Journal: Cell Death & Disease

Article Title: CRX is an intrinsic suppressor of epithelial‒mesenchymal transition in retinal pigment epithelial cells: a promising therapeutic avenue for subretinal fibrosis

doi: 10.1038/s41419-025-08352-y

Figure Lengend Snippet: A Uniform manifold approximation and projection (UMAP) dimensionality reduction plot showed the profiled cells including amacrine, astrocyte, bipolar, cone, horizontal, muller, myeloid, RGC, rod, RPE, vascular cells. B , C The violin plots of RPE65 and CRX expression in all cell types. D The co-expressions of RPE65 and CRX . Red indicates the expression of RPE65 (4359 RPE cells), green indicates the expression of CRX (2334 RPE cells); yellow indicates the co-expressions of RPE65 and CRX , the ratio of CRX + RPE cells was 53.54%.

Article Snippet: The samples were incubated with the primary antibodies against α-SMA (Abcam, Cambridge, UK), ZO-1 (Proteintech, Chicago, USA), CRX (Proteintech), YAP1 (Proteintech), β-catenin (Proteintech), and RPE65 (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: CRX is an intrinsic suppressor of epithelial‒mesenchymal transition in retinal pigment epithelial cells: a promising therapeutic avenue for subretinal fibrosis

doi: 10.1038/s41419-025-08352-y

Figure Lengend Snippet: A Changes in cell morphology, and the expressions of ZO-1 and α-SMA at different time points during the cell sub-culturing process. B Quantitative calculation of the percentage of α-SMA + cells ( n = 8). Detection of the mRNA and protein expression levels of CRX at different time points by ( C ) qRT-PCR, ( D ) Western blotting, and ( E ) quantitative analysis ( n = 3). F , G Nuclear retention ratio of CRX at different time points during the cell sub-culturing process ( n = 8). Scale bar = 50 μm. Data are presented as mean ± SD. Statistical significance was defined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant (using one-way ANOVA and post hoc Bonferroni’s test).

Article Snippet: The samples were incubated with the primary antibodies against α-SMA (Abcam, Cambridge, UK), ZO-1 (Proteintech, Chicago, USA), CRX (Proteintech), YAP1 (Proteintech), β-catenin (Proteintech), and RPE65 (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: CRX is an intrinsic suppressor of epithelial‒mesenchymal transition in retinal pigment epithelial cells: a promising therapeutic avenue for subretinal fibrosis

doi: 10.1038/s41419-025-08352-y

Figure Lengend Snippet: A, B The images of nuclear retention of YAP1 at different time points during sub-culturing of ESC-RPE cells and quantitative analysis ( n = 8). C The CRX expression level in ESC-RPE cells after being treated with the Hippo pathway inhibitor XMU-MP-1 for four days was detected by qRT-PCR ( n = 3). D After treatment with 5 μM XMU-MP-1 for four days, the nuclear retention of YAP1 was detected by immunostaining. E The quantitative analysis of YAP1 nuclear retention ratio ( n = 8). F , G The expression levels of p-YAP1, YAP1, and CRX in ESC-RPE cells after being treated with 5 μM XMU-MP-1 for four days were determined and quantified by Western blotting (n = 3). H The cellular localization of β-catenin at different time points during sub-culturing was demonstrated by immunostaining. I Co-IP analysis of the interaction between β-catenin and YAP1. J , K The protein level of β-catenin in the nucleus and cytoplasm of cells after being treated with 5 μM XMU-MP-1 for four days was determined and quantified by Western blotting (n = 3). L The CRX expression level in ESC-RPE cells after being treated with β-catenin inhibitor MSAB for four days was detected by qRT-PCR (n = 3). M , N The expression levels of β-catenin and CRX in ESC-RPE cells after being treated with 3 μM MSAB for four days were determined and quantified by Western blotting (n = 3). O Venn analysis was conducted on Hippo signaling pathway-related proteins ( https://www.genecards.org/ ), transcription factors predicted to bind to the CRX promoter in the UCSC database ( https://genome.ucsc.edu/ ) and the JAPSAR database ( https://jaspar.elixir.no/ ), the candidate protein TCF7 was screened out. P The regions of the CRX gene paired with qRT-PCR primers (#1–5: promoter, #6: intron). Q The matrices of β-catenin and TCF7 proteins binding to the CRX gene promoter predicted by the JASPAR database. R The binding sites of β-catenin and TCF7 to the CRX promoter were verified by ChIP-qRT-PCR ( n = 3). S CoIP analysis of the interaction between TCF7 and β-catenin. T-U TCF7 was knocked down in ESC-RPE cells, and the expression levels of TCF7 and CRX were determined and quantified by Western blotting ( n = 3). Scale bar = 50 μm. Data are presented as mean ± SD. Statistical significance was defined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001; ns not significant (using unpaired two-sided t-tests in E , K , and N and one-way ANOVA and post hoc Bonferroni’s test in the others).

Article Snippet: The samples were incubated with the primary antibodies against α-SMA (Abcam, Cambridge, UK), ZO-1 (Proteintech, Chicago, USA), CRX (Proteintech), YAP1 (Proteintech), β-catenin (Proteintech), and RPE65 (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Immunostaining, Western Blot, Co-Immunoprecipitation Assay, Binding Assay

Journal: Cell Death & Disease

Article Title: CRX is an intrinsic suppressor of epithelial‒mesenchymal transition in retinal pigment epithelial cells: a promising therapeutic avenue for subretinal fibrosis

doi: 10.1038/s41419-025-08352-y

Figure Lengend Snippet: CRX was overexpressed in ESC-RPE cells, and the expression level was detected by A qRT-PCR ( n = 3), B Western blotting, and C quantitative analysis ( n = 3). The expression levels of EMT-related genes in control- and OE-CRX-ESC-RPE cells after being sub-cultured for four days were determined by D qRT-PCR ( n = 3), E Western blotting, and F quantitative analysis ( n = 3). The sub-cultured control and OE-CRX-ESC-RPE cells were treated with 5 ng/mL TGF-β1 for 4 days. The expression levels of EMT-related genes were determined by G qRT-PCR ( n = 3), H Western blotting, and I quantitative analysis ( n = 3). J , K The protein level of β-catenin in the nucleus and cytoplasm of cells after being treated with 5 ng/mL TGF-β1 and 3 μM MSAB for four days was determined and quantified by Western blotting ( n = 3). L Representative images of sub-cultured ESC-RPE cells treated with TGF-β1 and MSAB for 4 days and 8 days. M , N The expression levels of α-SMA and CRX in sub-cultured ESC-RPE cells treated with TGF-β1 and MSAB for 8 days were determined and quantified by Western blotting ( n = 3). Scale bar = 50 μm. Data are presented as mean ± SD. Statistical significance was defined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001; (using unpaired two-sided t -tests in B , C , and one-way ANOVA and post hoc Bonferroni’s test in the others).

Article Snippet: The samples were incubated with the primary antibodies against α-SMA (Abcam, Cambridge, UK), ZO-1 (Proteintech, Chicago, USA), CRX (Proteintech), YAP1 (Proteintech), β-catenin (Proteintech), and RPE65 (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Cell Culture

Journal: Cell Death & Disease

Article Title: CRX is an intrinsic suppressor of epithelial‒mesenchymal transition in retinal pigment epithelial cells: a promising therapeutic avenue for subretinal fibrosis

doi: 10.1038/s41419-025-08352-y

Figure Lengend Snippet: A Schematic diagram of the laser-induced modeling and in vivo treatment in mice. Co-localization staining of GFP and CRX, RPE65 in the RPE cell layer 1 week after lentiviral infection in empty vector group ( B ) and lenti-CRX group ( C ). D Masson staining was performed to analyze the changes in the fibrotic area at different time points after laser induction. E Collagen 1 immunostaining demonstrated the fibrotic area in the choroidal flat-mount at different time points after laser induction. F The quantitative analysis of fibrotic area in the choroidal flat-mount ( n = 3). Scale bar = 50 μm. Data are presented as mean ± SD. Statistical significance was defined as follows: * p < 0.05, ** p < 0.01, not significant (using unpaired two-sided t -tests in (F).

Article Snippet: The samples were incubated with the primary antibodies against α-SMA (Abcam, Cambridge, UK), ZO-1 (Proteintech, Chicago, USA), CRX (Proteintech), YAP1 (Proteintech), β-catenin (Proteintech), and RPE65 (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C.

Techniques: In Vivo, Staining, Infection, Plasmid Preparation, Immunostaining

Journal: Cell Death & Disease

Article Title: CRX is an intrinsic suppressor of epithelial‒mesenchymal transition in retinal pigment epithelial cells: a promising therapeutic avenue for subretinal fibrosis

doi: 10.1038/s41419-025-08352-y

Figure Lengend Snippet: In normal and healthy RPE cells, the Hippo signaling pathway is activated. Under this condition, YAP1 is phosphorylated and subsequently degraded, while the TGF-β signaling pathway remains inactivated. The translocation of β-catenin into the nucleus is blocked, leading to the activation of CRX expression. This activation of CRX then promotes the expression of PPP2R2B and, in turn, inhibits the EMT. Conversely, in the RPE cells of wet AMD, the Hippo signaling pathway is inactivated, and the phosphorylation of YAP1 is blocked. This inactivation of the Hippo pathway, in synergy with the activated TGF-β signaling pathway, promotes the translocation of β-catenin into the nucleus. β-catenin binds to TCF7, which results in the inhibition of CRX expression. Subsequently, the repression of PPP2R2B expression promotes EMT.

Article Snippet: The samples were incubated with the primary antibodies against α-SMA (Abcam, Cambridge, UK), ZO-1 (Proteintech, Chicago, USA), CRX (Proteintech), YAP1 (Proteintech), β-catenin (Proteintech), and RPE65 (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C.

Techniques: Translocation Assay, Activation Assay, Expressing, Phospho-proteomics, Inhibition