Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: TRAP1 Improves Diabetic Retinopathy by Preserving Mitochondrial Function

doi: 10.2147/OPTH.S521660

Figure Lengend Snippet: HG promotes mitochondrial dysfunction in ARPE-19 cells, accompanied by a decrease in TRAP1 levels. ( A ) Cell viability of ARPE-19 cells cultured with different glucose concentrations for 4 days. (n=4) ( B ) Cell viability of ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=4) ( C ) Intracellular ROS levels in cells cultured with 50mM glucose for 1–6 days. (n=3) ( D ) Identification of Δψ m through JC-1 staining after culturing ARPE-19 cells with 50mM glucose for 6 days. Images captured under fluorescence microscope show red fluorescence representing polymer form, indicating intact Δψ m, and green fluorescence representing monomer form, indicating decreased Δψ m. Quantified data presented on the right. Scale bar=20μm. (n=3) ( E ) ARPE-19 cells cultured with 50mM glucose for 6 days and treated with Calcein AM (1X) and CoCl2 (1X). Images captured under fluorescence microscope shown on the left. Quantified data presented on the right. Scale bar=20μm. (n=3) ( F ) TEM images of mitochondrial ultrastructure in ARPE-19 cells cultured with 50mM glucose for 6 days and control ARPE-19 cells. Quantified data presented on the right. Scale bar=2μm. ( G ) Western blot analysis of TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( H ) Western blot analysis of intramitochondrial TRAP1 in ARPE-19 cells cultured with 50mM glucose for 1–6 days after mitochondrial extraction. (n=3) ( I ) Quantified data corresponding to Figure ( G ). ( J ) qRT-PCR analysis of TRAP1 mRNA expression in ARPE-19 cells cultured with 50mM glucose for 1–6 days. (n=3) ( K ) Quantified data corresponding to Figure ( H ). The error bars in the above histograms represent the mean±SD of independent experiments. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

Article Snippet: Fluorescence images were captured using a Zeiss AxioImager M2 fluorescence microscope (Zeiss, Germany) with the following parameters: Oxidized state (green fluorescence): Excitation wavelength of 488 nm, emission filter of 500–550 nm;- Reduced state (red fluorescence): Excitation wavelength of 581 nm, emission filter of 590–650 nm.Images were analyzed using Zen 3.1 software and quantified with ImageJ software.

Techniques: Cell Culture, Staining, Fluorescence, Microscopy, Polymer, Control, Western Blot, Extraction, Quantitative RT-PCR, Expressing