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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and <t>UCM-05194–treated</t> mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and <t>UCM-05194–treated</t> mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and <t>UCM-05194–treated</t> mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and <t>UCM-05194–treated</t> mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and <t>UCM-05194–treated</t> mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and <t>UCM-05194–treated</t> mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and <t>UCM-05194–treated</t> mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and <t>UCM-05194–treated</t> mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and UCM-05194–treated mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Journal: Investigative Ophthalmology & Visual Science

Article Title: LPA1-Induced EMT of Retinal Pigment Epithelial Cells Promotes Subretinal Fibrosis via USP1-Mediated Deubiquitination and Stabilization of ZEB1

doi: 10.1167/iovs.67.10.34

Figure Lengend Snippet: LPA1 promotes EMT in RPE cells. ( A ) Relative LPA1 mRNA levels in ARPE-19 cells transfected with an empty vector or the LPA1 overexpression plasmid, as measured by RT-qPCR. ( B , C ) WB analysis verifying LPA1 overexpression in ARPE-19 cells. ( D , E ) Scratch wound healing assays in ARPE-19 cells performed under the indicated conditions, photographed at 0 and 24 hours. ( F , G ) Transwell migration assays in ARPE-19 cells under the indicated conditions. Scale bar : 50 µm. ( H – J ) Protein levels of E-cadherin, fibronectin 1, collagen I, α-SMA, and LPA1 in ARPE-19 cells under the designated conditions. ( K , L ) IF staining for α-SMA in the RPE–choroid complex from control and UCM-05194–treated mice in the laser-induced SRF model. Scale bar : 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: One day after the initial laser treatment, mice in the indicated groups received intravitreal administration of UCM-05194 (HY-167862A; MedChemExpress, Monmouth Junction, NJ, USA), BMS-986278 (HY-139853; MedChemExpress), or aflibercept (EYLEA; Bayer, Leverkusen, Germany) at a volume of 1 μL per eye, corresponding to a final dose of 40 μg per eye.

Techniques: Transfection, Plasmid Preparation, Over Expression, Quantitative RT-PCR, Migration, Staining, Control