Fig 1: Overexpression of DAPL1 in the RPE of Dapl1−/− mice inhibits EMT in vivo.A At 2 weeks after infection with AAV9-NC or AAV9-DAPL1, western blotting showed the protein expression levels of HA tag, DAPL1, ZO-1, E-cadherin, ZEB1, N-cadherin, vimentin, and SNAIL in RPE cells of Dapl1−/− mice. B The bar graph shows quantification of the bands based on the results in (A). N = 3. C At 2 weeks after infection with AAV9 or AAV9-DAPL1, immunostaining images of ZO-1 in RPE flat mounts of 6-week-old Dapl1−/− mice. D At 2 weeks after the injection of AAV9-NC or AAV9-DAPL1, with the subsequent subretinal injection of sodium hyaluronate to cause retinal detachment for 10 days, histological images of H&E staining show retinal structures from 6-week-old Dapl1−/− mice. Note that subretinal membranes were observed in the control AAV9-NC- but not in the AAV9-DAPL1-injected eyes (black arrows). E The bar graph shows quantification of the area of the subretinal membrane based on the results from (D). F Immunohistochemistry for OTX2 (white arrow indicated) on the surfaces of the detached retinas of Dapl1−/− mice after the injection of AAV9-NC or AAV9-DAPL1 for 2 weeks. G Immunohistochemistry images showing α-SMA expression (white arrow indicated) on the surfaces of the detached retinas of Dapl1−/− mice after the injection of AAV9 or AAV9-DAPL1 for 2 weeks. N = 6, ** indicates P < 0.01. Scale bar: 50 μm.
Fig 2: Knockdown of P21 in ARPE-19-overexpressing DAPL1 cells promotes PVR progression in a rabbit model.A, B Western blots showing the expression of P21, ZO-1, vimentin, and SNAIL in ARPE-19 + DAPL1 cells after the knockdown of P21 via infection with sh-P21 lentivirus and related quantification. C Representative images showing fundus photographs captured at day 38. Note that severe retinal detachment with retinal folds was observed in ARPE-19 + DAPL1 + sh-P21-injected eyes. D Optical coherence tomography (OCT) scans were performed on day 38. Note that severe retinal detachment was observed in the ARPE-19 + DAPL1 + shP21-injected eyes. E Histological images of H&E staining of the rabbit eye in each indicated group showing severe retinal detachment (red stars) in the ARPE-19 + DAPL1 + sh-P21-injected eyes. F Images show that α-SMA expression was detected by immunohistochemistry around the retinas in the rabbits, and its positive signals could be observed in the epiretinal and subretinal membranes in ARPE-19 + DAPL1 + shP21-injected eyes. G PVR severity in each group was graded according to Fastenberg’s score at the indicated times. N = 6, ** or * indicates P < 0.01 or *P < 0.05, respectively.
Fig 3: DAPL1 inhibits PVR progression in a rabbit model.ARPE-19+EGFP or ARPE-19 + DAPL1 cells were injected into the vitreous body of pigmented rabbits to induce experimental PVR. A Representative images showing fundus photographs captured on day 38 in each group. Note that severe retinal detachment with retinal folds was observed in ARPE-19+EGFP-injected eyes but not in ARPE-19 + DAPL1-injected eyes. B Optical coherence tomography (OCT) scanning showing that severe retinal detachment could be observed in the ARPE-19+EGFP-injected eyes but not in the ARPE-19 + DAPL1-injected eyes after 38 days. C Representative histological images of H&E staining of the rabbit eye in each group were obtained and analyzed. Note that retinal folds (black triangles), retinal detachment, and the formation of the epiretinal and subretinal membranes (red stars) were observed in the ARPE-19+EGFP-injected eyes but not in the ARPE-19 + DAPL1-injected eyes. D Immunohistochemistry images showing the detection of α-SMA expression around the retinas in the rabbits; positive signals were observed in the epiretinal and subretinal membranes in ARPE-19+EGFP-injected eyes (yellow arrows). E The severity of PVR in each group was graded according to Fastenberg’s score at the indicated times. N = 6, Scale bar: 50 μm.
Fig 4: Overexpression of DAPL1 inhibits the EMT of RPE cells in vitro.A, B Western blotting showing the protein expression level of DAPL1 in ARPE-19 cells and its quantification. C Transwell assays for analyses of the migration of ARPE-19+EGFP or ARPE-19 + DAPL1 cells. D Quantification of the number of migrated cells based on the results from C. E, F Wound healing assay at 0, 24 and 48 h respectively in ARPE-19 + EGFP and ARPE-19 + DAPL1 cells and the wound healing percentage. Note that the cell migration was strongly inhibited by DAPL1 overexpression. G, H Western blotting showing the protein expression levels of ZO-1, E-cadherin, P-cadherin, ZEB1, N-cadherin, Vimentin, and SNAIL in ARPE-19+EGFP or ARPE-19 + DAPL1 cells and quantification. I The immunostaining images of ZO-1, Vimentin, and SNAIL in ARPE-19+EGFP or ARPE-19 + DAPL1 cells. N = 3, ** or * indicates P < 0.01 or *P < 0.05, respectively. Scale bar: 50 μm.
Fig 5: DAPL1 promotes P21 stability through NFκB.A Real-time PCR showing the mRNA expression level of P21 in ARPE-19 + DAPL1 cells. B Western blots showing the level of P21 protein in ARPE-19+EGFP or ARPE-19 + DAPL1 cells upon treatment with cycloheximide (CHX) for various times, as indicated. C Line chart showing the percentage of remaining P21 protein at different times based on the results from (B). (D, E) Western blots showing the protein level of P21 in ARPE-19+EGFP or ARPE-19 + DAPL1 cells upon treatment with proteasome inhibitor MG132 for 4 h or not, as indicated. F, G Western blots showing the expression of DAPL1, NFκB, p-P21 and p-P38 in ARPE-19 + DAPL1 cells and quantification of the bands. H, I Western blots showing the expression of NFκB, P21 and p-P21 in ARPE-19 + DAPL1 cells, where NFκB was knocked down, and quantification results. J After the knockdown of NFκB in ARPE-19-DAPL1 cells for 48 h and subsequent treatment with CHX for various times, as indicated, the expression level of P21 protein was analyzed by western blotting. K The line chart shows the percentage of remaining P21 protein at different times based on (J). N = 3, ** indicates P < 0.01 or *P < 0.05, respectively.
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