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

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: RNAi mediated down-regulation of IRAK1 expression. HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence or an IRAK1 specific shRNA. Geneticin selected cells were treated with medium only (Med) or 10 ng/ml IL-1β. After 24 hours cells were lysed in PBS with 2 M urea and 0.85% SDS and proteins examined by Western blotting. IRAK1 and GAPDH were detected using polyclonal anti-IRAK1 and anti-GAPDH rabbit antibodies, respectively.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Expressing, Transfection, Construct, Negative Control, shRNA, Sequencing, Western Blot

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: IRAK1 is required for CC chemokine secretion. (A and B), HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence (open bars) or IRAK1 specific shRNA (filled bars). (C and D), cells were transfected with either an empty (open bars) or IRAK1(1-217) (filled bars) expression construct. Geneticin selected cells were treated with medium only, 10 ng/ml IL-1β or 50 ng/ml TNF-α for 24 hours. Secretion of CCL5 (A and C) and CCL20 (B and D) was determined by ELISA. Expression of CCL5 and CCL20 in the control cells (open bars, scrambled shRNA (A and B) or empty vector (C and D)) was defined as 100 pct. Expression of CCL5 and CCL20 in the IRAK1 targeted cells (filled bars, IRAK1 specific shRNA (A and B) or IRAK1(1-217) expression vector (C and D)) is graphically represented relative to the control cells. * p < 0.05 compared to control cells transfected with scrambled shRNA plasmid (A and B) or empty vector (C and D) and receiving the same treatment.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Transfection, Expressing, Construct, Negative Control, shRNA, Sequencing, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: IL-1β enhancement of IFN-γ induced CXC chemokine expression is IRAK1 independent. A, HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence (open bars) or IRAK1 specific shRNA (filled bars). B, cells were transfected with either an empty (open bars) or IRAK1(1-217) (filled bars) expression construct. Geneticin selected cells were treated with 20 ng/ml IFN-γ or 20 ng/ml IFN-γ plus 10 ng/ml IL-1β for 2 hours. Expression of CXC chemokine mRNAs was examined using real-time RT-PCR. Expression of the individual CXC chemokine mRNA in response to co-treatment with IFN-β+ IL-1β is graphically represented relative to expression induced by IFN-γ alone. * p < 0.05 compared to cells transfected with the same vector and treated with IFN-γ only. ** p < 0.01 compared to cells transfected with the same vector and treated with IFN-γ only. # p < 0.05 compared to control cells transfected with empty vector and receiving the same treatment.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Expressing, Transfection, Construct, Negative Control, shRNA, Sequencing, Quantitative RT-PCR, Plasmid Preparation, Control