x
On day 1, HeLa cells stably expressing GFP_LC3 (kindly provided by Dr. Yu Li, Tsinghua University) were plated onto poly-lysine coated glass coverslips. On day 2, medium was changed with fresh medium containing DMSO or resveratrol at the indicated concentration for 2 h. The medium was then aspirated and cells were washed twice with PBS. Cells were fixed with 4% formaldehyde for 10 min at RT and stained with DAPI for 5 min at RT. Washing three times with PBS was conducted between each step. The coverslips were mounted on a slide glass and visualized using confocal microscopy (Carl Zeiss), and randomly captured images from various fields were used for autophagy formation analysis. GFP punctate positive cells were quantified as autophagy induced cells. The total number of cells was determined by measuring DAPI-positive cells.
Lentiviral shRNAs were prepared and transduced according to the Addgene protocol. Briefly, the pLKO empty vector or pLKO shULK1_8 (purchased from Addgene) was transfected with pMD2.G and psPAX2 (purchased from Addgene) using Lipofectamine according to the manufacturer’s instructions(Invitrogen). Media were collected at 24 h and 48 h after transfection. Collected media were centrifuged at 1200 rpm for 5 min and filtered with 0.45-μm filter. For virus infection, virus-containing media were diluted with growth medium and added to plated cells. Twenty-four hours after infection, the medium was replaced with fresh medium. Twenty-four hours after changing the medium, infected cells were used for the various experiments based on experimental purpose.
Article Produced By
Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 689-798, Republic of Korea
Department of Bioscience and Biotechnology, Sejong University, 98 Kunja-Dong, Kwangjin-Ku, Seoul 143-747, Republic of Korea
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763238/
(All these Articles can be read from this URL:
https://markethive.com/group/ama/blog/the-role-of-resveratrol-in-achieving-optimal-health-p5 )
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