Fig 2: DUSP3-deficiency is associated with increased OCLN phosphorylation in DelA cell lines. A Cell lysates collected from various DelA cell lines were subjected to 7% SDS-PAGE analyses containing 25 μM of Phos-Tag reagent plus MnCl2, followed by immunoblot analyses using indicated antibodies. Phosphorylated OCLN with slower mobility was indicated by an arrowhead. B The expression and phosphorylation levels of Src were analyzed by immunoblot assays. The bar graphs showed the means and standard deviations of three independent experiments. The value of DelA-F11 was assigned as 1. * p < 0.05. C and D Confluent DelA-F13, F14R and F16R cells were subjected to a 24 h treatment of an FAK inhibitor (Defactinib; 5 μM) and the cells were collected and analyzed by immunoblot assays (C) or IF staining (D) using indicated antibodies

Fig 3: Counteracting effects of DUSP3 and FAK regulates OCLN ubiquitination and degradation. A H1299-OCLN and OCLN-4YF cells were treated with cycloheximide (50 μg/ml) for indicated time periods and the cell lysates were subjected to immunoblot analyses. The arrowhead indicated the position of OCLN with slower mobility. B H1299-OCLN and OCLN-4YF cells were pre-treated with cycloheximide (50 μg/ml) for 30 min and then were treated with H2O2 (1 mM) for indicated periods. The cell extracts were then collected and subjected to immunoblot analyses. C H1299-OCLN and OCLN-4YF cells were pre-treated with MG132 (0 or 50 mM) for 1 h and then were treated with or without H2O2 (1 mM) for 1 h. The cell extracts were collected and subjected to immunoblot analyses. The graphs under the immunoblots showed the mean and standard deviations of three independent experiments (A-C). * p < 0.05, ** p < 0.01. D Cell extracts prepared from HA-ubiquitin (HA-Ub)-transfected H1299-OCLN and OCLN-4YF were subjected to immunoprecipitation of Flag-OCLN, and the immunocomplexes were then analyzed for the presence of indicated proteins. E and F H1299-OCLN and OCLN-4YF cells were treated with or without H2O2 (1 mM) for 30 min and were subjected to extract preparation. Flag-tagged OCLN and OCLN-4YF were immunoprecipitated and the immunocomplexes were then analyzed for the presence of indicated proteins. G H1299-DUSP3TR/OCLN and H1299-DUSP3-CSTR/OCLN cells were treated with or without Tet (1 mg/ml) for 12 h and then were treated with H2O2 (0.5 mM) for 30 min. Cell extracts were prepared and Flag-tagged OCLN and OCLN-4YF were immunoprecipitated. The immunocomplexes were then analyzed for the presence of indicated proteins. Cell extracts (2% percent input) were also examined for the relative amounts of individual proteins

Fig 4: Morphological and functional characteristics of the iHCPEnC(A) Experimental workflow for the generation of iHCPEnC and phase contrast image of primary HCPEnC, hTERT-expressing iHCPEnC, and primary senescent HCPEnC. The immortalized cell line grew as a monolayer with characteristic endothelial cellular morphology comparable to primary HCPEnC. Senescent primary HCPEnC revealed an enlarged and flattened morphology. Scale bar, 100 μm.(B) Phase contrast microscopy image of the capillary-like network of iHCPEnCs maintained 72 h on Matrigel. Scale bar, 100 μm.(C) Immunoblot analysis of primary and iHCPEnCs cells detecting expression of hTERT and endogenous c-Myc.(D) Detection of hTERT by indirect immunofluorescence. Primary HCPEnC and iHCPEnC grown on coverslips were stained for detection of hTERT. Only iHCPEnC showed a peri- and nuclear expression of the catalytic unit of hTERT. Cell nuclei were visualized using DAPI. As negative control, immunofluorescence without primary antibody was performed. Scale bar, 20 μm.(E) Immunofluorescence of pan-endothelial markers and tight junction proteins. iHCPEnC grown on coverslips were stained for detection of PECAM1 and VWF as endothelial markers and ZO1, OCLN, CLDN1, and CLDN5 as markers for tight junction proteins as indicated (green). Nuclei were visualized using DAPI (blue). As negative control, immunofluorescence without primary antibody was performed. Scale bar, 20 μm.(F) Agarose gel electrophoresis of RT-PCR products of tight junction protein transcripts. The indicated transcripts were detected in two independent reactions; a typical result is shown. Epithelial cadherin (CDH1), which is not expressed in endothelial cells, was used as negative control, GAPDH served as positive control.(G) TEER values of iHCPEnC seeded on filter inserts were monitored over a period of 5 days after confluence. Data are represented as mean ± SEM of three independent experiments performed in triplicates.(H) FITC-inulin flux across iHCPEnC seeded on filter inserts and grown to confluence was measured during a time-course of 5 days. The FITC-Inulin tracer solution was added to the upper compartment of the filter for 4 h. Data shown are represented as mean ± SEM of two independent experiments each performed in triplicates.

Fig 5: DUSP3 targets multiple Tyr residues at the carboxyl-terminus of OCLN. A and B H1299 cells expressing with wild-type OCLN or OCLN mutant (OCLN-Y398/402F) were treated with or without H2O2 (1 mM) for 30 min. Wild-type and mutated OCLN proteins were immunoprecipitated and analyzed for their Tyr-phosphorylation levels (A) or were subjected to in vitro DUSP3 phosphatase reactions and the subsequent immunoblot analyses (B). C H1299-OCLN cells or H1299 cells-expressing indicated individual OCLN mutants were treated with or without H2O2 (1 mM) for 30 min. Wild type and mutated OCLN proteins were immunoprecipitated and analyzed for their Tyr-phosphorylation levels. D and E H1299-OCLN, OCLN (Y315F), and OCLN (Y287/315/325/443F; named 4YF) cells were treated with or without H2O2 (1 mM) for 30 min. Wild-type and mutated OCLN proteins were immunoprecipitated and analyzed for their Tyr-phosphorylation levels (D) or were subjected to in vitro DUSP3 phosphatase reactions (E). F Affinity purified Flag-OCLN, OCLN-4YF, and OCLN (Y398/402F) proteins were subjected to in vitro FAK phosphorylation