 ng vs 5 µg p=0.006, 250 ng vs 1 μg p=0.0005, 250 ng vs 5 µg p=0.034, 500 ng vs 1 µg p<0.0001, 500 ng vs 5 µg p=0.0047, 1 µg vs 2.5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0081, Day 6: AraC vs 250 ng p=0.0385, AraC vs 1 µg p=0.0006, 50 ng vs 1 µg p=0.0016, 100 ng vs 1 µg p=0.0101, 100 ng vs 5 µg p=0.0173, 250 ng vs 5 µg p=0.0006, 500 ng vs 1 µg p=0.0099, 500 ng vs 5 µg p=0.0176, 1 µg vs 2.5 µg p=0.0144, 1 µg vs 5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0121. To reduce complexity, only comparisons with AraC are marked in the graph. N=6 biological replicates for each concentration; bar graph showing mean ± SEM.


B ) Bar graphs representing number of NeoAggpS403 only ( bars filled in orange ) , NeoAggpS409 only ( bars filled in purple ) double phosphorylated NeoAggpS403 - 409 ( unfilled bars ) for each condition . are labelled in different colored dots ( n = 3 biological replicates for each condition , graph bar showing mean ± ) . Two - way ANOVA repeated measures , with post - hoc Fisher ' s LSD , F ( 24 . 54 ) = 3 . 799 , p < 0 . 0001 , Day 4 : non - neurodegenerative control pS403 / 404 vs pS409 / 410 , p < 0 . 0001 , non - neurodegenerative control pS403 / 404 vs bi - phosphorylated , p < 0 . 0001 , FTLD - TDP - A pS403 / 404 vs pS409 / 410 , p < 0 . 0001 , FTLD - TDP - A pS403 / 404 vs bi - phosphorylated , p < 0 . 0001 FTLD - TDP - C pS403 / 404 vs pS409 / 410 , p < 0 . 0001 , FTLD - TDP - C pS403 / 404 vs bi - phosphorylated , p < 0 . 0001 . Day 5 : non - neurodegenerative control pS403 / 404 vs bi - phosphorylated , p = 0 . 0219 , FTLD - TDP - A pS403 / 404 vs double , p = 0 . 0650 , FTLD - TDP - C pS403 / 404 vs double , p = 0 . 0157 . Day 6 : Control bi - phosphorylated vs FTLD - TDP - A bi - phosphorylated , p = 0 . 5096 , control bi - phosphorylated vs FTLD - TDP - C , p = 0 . 1010 . Non - neurodegenerative control pS403 / 404 : day 3 vs 4 , p = 0 . 0559 , day 3 vs 6 , p = 0 . 0369 . FTLD - TDP - A pS404 / 404 , day 3 vs 4 , p = 0 . 0075 , day 4 vs 5 , p = 0 . 0714 . FTLD - TDP - C pS403 - 404 , day3 vs 4 , P = 0 . 0759 , day 4 vs 6 , 9 = 0 . 0305 . FTLD - TDP - A bi - phosphorylated : day 3 vs 5 , P = 0 . 0577 , day 3 vs 6 , p = 0 . 0609 . FTLD - TDP - C bi - phosphorylatede : day 3 vs 4 , p = 0 . 0522 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graphs representing the number of NeoAggpS403 only (bars filled in light orange), NeoAggpS409 only (bars filled in light purple) and double phosphorylated NeoAggpS403-409 (unfilled bars) for each condition. Individual patients are labelled in different colored dots (n=3 biological replicates for each condition, graph bar showing mean ± SEM). Two-way ANOVA repeated measures, with post-hoc Fisher's LSD test, F(24.54)=3.799, p<0.0001, Day 4: non-neurodegenerative control pS403/404 vs pS409/410, p<0.0001, non-neurodegenerative control pS403/404 vs bi-phosphorylated, p<0.0001, FTLD-TDP-A pS403/404 vs pS409/410, p<0.0001, FTLD-TDP-A pS403/404 vs bi-phosphorylated, p<0.0001 FTLD-TDP-C pS403/404 vs pS409/410, p<0.0001, FTLD-TDP-C pS403/404 vs bi-phosphorylated, p<0.0001. Day 5: non-neurodegenerative control pS403/404 vs bi-phosphorylated, p=0.0219, FTLD-TDP-A pS403/404 vs double, p=0.0650, FTLD-TDP-C pS403/404 vs double, p=0.0157. Day 6: Control bi-phosphorylated vs FTLD-TDP-A bi-phosphorylated, p=0.5096, control bi-phosphorylated vs FTLD-TDP-C, p=0.1010. Non-neurodegenerative control pS403/404: day 3 vs 4, p=0.0559, day 3 vs 6, p=0.0369. FTLD-TDP-A pS404/404, day 3 vs 4, p=0.0075, day 4 vs 5, p=0.0714. FTLD-TDP-C pS403-404, day3 vs 4, P=0.0759, day 4 vs 6, 9=0.0305. FTLD-TDP-A bi-phosphorylated: day 3 vs 5, P=0.0577, day 3 vs 6, p=0.0609. FTLD-TDP-C bi-phosphorylatede: day 3 vs 4, p=0.0522.


C ) Bar graph representing of NeoAggpS403 / 404 bearing double phosphorylation ( left side , bars filled in orange ) of NeoAggpS409 bearing double phosphorylation ( right side , bars filled in purple ) . Each is labelled in different colored dots . N = 3 biological replicates per condition , graph bar showing mean ± . Two - way ANOVA repeated measures , with post - hoc Fisher ' s LSD , pS403 / 404 also double positive : F ( 3 , 6 ) = 6 . 567 , p = 0 . 0253 : FTLD - TDP - A : Day 3 vs 5 , p = 0 . 0491 , day 4 vs 6 , p = 0 . 0321 , FTLD - TDP - C : ay 4 vs 6 , p = 0 . 037 . pS409 / 419 also double positive : F ( 3 , 6 ) = 1 . 246 , p = 0 . 3731 , FTLD - TDP - C : Day 4 vs 5 , p = 0 . 0144 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph representing the percentage of NeoAggpS403/404 bearing double phosphorylation (left side, bars filled in light orange) and percentage of NeoAggpS409 bearing double phosphorylation (right side, bars filled in light purple). Each individual patient is labelled in different colored dots. N=3 biological replicates per condition, graph bar showing mean ± SEM. Two-way ANOVA repeated measures, with post-hoc Fisher's LSD test, pS403/404 also double positive: F(3,6)=6.567, p=0.0253: FTLD-TDP-A: Day 3 vs 5, p=0.0491, day 4 vs 6, p=0.0321, FTLD-TDP-C: ay 4 vs 6, p=0.037. pS409/419 also double positive: F(3,6)=1.246, p=0.3731, FTLD-TDP-C: Day 4 vs 5, p=0.0144.


E ) Overexpression of GFP - TDP - 43 variants with different mutations that either block ( CC AA mutation , red labels ) or mimic phosphorylation ( DD EE , green label ) at S403 / 404 or S409 / 410 in motor neuron - like NSC - 34 . Western blot showed a specific signal for total TDP - 43 ( top panel ) , TDP - 43pS403 / 404 ( middle panel ) TDP - 43p409 / 410 ( lower panel ) load control SOD1 ( bottom 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

E) Overexpression of GFP-TDP-43 variants with different mutations that either block (CC and AA mutation, red labels) or mimic phosphorylation (DD and EE, green label) at S403/404 or S409/410 in motor neuron-like NSC-34 cells. Western blot showed a specific signal for total TDP-43 (top panel), TDP-43pS403/404 (middle panel) and TDP-43p409/410 (lower panel) and load control SOD1 (bottom panel).


A - C ) dSTORM of seeded for 6 days with non - neurodegenerative control ( non - ND control ) , FTLD - TDP - A FTLD - TDP - C seeds extracted from post - mortem brain tissue . shows localization of TDP - 43 ( HA tag , cyan ) along with LaminB1 ( yellow , A ) , nucleopore complexes ( nucleopores , yellow , B ) nucleolus ( yellow , C ) . seeded with FTLD - TDP seeds show coaggregation of upon neoaggregate formation . are presented at same scale , 2 μm as indicated in non - neurodegenerative control top panels . D ) dSTORM of containing after 6 days of seeding with SarkoSpin fractions of postmortem brain from with FTLD - TDP - A or FTLD - TDP - C . Red arrows point to . were labelled with TDP - 43 ( HA - tag , cyan ) phosphorylated TDP - 43 pS403 / 404 ( pTDP - 43 , magenta ) . are presented at same scale , 2 μm as indicated in FTLD - TDP - A top 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) dSTORM images of cells seeded for 6 days with non-neurodegenerative control (non-ND control), FTLD-TDP-A and FTLD-TDP-C seeds extracted from post-mortem brain tissue. Staining shows localization of TDP-43 (HA tag, cyan) along with LaminB1 (yellow, A), nucleopore complexes (nucleopores, yellow, B) and nucleolus (yellow, C). Cells seeded with FTLD-TDP seeds show coaggregation of nuclear membrane proteins upon neoaggregate formation. All images are presented at the same scale, 2 μm as indicated in the non-neurodegenerative control top panels. D) dSTORM images of cells containing aggregates after 6 days of seeding with SarkoSpin fractions of postmortem brain from patients with FTLD-TDP-A or FTLD-TDP-C. Red arrows point to cytoplasmic aggregates. Cells were labelled with TDP-43 (HA-tag, cyan) and phosphorylated TDP-43 pS403/404 (pTDP-43, magenta). All images are presented at the same scale, 2 μm as indicated in the FTLD-TDP-A top panel.


microscopy ( LM ) showing HA - immunoreactive inclusions ( green color , black arrowheads ) identified in ultrathin sections from resin - embedded monolayers . LM were overlaid with low magnification EM ( EM / LM overlay ) to localize same region within for EM imaging at higher magnification ( dotted black square , EM ) . A ) HA - immunopositive regions within seeded with FTLD - TDPA material showed accumulated filaments ( examples indicated with arrowheads ) . B ) HA - immunopositive regions within seeded with FTLD - TDP - C material showed aggregated amorphous material . Scale bars LM EM / LM overlay , 10 µm ; EM , 500

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

Light microscopy images (LM) showing the HA-immunoreactive inclusions (green color, black arrowheads) identified in ultrathin sections from resin-embedded cell monolayers. The LM images were overlaid with low magnification EM images (EM/LM overlay) to localize the same region within the cells for EM imaging at higher magnification (dotted black square, EM). A) HA-immunopositive regions within cells seeded with FTLD-TDPA patient material showed accumulated filaments (examples indicated with white arrowheads). B) HA-immunopositive regions within cells seeded with FTLD-TDP-C patient material showed aggregated amorphous material. Scale bars LM and EM/LM overlay, 10 µm; EM, 500 nm


A ) 2D ( first column ) shows an example of overall of neuronal inclusions from FTLD - TDP - A localized in postmortem human brain by correlative electron microscopy ( also shown in Fig . EV4D ) . 2D projections of central slices of reconstructed 3D tomograms ( middle column ) taken at higher magnification within aggregate show a dense accumulation of filaments seemingly enclosed by a boundary ( arrowheads ) . positions of filaments selected for subtomogram are shown within a single orthoslice from same tomogram ( colored , dots last column ) . B ) STED imaging of structures ( left column ) neuronal inclusions found in a postmortem tissue of a with FTLD - TDP - A . LaminB1 ( green ) was used as a marker along with TDP - 43 ( cyan ) TDP - 43pS403 / 404 ( magenta ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 2D TEM (first column) shows an example of the overall morphology of neuronal cytoplasmic inclusions from FTLD-TDP-A localized in postmortem human brain by correlative light and electron microscopy (also shown in Fig. EV4D). 2D projections of the central slices of reconstructed 3D tomograms (middle column) taken at higher magnification within the aggregate show a dense accumulation of filaments seemingly enclosed by a boundary membrane (white arrowheads). The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram (colored, dots last column). B) STED imaging of nuclear structures (left column) and neuronal cytoplasmic inclusions found in a postmortem tissue of a patient with FTLD-TDP-A. LaminB1 (green) was used as a nuclear membrane marker along with TDP-43 (cyan) and TDP-43pS403/404 (magenta).


C ) Confocal imaging of dystrophic neurites found in a postmortem tissue of a with FTLD - TDP - C . Dystrophic neurites were identified using TDP - 43 ( cyan ) , TDP - 43pS403 / 404 ( magenta ) . Neurite markers were used to visualize neuronal projections ( MAP2 for dendrites , PNF for axons ; yellow orange , respectively ) . D ) 2D ( first row ) of dystrophic neurites from FTLD - TDP - C , localized in postmortem human brain by correlative electron microscopy ( also shown in Fig . EV5D ) . 2D projections of central slices of reconstructed 3D tomograms ( middle row ) taken at higher magnification from regions within each aggregate show a sparse arrangement of filaments which are interspersed with vesicles ( asterisk ) . A similar arrangement of sparse filaments vesicles is seen in a cross - section of a dystrophic neurite from FTLD - TDP - C ( cross - section , also shown in Fig . EV5G ) . positions of filaments selected for subtomogram are shown within a single orthoslice from same tomogram ( colored dots , last row ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal imaging of dystrophic neurites found in a postmortem tissue of a patient with FTLD-TDP-C. Dystrophic neurites were identified using TDP-43 (cyan), and TDP-43pS403/404 (magenta). Neurite markers were used to visualize neuronal projections (MAP2 for dendrites, PNF for axons; yellow and orange, respectively). D) 2D TEM (first row) of dystrophic neurites from FTLD-TDP-C, localized in postmortem human brain by correlative light and electron microscopy (also shown in Fig. EV5D). 2D projections of the central slices of reconstructed 3D tomograms (middle row) taken at higher magnification from regions within each aggregate show a sparse arrangement of filaments which are interspersed with vesicles (white asterisk). A similar arrangement of sparse filaments and vesicles is seen in a cross-section of a dystrophic neurite from FTLD-TDP-C (cross-section, also shown in Fig. EV5G). The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram (colored dots, last row).


( A - C ) Atg7 + / + Atg7 - / - MEFs stably expressing GFP ‐ Gal3 ( A , C ) or GFP ‐ Ub ( B ) were treated with 1000 μM LLOMe or 250 μg / ml silica for 3 h . were subjected to immunocytochemistry using following antibodies : anti ‐ LC3 anti ‐ Lamp1 ( A ) , anti ‐ p62 anti ‐ Lamp1 ( B ) , or anti ‐ FK2 anti ‐ p62 ( C ) . Bars : 10 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Atg7+/+ and Atg7-/- MEFs stably expressing GFP‐Gal3 (A, C) or GFP‐Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti‐LC3 and anti‐Lamp1 (A), anti‐p62 and anti‐Lamp1 (B), or anti‐FK2 and anti‐p62 (C). Bars: 10 μm.