D . Progression - free survival of erlotinib - treated patients with EGFR - mutated lung tumors , according to RHOB expression , assessed by immunohistochemistry ( low RHOB group = negative + weak staining ; high RHOB group = moderate + high staining ) .

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

D. Progression-free survival of erlotinib-treated patients with EGFR-mutated lung tumors, according to RHOB expression, assessed by immunohistochemistry (low RHOB group = negative + weak staining; high RHOB group = moderate + high staining).


E . Progression - free survival of patients who received EGFR - TKi as first - line therapy ( n = 63 ) . F . Progression - free survival of patients who received EGFR - TKi as 2nd - line ( n = 28 ) , 3rd - line ( n = 3 ) or 4th - line ( n = 2 ) therapy .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Progression-free survival of patients who received EGFR-TKi as first-line therapy (n=63). F. Progression-free survival of patients who received EGFR-TKi as 2nd-line (n=28), 3rd-line (n=3) or 4th-line (n=2) therapy.


G . RHOB immunostaining score evolution in EGFR - mutated lung tumors before treatment and after EGFR - TKI relapse .

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

G. RHOB immunostaining score evolution in EGFR-mutated lung tumors before treatment and after EGFR-TKI relapse.


A . Representative H & amp ; amp ; E staining of whole lungs from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . Scale bar : 5 mm . B . Quantification of the tumor / lung ratio . n = 7 for each group except for EGFRL858R / Rhob + / - placebo ( n = 6 ) .

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

A. Representative H&amp;E staining of whole lungs from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. Scale bar: 5 mm. B. Quantification of the tumor/lung ratio. n=7 for each group except for EGFRL858R/Rhob+/- placebo (n=6).


C . Representative Ki67 immunostaining of EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days ( scale bar : 50 µm ) , and the corresponding quantification ( D ) . Three independent zones / mice lung were used for quantification . n = 21 ( 7 mice ) for each group except for EGFRL858R / Rhob + / - placebo ( n = 18 ; 6 mice ) .

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

C. Representative Ki67 immunostaining of EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). Three independent zones/mice lung were used for quantification. n=21 (7 mice) for each group except for EGFRL858R/Rhob+/- placebo (n=18; 6 mice).


E . Immunostaining of cleaved caspase - 3 in lung tumors from EGFRL858R / Rhob + / + or EGFRL858R / Rhob - / - mice treated for 24 h with erlotinib at 12 . 5 mg / kg . Scale bar : 50 µm . ( * * : p < 0 . 001 vs . placebo ; * * * : p < 0 . 0001 vs . placebo ) .

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

E. Immunostaining of cleaved caspase-3 in lung tumors from EGFRL858R/Rhob+/+ or EGFRL858R/Rhob-/- mice treated for 24 h with erlotinib at 12.5 mg/kg. Scale bar: 50 µm. (**: p<0.001 vs. placebo; ***: p<0.0001 vs. placebo).


HCC4006 cells were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells .

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

HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 cells were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 cells were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells .

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

HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 cells were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 cells were ( C ) both transfected with siRNA and transduced by adenoviruses ; then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells .

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

HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 cells were ( C ) both transfected with siRNA and transduced by adenoviruses ; then treated with increasing doses of erlotinib . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


Erlotinib IC50 values were quantified in RHOB - overexpressing or RHOB - depleted ( D ) HCC827 cells , ( E ) HCC2935 cells and ( F ) H3255 cells , as determined by an MTS assay after 72 h treatment . RHOB overexpression or inhibition was monitored by western blotting for each condition .

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

Erlotinib IC50 values were quantified in RHOB-overexpressing or RHOB-depleted (D) HCC827 cells, (E) HCC2935 cells and (F) H3255 cells, as determined by an MTS assay after 72 h treatment. RHOB overexpression or inhibition was monitored by western blotting for each condition.


G . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


G . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) , then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed .

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

G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed.


A . HCC4006 , HCC827 , HCC2935 and H3255 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line . The phosphorylation status of AKT , ERK1 / 2 and EGFR was assessed by western blotting and normalized according to total protein levels . RHOB overexpression was also monitored by western blotting .

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

A. HCC4006, HCC827, HCC2935 and H3255 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2 and EGFR was assessed by western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by western blotting.


B . Representative immunostaining of phospho - AKT ( Ser473 ) , phospho ERK1 / 2 and their total protein amounts in lung tumors from EGFRL858R / Rhob - / - or EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . The remaining hyperplastic areas were selected in erlotinib - treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes . Scale bar : 100 µm .

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

B. Representative immunostaining of phospho-AKT (Ser473), phospho ERK1/2 and their total protein amounts in lung tumors from EGFRL858R/Rhob-/- or EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. The remaining hyperplastic areas were selected in erlotinib-treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bar: 100 µm.


C . HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , and treated for 72 h with increasing concentrations of erlotinib . The surviving cell fraction was determined by an MTS assay . Data are representative of at least three independent experiments .

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

C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay. Data are representative of at least three independent experiments.


C . HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , and treated for 72 h with increasing concentrations of erlotinib . AKT overexpression and phosphorylation at Ser473 were assessed by western blotting . Data are representative of at least three independent experiments .

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

C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. AKT overexpression and phosphorylation at Ser473 were assessed by western blotting. Data are representative of at least three independent experiments.


A . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for four hours with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . The phosphorylation status of GSK3β ( Ser9 ) , ERK1 / 2 and EGFR ( Tyr1173 ) was assessed by western blotting and normalized according to the total protein levels . RHOB overexpression was also monitored by western blotting .

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

A. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting.


B . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 72 h with erlotinib alone ( black and red curves ) or in combination with the AKT inhibitor G594 at 100 nM ( green and blue curves ) . The surviving cell fraction was determined by an MTS assay .

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

B. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay.


C . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594 . The surviving cell fraction was determined by an MTS assay , and erlotinib IC50 values were determined for each condition . ( * * : p < 0 . 001 vs . AdCont cells ; * * * : p < 0 . 0001 vs . AdCont cells ) . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs .

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 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

C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs.


HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by quantification of the subG1 cell population ( D ) .

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

HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by quantification of the subG1 cell population (D).


HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by detection of cleaved PARP and caspase - 3 ( E ) .

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

HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by detection of cleaved PARP and caspase-3 (E).


A . Representative H & amp ; amp ; E staining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not during four days with erlotinib ( 12 . 5 mg / kg / day ) , with the AKT inhibitor G594 ( 25 mg / kg / day ) , or in combination with both drugs . Scale bar : 500 µm . B . Quantification of the tumor / lung ratio of mice treated or not with the individual drugs or with a combination of both .

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 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

A. Representative H&amp;E staining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not during four days with erlotinib (12.5 mg/kg/day), with the AKT inhibitor G594 (25 mg/kg/day), or in combination with both drugs. Scale bar: 500 µm. B. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both.


C . Representative Ki67 immunostaining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib alone ( 12 . 5 mg / kg / day ) or in combination with the AKT inhibitor G594 ( 25 mg / kg / day ) for four days ( scale bar : 50 µm ) , and the corresponding quantification ( D ) .

O O O B-MEASURED_VAR 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 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

C. Representative Ki67 immunostaining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D).


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 .

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

A.-E. 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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 ( 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 . ) .

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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 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 ) .

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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 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 .

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

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 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 ) .

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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 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 ) .

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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 . ) 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 ) .

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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).


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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 .

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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 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 .

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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 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 .

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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 ( 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 .

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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 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 .

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

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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 ] 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


. 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 .

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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 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 .

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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 - 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 .

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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 and 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 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 .

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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 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 .

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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 cells co - expressing the indicated C - terminal FLAG - and Myc - tagged proteins 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 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 .

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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 ( 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 .

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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 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 .

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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 proteins ( output ) were detected by immunobotting using α - Myc and 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 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 .

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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 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 .

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

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 ) 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 ) .

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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 ) and overlapping patterns for H2A ( C ) in mitotic prometaphase bam mutant cells 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 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 .

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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 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

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

(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 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 .

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 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

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 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 .

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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 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 .

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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 ) and Electrocardiography ( ECG ) , n = 15 f , 15 m Uqcrh - / - and 15 f , 15 m WT mice , 6 weeks old .

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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 ) 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 ) .

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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 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 ] )

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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 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 ) .

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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 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 .

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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 ) 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 ) .

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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 ) 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

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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

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

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 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 .

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 B-CONTROLLED_VAR 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.