Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 1. 14-3-3zeta expression in the cornea after alkali burn. (A) The localization and expression of 14-3-3zeta (green) and DAPI (blue) in mouse cornea tissues were detected by immunofluorescence staining. Scale bar: 30 μm. (B) mRNA expression of YWHAZ in the cornea under normal conditions and at different time points after alkali burn from qRT-PCR analysis (n = 4 per group). (C, D) Protein expression of 14-3-3zeta in the cornea form western blotting (n = 4 per group). Data were normalized against the expression of the control. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The membrane was blocked for 2 h with casein solution at 1:15 dilution at room temperature, incubated overnight at 4 ◦C with primary antibodies against 14-3- 3zeta (NBP1-31325, Novus Biologicals, Littleton, CO, USA), cyclin D1 (55506T, Cell Signaling Technology, Danvers, MA, USA), caspase-3 (9664S, Cell Signaling Technology), p-Akt (4060, Cell Signaling Technology), Akt (4691, Cell Signaling Technology), or β-actin (66009-1-1g, Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Immunofluorescence, Staining, Quantitative RT-PCR, Western Blot, Control

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 2. 14-3-3zeta overexpression promotes corneal wound healing after injury. (A) AAV was administered by subconjunctival injection. (B, C) Protein level of 14-3- 3zeta in the AAV-transfected cornea was measured by western blotting (n = 3 per group). (D, E) Fluorescein sodium staining on days 1 and 2 post-alkali burn (n = 6 per group). (F, G) Corneal opacity scoring on day 7 post-alkali burn (n = 6 per group). (H, I) Immunofluorescence staining for Ki67 on day 1 post-alkali burn (n = 4 per group). Scale bar: 30 μm. (J, K) HE staining on days 7 and 14 post-alkali burn (n = 4 per group). Scale bar: 50 μm.

Article Snippet: The membrane was blocked for 2 h with casein solution at 1:15 dilution at room temperature, incubated overnight at 4 ◦C with primary antibodies against 14-3- 3zeta (NBP1-31325, Novus Biologicals, Littleton, CO, USA), cyclin D1 (55506T, Cell Signaling Technology, Danvers, MA, USA), caspase-3 (9664S, Cell Signaling Technology), p-Akt (4060, Cell Signaling Technology), Akt (4691, Cell Signaling Technology), or β-actin (66009-1-1g, Proteintech, Rosemont, IL, USA).

Techniques: Over Expression, Injection, Transfection, Western Blot, Staining, Immunofluorescence

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 3. 14-3-3zeta knockdown inhibits the proliferation and migration of HCE-T cells and induces apoptosis. (A) mRNA levels of YWHAZ, CCND1, and caspase-3 in siRNA-transfected HCE-T cells were determined by qRT-PCR (n = 3 per group). (B, C) Protein levels of 14-3-3zeta, cyclin D1, and caspase-3 in siRNA-transfected HCE-T cells were determined by western blotting (n = 3 per group). (D) The viability of siRNA-transfected HCE-T cells was determined by the CCK-8 assay (n = 4 per group). (E, F) The proliferation ability of siRNA-transfected HCE-T cells was determined by the EdU incorporation assay (n = 3 per group). Scale bar: 100 μm. (G, H) Apoptosis of siRNA-transfected HCE-T cells was determined by TUNEL staining (n = 3 per group). Scale bar: 50 μm. (I, J) The migration ability of siRNA- transfected HCE-T cells was determined by the scratch test (n = 3 per group). Scale bar: 25 μm.

Article Snippet: The membrane was blocked for 2 h with casein solution at 1:15 dilution at room temperature, incubated overnight at 4 ◦C with primary antibodies against 14-3- 3zeta (NBP1-31325, Novus Biologicals, Littleton, CO, USA), cyclin D1 (55506T, Cell Signaling Technology, Danvers, MA, USA), caspase-3 (9664S, Cell Signaling Technology), p-Akt (4060, Cell Signaling Technology), Akt (4691, Cell Signaling Technology), or β-actin (66009-1-1g, Proteintech, Rosemont, IL, USA).

Techniques: Knockdown, Migration, Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, TUNEL Assay, Staining

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 4. 14-3-3zeta overexpression promotes the proliferation and migration of HCE-T cells. (A) mRNA levels of YWHAZ and CCND1 in plasmid-transfected HCE-T cells were determined by qRT-PCR analysis (n = 3 per group). (B, C) Protein expression of 14-3-3zeta and cyclin D1 in plasmid-transfected HCE-T cells was determined by western blotting (n = 3 per group). (D) Cell viability was determined by the CCK-8 assay (n = 4 per group). (E, F) The proliferation ability of plasmid- transfected HCE-T cells was determined by the EdU incorporation assay (n = 3 per group). Scale bar: 100 μm. (G, H) The migration ability of plasmid-transfected HCE-T cells was determined from a scratch test (n = 3 per group). Scale bar: 25 μm.

Article Snippet: The membrane was blocked for 2 h with casein solution at 1:15 dilution at room temperature, incubated overnight at 4 ◦C with primary antibodies against 14-3- 3zeta (NBP1-31325, Novus Biologicals, Littleton, CO, USA), cyclin D1 (55506T, Cell Signaling Technology, Danvers, MA, USA), caspase-3 (9664S, Cell Signaling Technology), p-Akt (4060, Cell Signaling Technology), Akt (4691, Cell Signaling Technology), or β-actin (66009-1-1g, Proteintech, Rosemont, IL, USA).

Techniques: Over Expression, Migration, Plasmid Preparation, Transfection, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 5. mRNA-seq analysis shows 14-3-3zeta’s involvement in regulating various biological processes and signaling pathways in HCE-T cells. (A) Volcano plot for differentially expressed genes in HCE-T cells transfected with si-14-3-3zeta versus si-NC. (B) GO annotation for differentially expressed genes in the si-14-3-3zeta group compared with the si-NC group (n = 3 per group). (C) KEGG pathway enrichment analysis for the differentially expressed genes. (D) Representative differentially expressed genes and related signaling pathways.

Article Snippet: The membrane was blocked for 2 h with casein solution at 1:15 dilution at room temperature, incubated overnight at 4 ◦C with primary antibodies against 14-3- 3zeta (NBP1-31325, Novus Biologicals, Littleton, CO, USA), cyclin D1 (55506T, Cell Signaling Technology, Danvers, MA, USA), caspase-3 (9664S, Cell Signaling Technology), p-Akt (4060, Cell Signaling Technology), Akt (4691, Cell Signaling Technology), or β-actin (66009-1-1g, Proteintech, Rosemont, IL, USA).

Techniques: Protein-Protein interactions, Transfection

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 1. 14-3-3zeta expression in the cornea after alkali burn. (A) The localization and expression of 14-3-3zeta (green) and DAPI (blue) in mouse cornea tissues were detected by immunofluorescence staining. Scale bar: 30 μm. (B) mRNA expression of YWHAZ in the cornea under normal conditions and at different time points after alkali burn from qRT-PCR analysis (n = 4 per group). (C, D) Protein expression of 14-3-3zeta in the cornea form western blotting (n = 4 per group). Data were normalized against the expression of the control. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: These sections were stained with an anti-14-3-3zeta antibody (NBP1-31325, Novus Biologicals, Littleton, CO, USA) or an anti-Ki67 antibody (ab16667, Abcam, Cambridge, UK) overnight at 4 ◦C.

Techniques: Expressing, Immunofluorescence, Staining, Quantitative RT-PCR, Western Blot, Control

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 2. 14-3-3zeta overexpression promotes corneal wound healing after injury. (A) AAV was administered by subconjunctival injection. (B, C) Protein level of 14-3- 3zeta in the AAV-transfected cornea was measured by western blotting (n = 3 per group). (D, E) Fluorescein sodium staining on days 1 and 2 post-alkali burn (n = 6 per group). (F, G) Corneal opacity scoring on day 7 post-alkali burn (n = 6 per group). (H, I) Immunofluorescence staining for Ki67 on day 1 post-alkali burn (n = 4 per group). Scale bar: 30 μm. (J, K) HE staining on days 7 and 14 post-alkali burn (n = 4 per group). Scale bar: 50 μm.

Article Snippet: These sections were stained with an anti-14-3-3zeta antibody (NBP1-31325, Novus Biologicals, Littleton, CO, USA) or an anti-Ki67 antibody (ab16667, Abcam, Cambridge, UK) overnight at 4 ◦C.

Techniques: Over Expression, Injection, Transfection, Western Blot, Staining, Immunofluorescence

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 3. 14-3-3zeta knockdown inhibits the proliferation and migration of HCE-T cells and induces apoptosis. (A) mRNA levels of YWHAZ, CCND1, and caspase-3 in siRNA-transfected HCE-T cells were determined by qRT-PCR (n = 3 per group). (B, C) Protein levels of 14-3-3zeta, cyclin D1, and caspase-3 in siRNA-transfected HCE-T cells were determined by western blotting (n = 3 per group). (D) The viability of siRNA-transfected HCE-T cells was determined by the CCK-8 assay (n = 4 per group). (E, F) The proliferation ability of siRNA-transfected HCE-T cells was determined by the EdU incorporation assay (n = 3 per group). Scale bar: 100 μm. (G, H) Apoptosis of siRNA-transfected HCE-T cells was determined by TUNEL staining (n = 3 per group). Scale bar: 50 μm. (I, J) The migration ability of siRNA- transfected HCE-T cells was determined by the scratch test (n = 3 per group). Scale bar: 25 μm.

Article Snippet: These sections were stained with an anti-14-3-3zeta antibody (NBP1-31325, Novus Biologicals, Littleton, CO, USA) or an anti-Ki67 antibody (ab16667, Abcam, Cambridge, UK) overnight at 4 ◦C.

Techniques: Knockdown, Migration, Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, TUNEL Assay, Staining

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 4. 14-3-3zeta overexpression promotes the proliferation and migration of HCE-T cells. (A) mRNA levels of YWHAZ and CCND1 in plasmid-transfected HCE-T cells were determined by qRT-PCR analysis (n = 3 per group). (B, C) Protein expression of 14-3-3zeta and cyclin D1 in plasmid-transfected HCE-T cells was determined by western blotting (n = 3 per group). (D) Cell viability was determined by the CCK-8 assay (n = 4 per group). (E, F) The proliferation ability of plasmid- transfected HCE-T cells was determined by the EdU incorporation assay (n = 3 per group). Scale bar: 100 μm. (G, H) The migration ability of plasmid-transfected HCE-T cells was determined from a scratch test (n = 3 per group). Scale bar: 25 μm.

Article Snippet: These sections were stained with an anti-14-3-3zeta antibody (NBP1-31325, Novus Biologicals, Littleton, CO, USA) or an anti-Ki67 antibody (ab16667, Abcam, Cambridge, UK) overnight at 4 ◦C.

Techniques: Over Expression, Migration, Plasmid Preparation, Transfection, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay

Journal: Experimental eye research

Article Title: Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

doi: 10.1016/j.exer.2024.109948

Figure Lengend Snippet: Fig. 5. mRNA-seq analysis shows 14-3-3zeta’s involvement in regulating various biological processes and signaling pathways in HCE-T cells. (A) Volcano plot for differentially expressed genes in HCE-T cells transfected with si-14-3-3zeta versus si-NC. (B) GO annotation for differentially expressed genes in the si-14-3-3zeta group compared with the si-NC group (n = 3 per group). (C) KEGG pathway enrichment analysis for the differentially expressed genes. (D) Representative differentially expressed genes and related signaling pathways.

Article Snippet: These sections were stained with an anti-14-3-3zeta antibody (NBP1-31325, Novus Biologicals, Littleton, CO, USA) or an anti-Ki67 antibody (ab16667, Abcam, Cambridge, UK) overnight at 4 ◦C.

Techniques: Protein-Protein interactions, Transfection