Cell culture and treatments
Bovine aortic endothelial cells (BAECs) obtained from VEC Technologies (Rensselaer, NY, USA) were cultured in Dulbecco Modified Eagle Medium (DMEM, Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone, GE Healthcare Life Sciences, Pistacaway, NJ, USA), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Thermo Fisher). Human umbilical vein endothelial cells (HUVECs) obtained from VEC Technologies were cultured in M199 media supplemented with 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and Endothelial Cell Growth Supplement (ECGS, Corning Life Sciences, Corning, NY, USA). Recombinant human VEGF-A was obtained from R&D System (Minneapolis, MI, USA). Sodium arsenite (NaAsO2), hydrogen peroxide H2O2 and MG-132 were from Millipore-Sigma (Burlington, MA, USA) and Thapsigargin was from Alomone labs (Jerusalem, Israel). Before each treatment, BAECs and HUVECs were serum starved for 6 hrs followed by a treatment. The doses and the duration of treatments are specified in the figure legends.
Cell migration and viability assays
Cell migration was measured in a wound healing assay. Briefly, BAECs plated in 6-well plates were transfected with siRNAs and allowed to reach 90% confluency over 48 hrs. Scratches on the monolayer were performed with a 200-µl pipette tip. Images of the wounded area were taken using a Zeiss Axio-Observer Z1 epifluorescent microscope (2.5×) at the beginning (t=0) and after 16 hrs of migration. Wound closure was measured using the draw spline contour tool on Zen Blue.
Cell viability was determined by Trypan blue exclusion. Viable cells were manually counted using an hemacytometer or using a Countess III cell counter (Life Technologies).
Plasmids and transfections
ZO-1, YB-1, β-catenin and VE-cadherin small interfering RNA (siRNA) as well as non-silencing control siRNA were obtained from Horizon Discoveries (Chicago, IL, USA). siCT AUG AAC GUG AAU UGC UCA AUU, siVE-cadherin (bovine) ACA AAG AAC UGG ACA GAG AUU, siVE-cadherin (human) GCA CAU UGA UGA AGA GAA A, siZO-1 (bovine) GCA GAG AGG AAG AGA GAA UUU, siZO-1 (human) UGG AAA UGA UGU UGG AAU A, siβ-catenin (human) CCA CUA AUG UCC AGC GUU U, siYB-1 (bovine) CAG CAG AAC UAC CAG AAU A, siYB-1 (human) CGG CAA UGA AGA AGA UAA A. BAECs and HUVECs were transfected with plasmids or siRNAs using Lipofectamine 2000 (Thermo Fisher) according to manufacturer’s instructions.
Immunoblotting and immunoprecipitation
Cells were solubilized with a lysis buffer containing 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.1% deoxycholic acid, 50 mM Tris (pH 7.4), 0.1 mM EGTA, 0.1 mM EDTA, 50 mM NaCl, 20 mM NaF, 1 mM Na4P2O7 and 1 mM Na3VO4. Lysate were incubated for 30 minutes at 4°C, centrifuged at 14000 g for 10 minutes and boiled in SDS sample buffer. For the immunoprecipitation, BAECs were treated (with VEGF or arsenite) and were lysed in the lysis buffer with NaCl adjusted to 75 mM. The lysates were centrifuged at 20,000g for 10 min and 1 mg of proteins was used for immunoprecipitation with 1 µg of antibody overnight. Immunoglobulin G (IgG) control IPs were used as a negative control. Immunocomplexes were incubated with protein A sepharose beads (Millipore-Sigma) for 1 hour, washed in lysis buffer and boiled in SDS sample buffer. The samples were separated by SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (Hybond-ECL, GE Healthcare Life Science), and western blotted. Detection was performed using HRP-coupled antibodies and an Image Quant LAS4000 chemiluminescence-based detection system (enhanced chemiluminescence) (GE Healthcare Life Science).
For immunoblots we used horseradish peroxidase (HRP)-coupled antibodies from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The primary antibodies used for immunoblots or immunofluorescence experiments were: rabbit anti-ZO-1 and mouse anti-ZO-1 (617300 and 339100, Thermo Fisher), rabbit anti-MYC-Tag (2278S), rabbit anti-phospho-Ser102-YB1 (2900S), rabbit-anti-YB1 (4202S for Immunofluorescence, 9744S for Immunoblotting), rabbit anti-caspase-3 (9665T) and mouse anti-actin (3700S ) (New England Biolabs, Ipswich MA, USA), mouse anti-G3BP1 (611126) and mouse anti-β-catenin (610153) (BD Biosciences, San Jose, CA, USA) goat anti-VE-cadherin (R&D Systems), mouse anti-EMAP II/AIMP1, mouse anti-FUS, anti-Ribosomal Protein L23a (sc-517097, Santa Cruz, CA, USA) and mouse anti- γ-catenin (JUP) (610253, BD Biosciences, San Jose, CA, USA).
Sample preparation for MS
For MS experiments, immunoprecipitates were washed three times with lysis buffer and then three times detergent free lysis buffer. Then, immunoprecipitated proteins were eluted in Urea 8 M, 50 mM Tris (pH 7.4), and proteases and phosphatases inhibitors. Beads were incubated with 50 µl elution buffer at room temperature for 30 minutes with frequent agitation. Eluted proteins were reduced at 37°C using dithiothreitol (DTT) for one hour and alkylated by iodoacetamide for 60 minutes at room temperature in the dark. The mixture was digested using trypsin (ratio enzyme/total protein of 1:50) followed by incubation at 37°C overnight. The tryptic digestion was quenched by adding 1% TFA (trifluoroacetic acid). Peptides were re-solubilized under agitation for 15 minutes in 21 µL of 1% ACN / 1% formic acid. The LC column was a PicoFrit fused silica capillary column (17 cm x 75 µm i.d; New Objective, Woburn, MA), self-packed with C-18 reverse-phase material (Jupiter 5 µm particles, 300 Å pore size; Phenomenex, Torrance, CA) using a high-pressure packing cell. This column was installed on the Easy-nLC II system (Proxeon Biosystems, Odense, Denmark) and coupled to the LTQ Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) equipped with a Proxeon nanoelectrospray Flex ion source. The buffers used for chromatography were 0.2% formic acid (buffer A) and 100% ACN / 0.2% formic acid (buffer B). Peptides were loaded on-column at a flow rate of 600 nL/minute and eluted with a 2 slopes gradient at a flow rate of 250 nL/minute. Solvent B first increased from 1 to 40% in 110 minutes and then from 40 to 80% B in 50 minutes.
MS data processing
Raw mass spectrometry data were processed using the MaxQuant software (version 188.8.131.52). Database searching was performed using the Andromeda search engine (version 184.108.40.206) integrated into MaxQuant against the bovine UniProt database and against the human UniProt database. MaxQuant default parameters were used with the exception of minimum ratio count and LFQ minimum ratio count set to 1. Enzyme specificity was set to trypsin and up to two missed cleavages was allowed. Cysteine carbamidomethylation (C) was set as fixed modification and oxidation (M) and phosphorylation (STY) were set as variable modification. The minimum required peptide length was 6 amino acids. Mass tolerances for precursor ions and fragment ions were set to 20 ppm and 0.5 Da, respectively. The “matching between runs” algorithm in MaxQuant was enabled. The false discovery rate (FDR) was estimated by searching against the databases with the reversed sequences. For protein and peptide identification, the maximum FDR was set to 1%. Three independent biological replicates and two technical replicates were performed. Correlations between the biological replicates are shown in Supplemental Fig. S1b. For proteins quantification, LFQ intensity values from biological and technical replicates that represent protein abundance were used for statistical analysis. Protein identification in at least two biological replicates was required for further analysis. Also, at least two peptides of a protein must be identified to be considered as a potential ZO-1 interacting protein. LFQ intensities across different samples were first normalized according to the intensities of the bait protein ZO-1 in each sample in order to have equal amounts of ZO-1 in each replicate. Then, intensities of ZO-1 interactors across different samples were adjusted according to the normalized ZO-1 intensities in each sample. Normalized LFQ intensities were used for determination of specific protein–protein interactions.
Significant interactors were determined by performing a statistical analysis of the bait IPs of each condition versus IgG control IPs. Datasets were log2 transformed and using Perseus tools, we imputed normal distributed values for missing values using a normal distribution with width of 0.3 and a downshift of the mean by 1.8 compared to distribution of all LFQ intensities. Then, we performed a student’s t-test–based comparisons of bait IPs versus IgG control IPs to identify significant interactors with false discovery threshold set at 0.05. Permutation-based FDR method in Perseus was used to perform multiple testing corrections. We calculated the average intensities of ZO-1 interacting proteins between the replicates and the treated/untreated ratio for each protein was determined. To determine the interactors that are modulated by each treatment, we compared statistically using a student’s t-test the bait IPs of each condition versus control condition. Only interactors with more than 2-fold change compared to control condition were considered as affected by VEGF treatment. Interactors that are statistically influenced by VEGF treatment with a p-value<0.05 were considered significantly modulated by VEGF. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD029332 64. The following secure account has been created to allow review while the data remain in private status: Username: [email protected] ; Password: 7qk7dVrX
Proteomics data analysis
Gene ontology annotations for biological processes were obtained from the Gene Ontology integrated in STRING database (version 11.0). Only GO annotations that were significantly enriched with a p value of less than 0.05 were used in the analysis. To generate protein interactions network, STRING interactions database was used. The published or informatic-predicted interactions were first determined using standard STRING-defined confidence (medium confidence 0.4). Protein enrichment in the protein interaction network was manually annotated based on the GO biological process enrichment. The obtained STRING network data were imported into the Cytoscape software for visualisation. For clustering analysis, The Markov Cluster Algorithm (MCL) algorithm with clusterMaker2 plugin in Cytoscape was used to identify functional protein clusters within the networks.
BAECs and HUVECs were transfected and then cultured on 0.1% gelatin-coated coverslips. Cells were washed with cold PBS and fixed for 20 minutes in 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100. Cells were rinsed with PBS and blocked with 1% BSA for 1 hour at room temperature. After blocking, cells were incubated with primary antibodies G3BP1 (dilution 1:100), ZO-1 (dilution 1:100), YB-1 (dilution 1:50) 2 hrs at room temperature in 0.1% BSA in PBS. Bound primary antibodies were visualized after 1 hour of incubation using Alexa Fluor 488-labeled donkey anti-goat, Alexa Fluor 568-labeled donkey anti-mouse, Alexa Fluor 568-labeled donkey anti-rabbit, Alexa Fluor 488 goat anti-mouse. Coverslips were mounted using Fluoromount (Millipore-Sigma) and observed using an LSM800 Zeiss confocal laser-scanning microscope (Carl Zeiss, Germany). Samples were viewed with a 63×/1.5 zoom oil objective. Images were assembled via ImageJ and Photoshop CC (Adobe Systems). Colocalization was analyzed by determining the Pearson colocalization coefficient using the Zen software (Zeiss).
All animal studies were approved by the Animal Care Committee of the University of Montreal in agreement with the guidelines established by the Canadian Council on Animal Care. C57BL/6J wildtype were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).
Dissection and whole mount staining of postnatal retinas of mice at the stage P5 were performed as described previously12. Retinas were fixed for 2 hrs on ice in 4% PFA. Dissected retinas were blocked overnight in 1% BSA, 0.3% Triton X-100 in PBS. For lectin I staining, retinas were equilibrated with Pblec buffer containing 1 mm CaCl2, 1 mm MgCl2, 1% Triton X-100 in PBS (pH 6.8) and then stained with Rhodamine conjugated Lectin I (dilution 1:100) overnight at 4°C. For G3BP1, YB-1 or ZO-1 staining, retinas were incubated with mouse anti-G3BP1 (dilution 1:100), rabbit anti-ZO-1 (dilution 1:100) and rabbit anti-YB-1 (dilution 1:50) in blocking buffer 2 hrs at 4°C. After primary incubation, retinas were labeled with Alexa-Fluor 488-labeled goat anti-mouse or anti-rabbit (dilution 1:100). Stained retinas were flat mounted using Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Z-stack confocal imaging was performed on Zeiss LSM800 confocal laser-scanning microscope using a 63× oil objective and a 2.5× digital zoom. All quantifications were performed on z-stack confocal images. Images were analyzed using Fiji software (NIH, Bethesda, MD, USA) or the built-in tools in Zen software (Zeiss).
Quantification of SGs staining
The number of SGs was quantified using the ImageJ software. IF images were randomly taken with a 63× objective lens in 10 different fields. The plug-in “Analyze particles” was used to count the number of SGs. The percentage of cells with SGs was calculated by counting the number of cells displaying SGs divided by the total number of cells.
Data are represented as the means ± SEM. Two-tailed independent Student’s tests were used when comparing two groups. Comparisons between multiple groups were made using one-way ANOVA followed by post-hoc Bonferoni’s multiple comparisons test among groups on using Prism 5 software (GraphPad, San Diego, CA, USA). P-value < 0.05 was considered statistically significant.