tandem fluorescent reporter  (Addgene inc)

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Protective Effect of Metformin against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial (RPE) Cells by Enhancing Autophagy through Activation of AMPK Pathway

doi: 10.1155/2020/2524174

Figure Lengend Snippet: Antibody information.

Article Snippet: Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) reporter, shRNA for LC3B, Beclin1, and AMPK shRNA were synthesized by Shanghai Genechem Co. Ltd (Shanghai, China).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Protective Effect of Metformin against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial (RPE) Cells by Enhancing Autophagy through Activation of AMPK Pathway

doi: 10.1155/2020/2524174

Figure Lengend Snippet: Metformin activated the autophagy signaling pathway in D407 cells. (a) Cells were transfected with tandem fluorescent-tagged LC3 reporter and treated with 60 μ M CQ and 1 mM metformin plus 60 μ M CQ for 2 h. Images were taken by a confocal microscope (scale bars, 20 μ m). (b) D407 cells were treated with 60 μ M CQ and 1 mM metformin plus 60 μ M CQ for 2 h. The expression of LC3B-I and LC3B-II was assessed by Western blot. (c) D407 cells were treated with different concentrations of metformin for 2 h, and the expression of LC3B, p62, P-Beclin1, and P-ULK1 was assessed by Western blot. (d) D407 cells were treated with 1 mM metformin for the periods of time indicated. The expression of LC3B, p62, P-Beclin1, and P-ULK1 was assessed by Western blot.

Article Snippet: Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) reporter, shRNA for LC3B, Beclin1, and AMPK shRNA were synthesized by Shanghai Genechem Co. Ltd (Shanghai, China).

Techniques: Transfection, Microscopy, Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Protective Effect of Metformin against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial (RPE) Cells by Enhancing Autophagy through Activation of AMPK Pathway

doi: 10.1155/2020/2524174

Figure Lengend Snippet: The protective effect of metformin was inhibited by autophagy blockage. D407 cells were pretreated with or without 2 mM 3-MA or 50 μ M CQ for 1 h before 1 mM metformin treatment for 2 h and then incubated with or without 200 μ M H 2 O 2 for further 24 h. (a) The effect of 3-MA or CQ pretreatment on the expression of LC3B and p62 was assessed using ICC (scale bars, 5 μ m). (b) Assessment of the effect of 3-MA pretreatment on cell viability was assessed using the MTT assay. (c) Assessment of the effect of CQ pretreatment on cell viability was assessed using the MTT assay (d) D407 cells were incubated with shBeclin1 plasmid for 24 h before 1 mM metformin treatment for 2 h and then incubated with or without 200 μ M H 2 O 2 for further 24 h. Cell viability was assessed using the MTT assay. (e) D407 cells were incubated with shLC3B plasmid for 24 h before 1 mM metformin treatment for 2 h and then incubated with or without 200 μ M H 2 O 2 for further 24 h. Cell viability was assessed using the MTT assay. (f) The effect of Beclin1 or LC3B knockdown on the oxidative damage was assessed by JC-1 and ROS staining (scale bars, 100 μ m). (g, h) Quantitative analysis of (d). Data are presented as the mean ± SD; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) reporter, shRNA for LC3B, Beclin1, and AMPK shRNA were synthesized by Shanghai Genechem Co. Ltd (Shanghai, China).

Techniques: Incubation, Expressing, MTT Assay, Plasmid Preparation, Knockdown, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Protective Effect of Metformin against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial (RPE) Cells by Enhancing Autophagy through Activation of AMPK Pathway

doi: 10.1155/2020/2524174

Figure Lengend Snippet: The activation of autophagy and the protective effect of metformin decreased after inhibition of the AMPK signaling pathway. D407 cells were pretreated with 2.5 μ M compound C for 30 min before 1 mM metformin treatment for 2 h and then incubated with or without 200 μ M H 2 O 2 for further 24 h. (a) The expression of LC3B and p62 was assessed using ICC (scale bars, 50 μ m). (b) The expression of LC3B, p62, P-Beclin1, and P-ULK1 was assessed by Western blot. (c) Cell viability was assessed using the MTT assay. (d) Cell cytotoxicity was measured by LDH assay. (e) Cells were pretreated with shAMPK plasmid, and after 48 h, cells were harvested and shAMPK interference efficiency was detected by Western blot. (f) Cells were pretreated with shAMPK3 plasmid and then treated as described above. Data are presented as the mean ± SD; ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) reporter, shRNA for LC3B, Beclin1, and AMPK shRNA were synthesized by Shanghai Genechem Co. Ltd (Shanghai, China).

Techniques: Activation Assay, Inhibition, Incubation, Expressing, Western Blot, MTT Assay, Lactate Dehydrogenase Assay, Plasmid Preparation