Journal: Journal of Translational Medicine

Article Title: Artemisinin conferred cytoprotection to human retinal pigment epithelial cells exposed to amiodarone-induced oxidative insult by activating the CaMKK2/AMPK/Nrf2 pathway

doi: 10.1186/s12967-024-05593-x

Figure Lengend Snippet: The Nrf2 pathway was involved in the cytoprotective effect of artemisinin on amiodarone-induced cell death in D407 cell cultures. ( a-b ) The cells were treated with artemisinin at various concentrations for 1 h. The Nrf2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( c ) The cell cultures were incubated with or without the Nrf2 inhibitor ML385 at concentrations ranging from 2.5 to 15 µM for 24 h, and cell viability was measured using MTT ( n = 4). ( d-e ) Cells attached to 12-well plates were treated with 5 or 10 µM ML385 for 24 h. The Nrf2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 4). ( f-g ) The cell cultures were pretreated with 5 µM ML385 for 6, 12, or 24 h, followed by 20 µM artemisinin for 1 h and 5 µM amiodarone for another 24 h. Cell viability was determined by the MTT assay ( n = 4), and cell death was detected through the LDH cytotoxicity assay ( n = 4). ( h-j ) Cells attached to 24-well plates were transfected with control shRNA (shCTL) or Nrf2 shRNA (shNrf2) plasmids (0.5 µg/well) for 72 h. The Nrf2, HO-1, and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( k ) Cells transfected with shCTL or shNrf2 plasmids were attached to 96-well plates, and pretreated with artemisinin for 1 h, then treated with amiodarone for another 24 h and processed for LDH assay ( n = 4)

Article Snippet: The human AMPKɑ2 shRNA plasmids (target sequence: TGTGAAAGAAGTGTGTGAA), control shRNA plasmids (CON207, target sequence: TTCTCCGAACGTGTCACGT), the AMPKɑ2-overexpressing plasmids, and control overexpressing plasmids (CON238) were purchased from GenePharma Co., Ltd. (Shanghai, China).

Techniques: Western Blot, Incubation, MTT Assay, LDH Cytotoxicity Assay, Transfection, Control, shRNA, Lactate Dehydrogenase Assay

Journal: Journal of Translational Medicine

Article Title: Artemisinin conferred cytoprotection to human retinal pigment epithelial cells exposed to amiodarone-induced oxidative insult by activating the CaMKK2/AMPK/Nrf2 pathway

doi: 10.1186/s12967-024-05593-x

Figure Lengend Snippet: AMPK-mediated activation of the Nrf2/HO-1 pathway was linked to the cytoprotective effect of artemisinin on amiodarone-induced apoptosis in D407 cell cultures. ( a-d ) The cell cultures were pretreated with 1 µM STO-609 for 30 min or 1 µM Compound C for 30 min, followed by 20 µM artemisinin for 1 h and 5 µM amiodarone for another 24 h. The HO-1, cleaved caspase-3, and GAPDH protein levels were detected by western blotting, and the protein bands were quantified by Image J ( n = 3). ( e-h ) Cells attached to 12-well plates were transfected with shCTL or AMPKɑ2 shRNA (shAMPKɑ2) plasmids (2 µg/well) for 48 h. The AMPKɑ2, HO-1, cleaved caspase-3, and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( i-j ) Cells attached to 24-well plates were transfected with the AMPKɑ2-overexpressing (OE-AMPKɑ2) or control-overexpressing (OE-CTL) plasmids (0.5 µg/well) for 24 h. The AMPKɑ2 and GAPDH protein levels were detected by western blotting, and protein bands were quantified by Image J ( n = 3). ( k ) The cell cultures transfected with OE-CTL or OE-AMPKɑ2 plasmids for 24 h were attached to 96-well plates and treated with 5 µM amiodarone for 24 h, and processed for the LDH assay ( n = 4)

Article Snippet: The human AMPKɑ2 shRNA plasmids (target sequence: TGTGAAAGAAGTGTGTGAA), control shRNA plasmids (CON207, target sequence: TTCTCCGAACGTGTCACGT), the AMPKɑ2-overexpressing plasmids, and control overexpressing plasmids (CON238) were purchased from GenePharma Co., Ltd. (Shanghai, China).

Techniques: Activation Assay, Western Blot, Transfection, shRNA, Control, Lactate Dehydrogenase Assay