EMBO/SourceData huggingface dataset:
Figure 4(A) Knockdown of SIRT1 abolished CSAG2-mediated decrease in ac-p53 K382. CSAG2 over-expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin/TSA treatment for 6 hr. Cell lysates were blotted for the indicated proteins.(B) Inhibition of SIRT1 enzymatic activity blocked CSAG2-induced decrease in p53 ac-K382. CSAG2 over-expressing HCT116 cells were treated with 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cell lysates were blotted for the indicated proteins.Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins (C)Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins and quantitated (D; n=3 biological replicates). Data shown as mean ± SD.(E) CSAG2 binds catalytically inactive SIRT1 H363Y. Cells were transfected with the indicated constructs before anti-Myc IP and blotting for the indicated proteins.(F) SIRT1 H363Y blocks CSAG2 regulation of p53 ac-K382 levels. HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins.(G) Inhibition of SIRT1 activity abolished CSAG2-induced anchorage-independent growth. Soft agar colony formation assays were performed in control or CSAG2 over-expressing HCT116 cells with or without EX-527 treatment. Data are represented as the mean ± SD, n=3 biological replicates.(H) Summary of region on CSAG2 required for SIRT1 interaction. HEK293FT cells stably expressing HA-SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.(I) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro. Myc-tagged CSAG2 38-127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST-SIRT1, SDS-PAGE, and immunoblotting for anti-Myc.(J) CSAG2 Δ37 mutant does not regulate p53 ac-K382 levels. Cells were transfected with the indicated Myc-CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin/ 0.4 μM TSA. Cell lysates were blotted for the indicated proteins.(K) CSAG2 Δ37 fails to promote anchorage-independent growth of H460 cells. Soft agar colony formation assays were performed in CSAG2-negative H460 cells stably expressing CSAG2 wild-type or CSAG2 Δ37 mutant (n=3 biological replicates). Data are represented as the mean ± SD.
Figure 5Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 (B) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of H2O2 (D) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).(E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F).(G) H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 hr. Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).(H) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 over-expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX-527 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (J) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (K) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (L) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H2O2 (M) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of doxorubicin (N) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).
Figure 6(A) CSAG2 does not alter SIRT1 protein levels. HEK293FT cells were transfected with different amounts of Myc-CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins.(B) CSAG2 does not alter SIRT1 subcellular location. U2OS cells were transfected with or without mcherry-CSAG2 for 48 hr before fixation, SIRT1 immunostaining, and imaging. Scale bar: 10 µm.(C) Summary of results mapping interaction region of SIRT1 recognized by CSAG2. HEK293FT cells stably expressing HA-CSAG2 were transfected with indicated Myc-SIRT1 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.(D) CSAG2 directly binds SIRT1 catalytic domain in vitro. HA-tagged SIRT1 fragments were in vitro translated. In vitro binding assays with recombinant GST or GST-CSAG2 were performed followed by SDS-PAGE and immunoblotting for anti-HA.(E) CSAG2 enhances SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST or GST-CSAG2, n=3 biological replicates. Nicotinamide (1 mM) was added to the reaction were indicated to block SIRT1 activity.(F) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST, GST-CSAG2 or GST-CSAG2 38-127, n=3 biological replicates.(G) CSAG2 enhances SIRT1 deacetylation of p53 ac-K382 peptide through increasing kcat, but not Km. not affecting the binding ability with its substrate p53. SIRT1 activity assays were performed as described above, except varying amounts of p53 ac-K382 fluorometric peptide were used in the presence of saturating NAD+ (3 mM), n=3 biological replicates.(H-I) U2OS cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr before being treated with or without 0.4 μM TSA for 6 hr. Cell lysates were blotted for the indicated proteins (H). Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.(J) HeLa, U2OS, or HCT116 cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.(K) Knockdown of endogenous CSAG2 in A375 cells increased ac-H4K16 levels. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of ac-H4K16 relative to total H4 is shown as the mean ± SD, n=3 biological replicates.
Figure 1A. Four representative images of RHOB immunostaining in human non-small-cell lung cancers. Figures correspond to the percentage of tumor analyzed.B. Characteristics of 96 patients with an EGFR-activating mutation who were treated with EGFR-TKI. Each column corresponds to one patient. Data include general characteristics (stage at diagnosis, age, sex, tobacco usage, position of EGFR mutation, TKI type), progression-free survival (PFS; lower graph) and intensity of RHOB staining as determined by immunohistochemistry (null: 0, weak: +, moderate: ++ and high: +++).C. Representative scans of patients with low or high RHOB-expressing lung tumors, before and after erlotinib treatment. Red arrows indicate lung tumors.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.G. RHOB immunostaining score evolution in EGFR-mutated lung tumors before treatment and after EGFR-TKI relapse.
Figure 2A. 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).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).
Figure 3HCC4006 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.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.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.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.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.
Figure 4A. 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.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.
Figure 5A. 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.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).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).
Figure 6A. 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.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).
Figure 1A.-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.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.
Figure 2A. 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.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.
Figure 3A. 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).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).
Figure 4A. 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).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).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).
Figure 5A-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.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.