As₂O₃ induces cell death. (a) Cell viability was assessed using the CellTiter-Glo assay in T80, HEY, and SKOV3 cells treated for 18 hours with varying doses of As₂O₃ (2–50µM). Results are presented as % cell viability relative to control cells (0µM). The data shown are representative of 3 independent experiments. (b) T80, HEY, and SKOV3 cells were seeded at 250,000 cells per well. After 24 hours, the cells were treated with varying concentrations of As₂O₃ (5–50µM). Following 18 hour treatment, images were captured at 40× magnification. The data shown are representative images. (c) HEY cells were seeded at 250,000 cells per well in 6-well plates. Following cell attachment, cells were treated with 10µM As₂O₃ 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. (Top panels) Raw data plots are shown as log fluorescence values of annexin V-FITC and PI on the X and Y axis, respectively. (Bottom panels) The data is displayed in bar graphs as the percentage of viable, early apoptotic, and late apoptotic/necrotic cells. The data shown are representative of 2 independent experiments. (d) GSH levels were quantified in T80, HEY, and SKOV3 cells using the GSH-glo assay kit. Results are presented as % GSH relative to untreated cells. The data shown are representative of 3 independent experiments. (e) T80, HEY, and SKOV3 cells were grown to confluence in T-25 flasks. Cell lysates were harvested into 200µl of lysis buffer containing 10mM sodium orthovanadate, 100mM sodium fluoride, and protease cocktail inhibitors. Western analysis was performed using the following antibodies: (1) MRP1 and (2) GAPDH. The data are representative of 2 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.

Knockdown of SnoN modulates the sensitivity of ovarian cancer cells to As₂O₃. (a) HEY cells were plated at 250,000 cells per well in 6-well plate. After overnight attachment, the cells were transiently transfected with (1) non-targeting (control) siRNA or (2) SnoN siRNA. Following 24 hours post-transfection, the cells were treated with 10µM As₂O₃. After 18 hours, light microscope images were captured at 40× magnification. The data shown are representative images. (b) (Left panel) HEY cells were seeded at 250,000 cells per well in 6-well plates. After 24 hours, the cells were transiently transfected with (1) non-targeting (control) siRNA or (2) SnoN siRNA. Following 24 hours post-transfection, the cells were treated with 25µM As₂O₃. After 18 hour incubation, cell lysates were harvested and western analysis was performed using the following antibodies: (1) SnoN, (2) PARP, (3) LC3, (4) p62, and (4) GAPDH as a loading control. The data shown are representative of 2 independent experiments. (Right panel) Densitometric analyses of selected western data (only As₂O₃-treated samples) which include SnoN, cleaved PARP, LC3-I, LC3-II, and p62. (c) (Left panel) 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) non-targeting (control) siRNA or (2) SnoN siRNA followed by cell treatment with 10µM As₂O₃. Immunofluorescence images are representative and were obtained at 40× magnification. (Right panel) Immunofluorescence data was quantitated and 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 transfected with control or SnoN siRNA followed by treatment for 18 hours with 10µM As₂O₃. Results are presented as % cell viability relative to non-targeting (control) siRNA treated cells. The data shown are representative of 2 independent experiments. (e) (Top panel) HEY cells were seeded at 250,000 cells per well in 6-well plates. Following 24 hours, cells were treated with (1) non-targeting (control) siRNA or (2) SnoN siRNA. Twenty-four hours post-transfection, cells were treated for 48 hours with 10µM As₂O₃ 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 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.

Therapeutic potential for SnoN in mediating As₂O₃-induced autophagic cell survival. As₂O₃ diffuses into the cell through the cell membrane leading to a reduction in intracellular glutathione (GSH) levels and an increase in reactive oxygen species (ROS) which through unknown mechanisms lead to increases in SnoN levels. SnoN alters LC3-II expression, a marker of autophagosome maturation, suggesting that SnoN can induce autophagy. Induction of autophagy appears to be a protective mechanism of cell survival following As₂O₃ treatment in ovarian cells. In high MRP1 expressing cell lines, As₂O₃ is effluxed through these channels resulting in reduced sensitivity to As₂O₃-induced apoptosis.

As₂O₃ alters expression of TGFβ signaling mediators in HEY ovarian cancer cell line. (a) HEY cells were seeded at 250,000 cells per well in 6-well plates. After overnight attachment, the cells were treated with varying concentrations of As₂O₃ (2–50µM). After 18 hour incubation, the cell lysates were harvested and western analysis was performed using the following antibodies: (1) EVI1, (2) SnoN, (3) TAK1, (4) TGFβRII, (5) SMAD2/3, (6) AKT, and (7) GAPDH as a loading control. The data shown are representative of 4 independent experiments. (b) HEY cells were seeded at 250,000 cells per well in 6-well plates. After overnight attachment, the cells were treated with (1) 5µM As₂O₃, (2) 5µM MG132, or (3) 5µM As₂O₃ and 5µM MG132. After 6 or 18 hour treatment, the cell lysates were harvested and western analysis was performed using the following antibodies: (1) EVI1, (2) SnoN, (3) TGFβRII, (4) SMAD2/3, (5) AKT, and (6) GAPDH as a loading control. The data shown are representative of 3 independent experiments. (c) HEY cells were seeded at 250,000 cells in 6-well plates. After overnight attachment, the cells were treated with (1) 10µM As₂O₃, (2) 500nM PS-341, or (3) 10µM As₂O₃ and 500nM PS-341. After 18 hour treatment, the cell lysates were harvested and western analysis was performed using the following antibodies: (1) EVI1, (2) SnoN, (3) TGFβRII, (4) SMAD2/3, (5) AKT, and (6) GAPDH as a loading control. The data shown are representative of 3 independent experiments. (d) HEY cells were seeded at 250,000 cells per well in 6-well plates. After 24 hours, the cells were treated at different time points (1, 3, 18h) with 5µM MG132. Cell lysates were harvested and western analysis was performed using the following antibodies: (1) EVI1, (2) SnoN, (3) TGFβRII, (4) SMAD2/3, (5) AKT, and (6) GAPDH as a loading control. The data shown are representative of 3 independent experiments. (e) HEY cells were seeded at 500,000 cells per well in 6-well plates. Following 24 hours, the cells were treated with (1) DMSO, (2) 5µM As₂O₃ and DMSO, or (3) 5µM MG132 for 18 hours. RNA was then isolated and used for qPCR. Relative RNA-fold changes are presented for EVI1, SnoN, and TGFβRII. The data shown are representative of 3 independent experiments.

As₂O₃ induces autophagy in ovarian cells. (a) T80 (left panel), HEY (middle panel), and SKOV3 (right panel) cells were seeded at 250,000 cells per well in 6-well plates. After overnight attachment, the cells were treated with varying concentrations of As₂O₃ (2–50µM). After 18 hour incubation, the cell lysates were harvested and western analysis was performed using the following antibodies: (1) SnoN, (2) PARP, (3) Procaspase-3, (4) LC3, (5) p62, and (6) GAPDH as a loading control. The data shown are representative of 3 independent experiments. (b) HEY cells were plated at 250,000 cells per well in 6-well plate. After overnight attachment, cells were treated with 25µM As₂O₃ for varying times (0, 3, 9, 18, 24, and 30 hours) and light microscope images were captured at 40× magnification. The data shown are representative images. (c) HEY cells were seeded at 250,000 cells per well. Following overnight attachment, the cells were treated with 25µM As₂O₃ at various time points (3, 9, 18, 24, 30h). Cell lysates were harvested and western analysis was performed using the following antibodies: (1) EVI1, (2) SnoN, (3) TAK1, (4) TGFβRII, (5) SMAD2/3, (6) PARP, (7) Procaspase-3, (8) LC3, (9) Beclin-1, (10) p62, and (11) GAPDH as a loading control. The data shown are representative of 2 independent experiments. (d) and (e), Confluent HEY cells grown in T-75 flasks were treated with 25µM As₂O₃ for 3, 9, 18 hours and prepared for TEM. Images were captured at varying magnifications as indicated. Representative images are shown.

As₂O₃ induces autophagy via a Beclin-1 independent pathway. (a) 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 increasing doses of As₂O₃ (2–50µM) or 25µM As₂O₃ in the presence of 5mM 3-MA for 18 hours. Immunofluorescence images were obtained at 40× magnification. The data shown are representative of 2 independent experiments. (b) Immunofluorescence data from (a) was quantitated and presented as the percentage of EGFP positive cells with punctate LC3 expression. (c) HEY cells were seeded at 250,000 cells per well. After 24 hours, the cells were treated with: (1) 25µM As₂O₃, (2) 25µM As₂O₃ and 0.1mM 3-MA, (3) 25µM As₂O₃ and 1.0mM 3-MA, (4) 25µM As₂O₃ and 2.0mM 3-MA, (5) 25µM As₂O₃ and 5.0mM 3-MA, and (6) 5.0mM 3-MA only for 18 hours. Cell lysates were harvested and western analysis was performed using the following antibodies: (1) SnoN, (2) PARP, (3) Procaspase-3, (4) Beclin-1, (5) LC3, and (6) GAPDH as a loading control. The data shown are representative of 4 independent experiments. (d) HEY cells were seeded at 250,000 cells per well. (Left panel): Following overnight attachment, the cells were treated with: (1) 25µM As₂O₃ (2) 25µM As₂O₃ and 25nM Bafilomycin A (BAF), (3) 25µM As₂O₃ and 50nM Bafilomycin A, (4) 25µM As₂O₃ and 75nM Bafilomycin A, (5) 25µM As₂O₃ and 100nM Bafilomycin A, and (6) 100nM Bafilomycin A only. (Right panel): After 24 hours, the cells were treated with: (1) 25µM As₂O₃, (2) 25µM As₂O₃ and 25µM zVAD-Fmk (3) 25µM As₂O₃ and 50µM zVAD-Fmk (4) 25µM As₂O₃ and 75µM zVAD-Fmk (5) 25µM As₂O₃ and 100µM zVAD-Fmk, and (6) 100µM zVAD-Fmk only. After 18 hour incubation, cell lysates were harvested and western analysis was performed using the following antibodies: (1) SnoN, (2) PARP, (3) Procaspase-3, (4) LC3, and (5) GAPDH as a loading control. The data shown are representative of 2 independent experiments. (e) Ovarian cells (T80, T29, OVCAR8, HEY, and SKOV3 cells) were seeded at 500,000 cells per well in T25 flasks. After overnight attachment, cell lysates were harvested and western analysis was performed using the following antibodies: (1) ATG5, (2) Beclin-1, (3) ATG7, (4) (5) hVps34, and (6) GAPDH as a loading control. (f) HEY cells were seeded at 250,000 cells per well in 6-well plates. After 24 and 48 hours, the cells were treated with (1) non-targeting (control) siRNA, (2) ATG5 siRNA, (3) Beclin-1 siRNA, (4) ATG7 siRNA, and (5) hVps34 siRNA. Seventy-two hours post-transfection, cells were treated for 18 hours with 25µM As₂O₃. Cell lysates were harvested and western analysis was performed using the following antibodies: (1) ATG5, (2) Beclin-1, (3) ATG7, (4) hVps34, (5) LC3, (6) PARP, (7) SnoN, and (8) GAPDH as a loading control. The data shown are representative of 2 independent experiments. (g) HEY cells were seeded at 250,000 cells per well in 6-well plates. After 24 and 28 hours, the cells were treated with (1) non-targeting (control) siRNA, (2) ATG5 siRNA, (3) Beclin-1 siRNA, (4) ATG7 siRNA, and (5) hVps34 siRNA. Seventy-two hours post-transfection, cells were treated for 24 hours with 10µM As₂O₃. Cells were stained with LC3B rabbit polyclonal antibody and counterstained with DAPI. Immunofluorescence images are representative and were obtained at 63× magnification. (h) Immunofluorescence data was quantitated and presented as the percentage of positive cells with punctate LC3 expression. The data shown are representative of 2 independent experiments. (i) HEY cells were seeded at 250,000 cells per well in 6-well plates. After 24 and 28 hours, the cells were treated with (1) non-targeting (control) siRNA, (2) ATG5 siRNA, (3) Beclin-1 siRNA, (4) ATG7 siRNA, and (5) hVps34 siRNA. Seventy-two hours post-transfection, cells were treated for 48 hours with 10µM As₂O₃ 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 (Top panel). The data is also displayed as a bar graph as the percentage of viable, early apoptotic, and late apoptotic/necrotic cells. The data shown are representative of 2 independent experiments (bottom panel).

AKT knockdown modulates apoptosis in response to As₂O₃. (a) HEY cells were seeded at 375,000 cells per well in 6-well plates. Following 24 hour attachment, cells were transfected with either non-targeting (control) or TAK1 siRNA. Transfected cells were then treated for 18 hours with (1) 10µM As₂O₃, (2) 10µM As₂O₃ with 1mM NAC, and (3) 50µM As₂O₃. Cell lysates were harvested and western analysis was performed using the following antibodies (1) EVI1, (2) SnoN, (3) TAK1, (4) PARP, (5) LC3, (6) p62, and (7) GAPDH as a loading control. The data shown are representative of 2 independent experiments. (b) HEY cells were seeded at 375,000 cells per well in 6-well plates. Following 24 hour attachment, cells were transfected with either non-targeting (control) or AKT siRNA. Transfected cells were then treated for 18 hours with (1) 10µM As₂O₃, (2) 10µM As₂O₃ with 1mM NAC, and (3) 50µM As₂O₃. Cell lysates were harvested and western analysis was performed using the following antibodies: (1) EVI1, (2) SnoN, (3) TAK1, (4) AKT, (5) PARP, (6) LC3, (7) p62, and (8) GAPDH as a loading control. The data shown are representative of 2 independent experiments.