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

Journal: Cell reports

Article Title: Gut microbiota-mediated secondary bile acids regulate dendritic cells to attenuate autoimmune uveitis through TGR5 signaling.

doi: 10.1016/j.celrep.2021.109726

Figure Lengend Snippet: Figure 6. TGR5 signaling inhibits NF-kB activation via the cAMP-PKA pathway (A, C, and E) The phosphorylation levels of p65 and Ikba in LPS-primed BMDCs treated by INT-777 in the presence of forskolin (A), KH7 and MDL12300A (C), and PKI and H89 (E) were assayed by western blot. (B, D, and F) The expression levels of IL-1b, IL-6, IL-12p70, and TNF-a in LPS-primed BMDCs treated by INT-777 in the presence of forskolin (B), KH7 and MDL12300A (D), PKI and H89 (F) were assayed by ELISA. n = 4 per group. Data are shown as mean ± SD. A one-way ANOVA was used. *p < 0.05, **p < 0.01; ns, not significant (p > 0.05). See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER mouse GM-CSF R&D Cat# 415-ML-50 mouse IL-4 R&D Cat# 404-ML-50 Lipopolysaccharide Sigma Cat# L2880 DCA Sigma Cat# L6250 INT-777 MedChemExpress Cat# HY-15677 Forskolin MedChemExpress Cat# HY-15371 IBMX MedChemExpress Cat# HY-12318 KH7 MedChemExpress Cat# HY-103194 MDL12330A Sigma Cat# 444200 PKI MedChemExpress Cat# HY-P0222 H89 MedChemExpress Cat# HY-15979 Foxp3 / Transcription Factor Staining Buffer Set Kit Invitrogen Cat# 00-5523-00 PMA Sigma Cat# P1585 Ionomycin Sigma Cat# I3909 brefeldin A Sigma Cat# B6542 human GM-CSF AcroBiosystems Cat# GMF-H8214 human GM-IL-4 AcroBiosystems Cat# IL-4-H4218 Mouse TNF-a DuoSet ELISA R&D Cat# DY410-05 Mouse IL-6 DouSet ELISA R&D Cat# DY406-05 Mouse IL-1b DouSet ELISA R&D Cat# DY401-05 Mouse IL-17 DouSet ELISA R&D Cat# DY421-05 Mouse IFN-g DouSet ELISA R&D Cat# DY485-05 Mouse IL-10 DouSet ELISA R&D Cat# DY-417-05 Mouse IL-12/P70 DouSet ELISA R&D Cat# DY-419-05 Mouse IL-23 DouSet ELISA R&D Cat# DY1887-05 Human IL-1b DouSet ELISA R&D Cat# DY201 Human IL-6 DouSet ELISA R&D Cat# DY206 Human IL-12/P70 ELISA KIT Invitrogen Cat# BMS238 Human TNF-a DouSet ELISA R&D Cat# DY210 Deposited data Microbiota 16S rRNA gene sequences (NCBI GenBank) This paper PRJNA718486 Oligonucleotides Mouse PXR F: 50-GACCTGCCTATTG AGGACCA-30 Sangon Biotech N/A Mouse PXRR: 50-TTCTGGAAGCCACCATTAGG-30 Sangon Biotech N/A Mouse a5b1 F: 50-AGCGACTGGAATCC TCAAGA-30 Sangon Biotech N/A Mouse a5b1 R: 50-TGCTGAGTCCTGT CACCTTG-30 Sangon Biotech N/A Mouse GR F: 50-AGGCCGCTCAGTGTTTTCTA-30 Sangon Biotech N/A Mouse GR R: 50-TACAGCTTCCACACGTCAGC-30 Sangon Biotech N/A Mouse TGR5 F: 50-TTCTCTCTGTCCG CGTGTTG-30 Sangon Biotech N/A Mouse TGR5 R: 50-GGTGCTGCCCAA TGAGATGA-30 Sangon Biotech N/A Mouse VDR F: 50-GATGAGGAGGTGC AGCGTAA-30 Sangon Biotech N/A (Continued on next page) Cell Reports 36, 109726, September 21, 2021 e2

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Hippocampus

Article Title: Somatostatin Depresses the Excitability of Subicular Bursting Cells: Roles of Inward Rectifier K + Channels, KCNQ channels and Epac

doi: 10.1002/hipo.22744

Figure Lengend Snippet: PKA and PKC are not required for SST-elicited outward HCs, but Epac2 is partially involved. A, Application of Rp-cAMPS (200 μM) via the recording pipettes did not block SST-mediated outward HCs. B, Inclusion of PKI(5-24) (2 μM) in the recording pipettes failed to alter SST-induced outward HCs. C, Pretreatment of slices with and inclusion of H89 (10 μM) in the recording pipettes did not block SST-evoked outward HCs. D, Pretreatment of slices with and inclusion of KT 5720 (5 μM) in the recording pipettes did not block SST-elicited outward HCs. E, Pretreatment of slices with and inclusion of GF109203X (2 μM) in the recording pipettes failed to block SST-elicited outward HCs. F, Pretreatment of slices with and inclusion of HJC0350 (100 μM) in the recording pipettes reduced SST-induced outward HCs. G, Summery graph.

Article Snippet: PKI(5-24) was provided by Enzo Life Sciences.

Techniques: Blocking Assay