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. Author manuscript; available in PMC: 2011 Jun 1.

Published in final edited form as: Cell Death Differ. 2010 May 28;17(12):1867–1881. doi: 10.1038/cdd.2010.53

Effects of As₂O₃ are reversed by antioxidant. (a) HEY cells were seeded at 250,000 cells per well in a 6-well plate. Following 24 hours, cells were treated with (1) 10µM As₂O₃, (2) 1000µM NAC, or (3) 10µM As₂O₃, and 1000µM NAC. After 18 hour incubation, the cells were photographed using a light microscope at 40× magnification. The data shown are representative images. (b) HEY cells were seeded at 250,000 cells per well in a 6-well plate. Following 24 hour cell attachment, cells were treated with (1) 10µM As₂O₃ alone, (2) 10µM As₂O₃ with 100µM NAC, (3) 10µM As₂O₃ with 500µM NAC, (4) 10µM As₂O₃ with 1000µM NAC, or (5) 1000µM NAC only. Following 18 hours incubation, cell lysates were harvested and western analysis was performed using the following antibodies (left panel): (1) EVI1, (2) SnoN, (3) TAK1, (4) TGFβRII, (5) SMAD2/3, and (6) GAPDH as a loading control as well as (right panel): (1) PARP, (2) LC3, (3) p62, and (4) GAPDH. The data shown are representative of 3 independent experiments. (c) HEY cells were plated at 250,000 cells per well in 6-well plate onto glass coverslips. After overnight attachment, cells were transfected with EGFP-LC3. Following recovery for 24 hours, cells were treated with: (1) 10µM As₂O₃, (2) 10µM As₂O₃ and 1000µM NAC, or (3) 1000µM NAC for 18 hours. (Top panel) Immunofluorescence images were obtained at 40× magnification. (Bottom panel) The displayed graph is the quantification of the data presented as the percentage of EGFP positive cells with punctate LC3 expression. The data shown are representative of 2 independent experiments. (d) Cell viability was assessed using the CellTiter-Glo assay in HEY cells treated for 18 hours with 10µM As₂O₃ in the absence or presence of 1000µM NAC or 1000µM NAC alone. Results are presented as % cell viability relative to control cells. The data shown are representative of 3 independent experiments. (e) (Top panel) HEY cells were seeded at 250,000 cells per well in 6-well plates. Following cell attachment, cells were treated with (1) 10µM As₂O₃, (2) 10µM As₂O₃ and 1000µM NAC, and (3) 1000µM NAC, at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown as log fluorescence values of annexin V-FITC and PI on the X and Y axis, respectively. (Bottom panel) The data is also displayed in a bar graph as the percentage of viable, early apoptotic, and late apoptotic/necrotic cells. The data shown are representative of 3 independent experiments.

Effects of As₂O₃ are reversed by antioxidant. (a) HEY cells were seeded at 250,000 cells per well in a 6-well plate. Following 24 hours, cells were treated with (1) 10µM As₂O₃, (2) 1000µM NAC, or (3) 10µM As₂O₃, and 1000µM NAC. After 18 hour incubation, the cells were photographed using a light microscope at 40× magnification. The data shown are representative images. (b) HEY cells were seeded at 250,000 cells per well in a 6-well plate. Following 24 hour cell attachment, cells were treated with (1) 10µM As₂O₃ alone, (2) 10µM As₂O₃ with 100µM NAC, (3) 10µM As₂O₃ with 500µM NAC, (4) 10µM As₂O₃ with 1000µM NAC, or (5) 1000µM NAC only. Following 18 hours incubation, cell lysates were harvested and western analysis was performed using the following antibodies (left panel): (1) EVI1, (2) SnoN, (3) TAK1, (4) TGFβRII, (5) SMAD2/3, and (6) GAPDH as a loading control as well as (right panel): (1) PARP, (2) LC3, (3) p62, and (4) GAPDH. The data shown are representative of 3 independent experiments. (c) HEY cells were plated at 250,000 cells per well in 6-well plate onto glass coverslips. After overnight attachment, cells were transfected with EGFP-LC3. Following recovery for 24 hours, cells were treated with: (1) 10µM As₂O₃, (2) 10µM As₂O₃ and 1000µM NAC, or (3) 1000µM NAC for 18 hours. (Top panel) Immunofluorescence images were obtained at 40× magnification. (Bottom panel) The displayed graph is the quantification of the data presented as the percentage of EGFP positive cells with punctate LC3 expression. The data shown are representative of 2 independent experiments. (d) Cell viability was assessed using the CellTiter-Glo assay in HEY cells treated for 18 hours with 10µM As₂O₃ in the absence or presence of 1000µM NAC or 1000µM NAC alone. Results are presented as % cell viability relative to control cells. The data shown are representative of 3 independent experiments. (e) (Top panel) HEY cells were seeded at 250,000 cells per well in 6-well plates. Following cell attachment, cells were treated with (1) 10µM As₂O₃, (2) 10µM As₂O₃ and 1000µM NAC, and (3) 1000µM NAC, at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown as log fluorescence values of annexin V-FITC and PI on the X and Y axis, respectively. (Bottom panel) The data is also displayed in a bar graph as the percentage of viable, early apoptotic, and late apoptotic/necrotic cells. The data shown are representative of 3 independent experiments.