Journal: International Journal of Ophthalmology

Article Title: Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway

doi: 10.18240/ijo.2020.04.04

Figure Lengend Snippet: Cells were seeded into 12-well plates and cultured. Various factors were added into the culture medium. After 24h incubation, microphotographs were taken by phase-contrast microscopy at high magnification (×200). 0: Control, cells not treated with any of these factors; H: Treated with HCQ 30 µmol/L; H+S: Treated with HCQ and salbutamol (10−5 mol/L); H+S+PI: Treated with HCQ, salbutamol and PKA inhibitor (10 µmol/L). In HCQ treated cells, cells were enlarged, numerous vacuoles appeared in the cytoplasm with slightly decrease of cell numbers. Salbutamol treatment significantly decreased the number of vacuoles. PKA inhibitor increased the number of vacuoles as compared with cells treated with HCG and salbutamol only.

Article Snippet: PKA inhibitor (PKA inhibitor 5-24) was obtained from TOCRIS Bioscience (Minneapolis, MN, USA).

Techniques: Cell Culture, Incubation, Microscopy, Control

Journal: International Journal of Ophthalmology

Article Title: Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway

doi: 10.18240/ijo.2020.04.04

Figure Lengend Snippet: Cells were treated as described in Figure 1. After 24h incubation, viable cells (non-stained) and nonviable cells (stained blue by trypan blue) were counted separately. Viable cells in HCQ (H) and PKA inhibitor (H+S+PI) were significantly less than that of the controls (aP<0.05). Salbutamol (H+S) significantly increased the number of viable cells (cP<0.05) and PKA inhibitor (H+S+PI) significantly decreased the viable cells (cP<0.05).

Article Snippet: PKA inhibitor (PKA inhibitor 5-24) was obtained from TOCRIS Bioscience (Minneapolis, MN, USA).

Techniques: Incubation, Staining

Journal: International Journal of Ophthalmology

Article Title: Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway

doi: 10.18240/ijo.2020.04.04

Figure Lengend Snippet: Cells were seeded into 12-well plates and cultured. Various factors were added into the culture medium. After 24h incubation, microphotographs were taken by phase-contrast microscopy at low magnification (×100). 0: Control, cells not treated; H: Treated with HCQ 100 µmol/L; H+S: Treated with HCQ and salbutamol (10−5 mol/L); H+S+PI: Treated with HCQ, salbutamol and PKA inhibitor (10 µmol/L). In all three treated groups, cells were transformed into spindle shape, viable cells were significantly decreased and many nonviable cells floated in the medium or sill attached to the culture dish, especially in cultures treated with H and H+S+PI.

Article Snippet: PKA inhibitor (PKA inhibitor 5-24) was obtained from TOCRIS Bioscience (Minneapolis, MN, USA).

Techniques: Cell Culture, Incubation, Microscopy, Control, Transformation Assay

Journal: International Journal of Ophthalmology

Article Title: Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway

doi: 10.18240/ijo.2020.04.04

Figure Lengend Snippet: Cells were treated as described in Figure 3. After 24h incubation, viable cells (non-stained) and nonviable cells (stained blue by trypan blue) were counted separately. A: Viable cells in cultures treated with HCQ (H), HCQ with salbutamol (H+S) and PKA inhibitor (H+S+PI) were significantly less than that of the controls (aP<0.05). Salbutamol (H+S) significantly increased the number of viable cells (cP<0.05) and PKA inhibitor (H+S+PI) significantly decreased the viable cells (cP<0.05). B: Nonviable cells in H, H+S and H+S+PI groups were significantly greater than that of the controls (aP<0.05). Salbutamol (H+S) significantly decreased the number of nonviable cells (cP<0.05) and PKA inhibitor (H+S+PI) significantly increased the viable cells (cP<0.05).

Article Snippet: PKA inhibitor (PKA inhibitor 5-24) was obtained from TOCRIS Bioscience (Minneapolis, MN, USA).

Techniques: Incubation, Staining

Journal: International Journal of Ophthalmology

Article Title: Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway

doi: 10.18240/ijo.2020.04.04

Figure Lengend Snippet: Cells were treated as described in Figure 1. Microphotographs were taken by phase-contrast microscopy at high magnification (×200). Selected cells in the microphtographs were outlined with exclusion of the nuclei. The vacuoles were thresholded using the BW mode of the Image J software. Cells were black in color and the vacuoles were white in color. Examples of cells treated by Image J software. 0: Control, cells not treated with any of these factors. No vacuole could be detected. H: Cells treated with HCQ 30 µmol/L. Numerous vacuoles appeared in the cytoplasm. H+S: Cells treated with HCQ and salbutamol (10−5 mol/L). Very few vacuoles appeared in the cytoplasm, which were significantly less than that in the H. H+S+PI: Cells treated with HCQ, salbutamol and PKA inhibitor (10 µmol/L). Numerous vacuoles appeared in the cytoplasm.

Article Snippet: PKA inhibitor (PKA inhibitor 5-24) was obtained from TOCRIS Bioscience (Minneapolis, MN, USA).

Techniques: Microscopy, Software, Control

Journal: International Journal of Ophthalmology

Article Title: Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway

doi: 10.18240/ijo.2020.04.04

Figure Lengend Snippet: Cells were treated as described in Figure 1. After 24h incubation, photomicrographs were taken. Ten cells were randomly selected from each group. The size of the vacuoles and cell cytoplasm were measured and compared by Image J (the ratio of total vacuoles/cytoplasm size) and expressed as the percentage of the control. HCQ at 30 µmol/L (H) and HCQ with salbutamol and PKA inhibitor (H+S+PI) significantly increased the size of vacuolation, aP<0.05. Salbutamol (H+S) significantly decreased the size of vacuolation (cP<0.05), whereas PKA inhibitor (H+S+PI) significantly increased the size of vacuolation, cP<0.05.

Article Snippet: PKA inhibitor (PKA inhibitor 5-24) was obtained from TOCRIS Bioscience (Minneapolis, MN, USA).

Techniques: Incubation, Control

Journal: International Journal of Ophthalmology

Article Title: Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway

doi: 10.18240/ijo.2020.04.04

Figure Lengend Snippet: A: Western blot photos prepared from the cell lysates. 0: Control, cells not treated; H: Cells treated with HCQ alone; H+S: Treated with HCQ and salbutamol; H+S+PI: Treated with HCQ, salbutamol and PKA inhibitor. B: Western blot band relative density analyzed by using Image J. p-PKA levels in cell lysates treated with HCQ were significantly decreased as compared with the controls (0), aP<0.05. p-PKA levels in HCQ and salbutamol (H+S) group were significantly elevated than that of cells treated with HCQ alone (H), cP<0.05. p-PKA levels in cells treated with HCQ, salbutamol and PKA inhibitor (H+S+PI) were significantly lower than that of cells treated with HCQ and salbutamol, (cP<0.05) and than that of the cells treated with HCQ alone P<0.05.

Article Snippet: PKA inhibitor (PKA inhibitor 5-24) was obtained from TOCRIS Bioscience (Minneapolis, MN, USA).

Techniques: Western Blot, Control

Journal: International Journal of Ophthalmology

Article Title: Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway

doi: 10.18240/ijo.2020.04.04

Figure Lengend Snippet: PKA kinase activity levels were measured by PKA kinase activity kits in cell lysates from four different groups. 0: Control, cells not treated; H: Cells treated with HCQ alone; H+S: Treated with HCQ and salbutamol; H+S+PI: Treated with HCQ, salbutamol and PKA inhibitor. PKA kinase activity levels in the controls (0) were significantly reduced by the addition of HCQ, aP<0.05. Salbutamol significantly increased PKA kinase activity levels as compared with HCQ, +P<0.05; but still lower than that of the control, aP<0.05. Addition of PKA inhibitor inhibited the PKA kinase activity levels in cells treated with HCQ and salbutamol, cP<0.05; and lower than that of cells treated with HCQ alone, P<0.05.

Article Snippet: PKA inhibitor (PKA inhibitor 5-24) was obtained from TOCRIS Bioscience (Minneapolis, MN, USA).

Techniques: Activity Assay, Control