Solated from mouse eyes following previously published methods [33]. In brief, after induction of euthanasia, the whole eyes were removed and washed profusely first in PBS and gentamicin reagent solution 50 mg/ml (Life Technologies, Gibco, Grand Island, NY, 1:5000 in PBS) for 5 minutes followed by 5 minutes of rinsing with PBS for four times. The globes were then submerged in 10 mg/ml 11089-65-9 Dispase (Life technologies) solution in DMEM/F12 (1:1) with 36 mg/ml sorbitol (Fisher Scientific, Pittsburgh, PA) and kept in 4 for 16 hours. To separate the epithelial sheets, the corneal epithelium was BIBS39 peeled off using a small scraper, then the epithelial sheets were digested in 0.25 trypsin without EDTA (Life Technologies) at 37 for 10 minutes and neutralized with soybean trypsin inhibitor 24195657 2 mg/ml solution (Life Technologies). The sheets were pipetted multiple times before centrifugation in 4 at 800 g for 5 minutes. The supernatant was removed and the cells were re-suspended in defined keratinocyte serum-free medium (D-KSFM, Life Technologies) and plated in collagenResultsNotch1 deletion in the corneal epithelium leads to progressive barrier impairmentNotch1 was conditionally deleted in 2-4 months old Notch1flox/ , K14-Cre-ERT+/- mice following intra-peritoneal injection of 4OHT for 3-5 consecutive days. Efficiency of Notch1 deletion was estimated by western blot on a pooled sample of corneal epithelial sheets (N = 8 per group) and found to be 64-70 (Figure 1A). No loss of Notch1 or any phenotype was ever observed in untreated mice confirming that Cre-ERT was notfloxNotch1 and Corneal Epithelial BarrierFigure 1. Notch1 deletion in the corneal epithelium leads to progressive barrier impairment. Notch1 1315463 knockout efficiency was evaluated by western blotting of pooled corneal epithelial sheets (N=8 per group) from conditional Notch1-/- mice showing 64 to 70 decrease in Notch 1 expression comparing to epithelial sheets from WT littermates (A). Compared to WT (B1, B2), Notch1 deleted corneas demonstrate increased fluorescein staining at 2 (B3) and 6 (B4) weeks after 4-OHT treatment. LC-biotin (stained red with rhodamine) could not penetrate beyond the top few layers of the epithelium in WT mice (C) while it passed the epithelium and reached the stroma in Notch1-/- mice at 4 weeks after Notch1 deletion (D). Red: rhodamine. Blue: DAPI; Scale bar: 50 .doi: 10.1371/journal.pone.0069113.gspontaneously activated. Likewise, no phenotype was ever noted in Notch1+/- heterozygotes (Figure 2B). The mice were examined weekly after completing the treatment with 4-OHT. Typically by 2 weeks after Notch1 deletion, the mice continued to demonstrate clear corneas by slit lamp examination. However, fluorescein staining, which measures the integrity of the corneal epithelial barrier, began to show increased staining (uptake) compared to control littermates — which as a control measure had also been treated with 4-OHT (Figure 1 B1 4). The corneal staining progressively increased every week. The degree of staining after Notch1 deletion was further quantified in the central 1.5 mm of the cornea on a scale of 0 to 3 [35]. At 2 weeks aftertamoxifen injection 62.5 (5/8) eyes and at 6 weeks 100 (8/8) had developed variable degrees of scattered corneal fluorescein staining which was highly significant compared to WT eyes with no staining in all subjects (N = 8) after 2 or 6 weeks (P= 0.026). The mean fluorescein staining score at 6 weeks was and found to be 2.5 ?0.6 in Notch1-.Solated from mouse eyes following previously published methods [33]. In brief, after induction of euthanasia, the whole eyes were removed and washed profusely first in PBS and gentamicin reagent solution 50 mg/ml (Life Technologies, Gibco, Grand Island, NY, 1:5000 in PBS) for 5 minutes followed by 5 minutes of rinsing with PBS for four times. The globes were then submerged in 10 mg/ml Dispase (Life technologies) solution in DMEM/F12 (1:1) with 36 mg/ml sorbitol (Fisher Scientific, Pittsburgh, PA) and kept in 4 for 16 hours. To separate the epithelial sheets, the corneal epithelium was peeled off using a small scraper, then the epithelial sheets were digested in 0.25 trypsin without EDTA (Life Technologies) at 37 for 10 minutes and neutralized with soybean trypsin inhibitor 24195657 2 mg/ml solution (Life Technologies). The sheets were pipetted multiple times before centrifugation in 4 at 800 g for 5 minutes. The supernatant was removed and the cells were re-suspended in defined keratinocyte serum-free medium (D-KSFM, Life Technologies) and plated in collagenResultsNotch1 deletion in the corneal epithelium leads to progressive barrier impairmentNotch1 was conditionally deleted in 2-4 months old Notch1flox/ , K14-Cre-ERT+/- mice following intra-peritoneal injection of 4OHT for 3-5 consecutive days. Efficiency of Notch1 deletion was estimated by western blot on a pooled sample of corneal epithelial sheets (N = 8 per group) and found to be 64-70 (Figure 1A). No loss of Notch1 or any phenotype was ever observed in untreated mice confirming that Cre-ERT was notfloxNotch1 and Corneal Epithelial BarrierFigure 1. Notch1 deletion in the corneal epithelium leads to progressive barrier impairment. Notch1 1315463 knockout efficiency was evaluated by western blotting of pooled corneal epithelial sheets (N=8 per group) from conditional Notch1-/- mice showing 64 to 70 decrease in Notch 1 expression comparing to epithelial sheets from WT littermates (A). Compared to WT (B1, B2), Notch1 deleted corneas demonstrate increased fluorescein staining at 2 (B3) and 6 (B4) weeks after 4-OHT treatment. LC-biotin (stained red with rhodamine) could not penetrate beyond the top few layers of the epithelium in WT mice (C) while it passed the epithelium and reached the stroma in Notch1-/- mice at 4 weeks after Notch1 deletion (D). Red: rhodamine. Blue: DAPI; Scale bar: 50 .doi: 10.1371/journal.pone.0069113.gspontaneously activated. Likewise, no phenotype was ever noted in Notch1+/- heterozygotes (Figure 2B). The mice were examined weekly after completing the treatment with 4-OHT. Typically by 2 weeks after Notch1 deletion, the mice continued to demonstrate clear corneas by slit lamp examination. However, fluorescein staining, which measures the integrity of the corneal epithelial barrier, began to show increased staining (uptake) compared to control littermates — which as a control measure had also been treated with 4-OHT (Figure 1 B1 4). The corneal staining progressively increased every week. The degree of staining after Notch1 deletion was further quantified in the central 1.5 mm of the cornea on a scale of 0 to 3 [35]. At 2 weeks aftertamoxifen injection 62.5 (5/8) eyes and at 6 weeks 100 (8/8) had developed variable degrees of scattered corneal fluorescein staining which was highly significant compared to WT eyes with no staining in all subjects (N = 8) after 2 or 6 weeks (P= 0.026). The mean fluorescein staining score at 6 weeks was and found to be 2.5 ?0.6 in Notch1-.
