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Results: 9

1.
Figure 7

Figure 7. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

Active caspase-3 ELISA analysis of possible apoptosis in HLE-B3 cells treated with SB216763, UO126, staurosporine, or DMSO. The possibility of the onset of apoptosis was determined using an active capase-3 ELISA with HLE-B3 cells treated with SB216763; 12 µM), UO126; 10 µM), staurosporine (100 nM), or 0.05% DMSO vehicle. Treated and mock-treated HLE-B3 cells were incubated with serum-free minimal essential media (MEM) for 90 min in atmospheric oxygen. The cells were then switched to hypoxia for 180 min in the continued presence of each individual treatment. At the end of the hypoxic exposure, the media were removed and replaced with fresh, oxygenated media still containing SB216763, UO126, staurosporine, or DMSO vehicle. The cells were placed in atmospheric oxygen for 60 min and subsequently lysed, and the quantity of protein determined per treatment. Caspase-3 activity was determined using 10 µg of protein following the manufacturer’s instructions. Data are based upon results from three independent cell populations and were analyzed using GraphPad Prism 5. The error bars represent the standard error. Only treatment with staurosporine indicated a marked increase in activation of caspase-3.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.
2.
Figure 9

Figure 9. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

JC-1 analysis of bovine lens epithelial cells treated with UO126 inhibitor. Secondary cultures of bovine lens epithelial cells were incubated for 90 min with serum-free minimal essential media (MEM), under atmospheric condition, containing 10 µM UO126 or 0.05% DMSO vehicle. Cells were switched to hypoxia for 3h in the continued presence or absence of UO126. At the end of the hypoxic exposure, the cell media were removed, and fresh, oxygenated serum-free MEM containing 5 µg/ml JC-1 and either UO126 or DMSO added for 30 min in atmospheric oxygen. At the end of the 30 min incubation period, the media were again switched to fresh serum-free MEM containing UO126 or DMSO in the absence of the JC-1 dye. A random field of cells was chosen, and that field of cells was imaged every 150 s for 60 min. Serial confocal imaging of mitochondrial depolarization of the secondary cultures of normal bovine lens epithelial cells in the presence of UO126 demonstrated significant depolarization compared to control cells (top panel). Images of the red and green intensity (bottom panel) for the UO126- and DMSO-treated cells at t=60 min (bar=20 µm) are shown (bottom panel). Note the marked intensity of the green channel with the UO126-treated cells relative to DMSO mock treatment at the completion of the 60 min analysis, which indicates a propensity toward a significant degree of mitochondrial depolarization.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.
3.
Figure 1

Figure 1. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

JC-1 stained mock treated (DMSO) HLE-B3 cells under several adaptations of hypoxic and atmospheric oxygen exposure. Human lens epithelial (HLE-B3) cells were stained with JC-1 (5 µg/ml) and observed with confocal microscopy with images captured every 2.5 min over a 60 min period, under the following conditions: (1) continuous hypoxia, (2) continuous atmospheric oxygen, (3) switching from hypoxia to atmospheric oxygen, or (4) switching from atmospheric oxygen to hypoxia. Top row: (continuous oxygen) HLE-B3 cells were maintained in atmospheric oxygen and stained for 30 min in atmospheric oxygen. At the end of the 30 min staining period, fresh oxygenated media without the JC-1 dye were added to the dishes. A random field of cells was then imaged. Note to the reader: The term “random field of cells” here and with all successive JC-1 analyses is meant to infer an arbitrary field of cells is selected, but once chosen, the same field of cells is photographed throughout the 60 min image capture. Second row: (atmospheric oxygen to hypoxia) HLE-B3 cells were maintained in atmospheric oxygen and stained for 30 min in atmospheric oxygen. At the end of the 30 min staining period, the cells were switched to hypoxic media (i.e., medium that had been preincubated at 1% oxygen). A random field of cells was immediately imaged. Third row: (hypoxia to atmospheric oxygen) HLE-B3 cells were placed in hypoxic conditions for 180 min. At the end of the hypoxic exposure, the media were removed and replaced with fresh oxygenated media containing JC-1. The cells were stained for 30 min in atmospheric oxygen. After this 30 min period, the media were again removed, and fresh oxygenated media were added without the JC-1 dye. A random field of cells was then imaged. Fourth row: (continuous hypoxia) HLE-B3 cells were stained with serum-free minimal essential media (MEM) cotaining JC-1 for 30 min in atmospheric oxygen. At the end of this 30 min period, the media were removed, and fresh medium that had been preincubated at 1% oxygen was added without the JC-1 dye. The cells were then switched into the hypoxic conditions for 180 min. The cells were imaged following the 3 h hypoxic exposure. Under all experimental conditions, there was no evidence of loss of membrane potential throughout the 60 min image capture.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.
4.
Figure 4

Figure 4. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

Western blot analysis of BAX, Bcl-2, pBcl-2, and phospho-c-Jun in HLE-B3 cells in the presence or absence of SB216763. Total cell lysates were collected from >85% confluent HLE-B3 cell cultures that were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either 12 µM SB216763) or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h in the continued presence of SB216763 or DMSO vehicle. At the end of the hypoxic incubation period, the hypoxic media were removed, and fresh, oxygenated serum-free MEM with SB216763 or DMSO were added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after (1) continuous normoxic exposure (about 21% oxygen), (2) after the 3 h hypoxic exposure (about 1% oxygen), or (3) after reintroduction of atmospheric oxygen (about 21%) for 1, 2, or 3 h subsequent to the 3 h hypoxic exposure. Total cell lysates were analyzed with immunoblots using 25 µg of protein per lane. Anti-actin was used to normalize the bands to ensure equivalent lane loading. Three experiments, using independent cell populations, were quantified using GraphPad Prism 5 and the relative densities plotted for BAX/actin, Bcl-2/actin, pBcl-2/actin, and phospho-c-Jun/Actin. No change was evident in the ratio of BAX/actin, Bcl-2/actin, pBcl-2/actin, or phospho-c-Jun/actin by treatment with SB216763. Error bars represent standard error, Student t test.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.
5.
Figure 2

Figure 2. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

Western blot analysis of glycogen synthase kinase 3β and glycogen synthase phosphorylation in HLE-B3 cells in the presence or absence of SB216763. Total cell lysates were collected from >85% confluent human lens epithelial (HLE-B3) cell cultures that were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either 12 µM SB216763 or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h in the continued presence of SB216763. At the end of the hypoxic incubation period, the hypoxic media were removed, and fresh, oxygenated serum-free MEM with SB216763 or DMSO vehicle were added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after (1) continuous normoxic exposure (about 21% oxygen), (2) after the 3 h hypoxic exposure (about 1% oxygen), or (3) after reintroduction of atmospheric oxygen (about 21%) for 1, 2, or 3 h subsequent to the 3 h hypoxic exposure. Total cell lysates were analyzed with immunoblots using 25 µg of protein per lane. Anti-actin was used to normalize the bands to ensure equivalent lane loading. Three experiments, using independent cell populations, were quantified using GraphPad Prism 5 and the relative densities plotted for pGSK-3β/GSK-3β and pGS/GS. No change was evident in the ratio of pGSK-3β/GSK-3β while significant inhibition of the phosphorylation of glycogen synthase by SB216763 was noted. Error bars represent standard error. The asterisks (***) indicate p<0.001, Student t test.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.
6.
Figure 6

Figure 6. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

Western blot analysis of phospho-c-Jun in the presence of SP600125 or AS601245. HLE-B3 cell cultures were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either SP600125 (5 µM, 10 µM, and 20 µM), AS601245 (5 µM, 10 µM, and 20 µM), or DMSO (0.05%) vehicle. Cells were then exposed to hypoxia for 3 h in the continued presence of inhibitors. At the end of the hypoxic exposure, the media were removed, and fresh, oxygenated serum-free MEM with either inhibitor or DMSO were added to the cultures. Cells were subsequently placed in atmospheric oxygen for 3 h. Whole cell lysates were collected at the end of the 3 h reintroduction of atmospheric oxygen. Lysates were analyzed with western blot for phospho-c-Jun, pERK, BAX, Bcl-2, and p-BCL-2 using 25 µg of protein per lane. Anti-actin was used to normalize the bands to ensure equivalent lane loading. The inhibition of the phosphorylation of c-Jun by either JNK inhibitor indicates the inactivation of JNK activity, while no loss of BAX, Bcl-2, or pBcl-2 was noted. Under this condition, the phosphorylation of ERK was unimpeded. This experiment was performed twice with two independent cell populations with identical results. Treatment with either JNK inhibitor, SP600125 or AS601245, resulted in a marked decrease in the phosphorylation of c-Jun, indicating that both inhibitors inhibited JNK activation. The asterisks (**) indicate p<0.01, Student t test. BAX, Bcl-2, and pBcl-2 were not significantly diminished relative to the DMSO control, indicating that the ERK pathway, but not the JNK pathway, is involved in the loss of Bcl-2 and pBcl-2 (refer to Figure 5). Two experiments, using independent cell populations, were quantified using GraphPad Prism 5. Reader note: The 20 µM AS601245 was run only once as reflected in the western blot but not in the densitometry plots because we cannot generate statistics on one run.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.
7.
Figure 8

Figure 8. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

Western blot analysis of GSK-3β and GS phosphorylation and BAX, Bcl-2, pBcl-2, and phospho-c-Jun in secondary cultures of normal bovine cells treated with UO126 inhibitor. Bovine cell cultures were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either 10 µM UO126 or 0.05% DMSO) vehicle. Cells were then exposed to hypoxia for 3 h in the presence of either UO126 or DMSO vehicle. At the end of the hypoxic incubation period, the hypoxic media were removed, and fresh, oxygenated serum-free MEM with UO126 or DMSO were added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after (1) continuous normoxic exposure (about 21% oxygen), (2) after the 3 h hypoxic exposure (about 1% oxygen), or (3) after reintroduction of atmospheric oxygen (about 21%) for 1, 2, or 3 h subsequent to the 3 h hypoxic exposure. Total cell lysates were analyzed with immunoblots using 25 µg of protein per lane. Anti-actin was used to normalize the bands to ensure equivalent lane loading. Prevention of phosphorylation of ERK with UO126 treatment was noted (top panel), as was the inhibition of phosphorylated glycogen synthase with western blot analysis (middle panel). The levels of GSK-3β) and pGSK-3β were consistent in the presence and absence of UO126. The phosphorylation of c-Jun, as well as that of Bcl-2, was blocked by treatment with UO126 under all conditions as determined with western blot analysis (bottom panel). Of particular note, Bcl-2 levels were unaffected by UO126 treatment compared to HLE-B3 cells (refer to Figure 5) where a significant diminution of Bcl-2 was observed but only upon reintroduction to atmospheric oxygen. No change in the relative levels of BAX was evident by treatment with UO126 under any condition. The experiment with normal, secondary cultures of bovine cells was run once, to confirm that a similar pattern of biochemical modifications by treatment with UO126 was reproducible with normal bovine cell cultures as was observed with HLE-B3, affirming that the former results were not influenced by viral transformation.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.
8.
Figure 5

Figure 5. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

Western blot analysis of BAX, Bcl-2, pBcl-2, and phospho-c-Jun in HLE-B3 cells in the presence or absence of UO126. Total cell lysates were collected from >85% confluent human lens epithelial (HLE-B3) cell cultures that were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either 10 µM UO126 or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h in the continuous presence of UO126 or DMSO vehicle. At the end of the hypoxic incubation period, the hypoxic media were removed, and fresh, oxygenated serum-free MEM with UO126 or DMSO were added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h in the presence or absence of SB216763. Cultures were collected after (1) continuous normoxic exposure (about 21% oxygen), (2) after the 3 h hypoxic exposure (about 1% oxygen), or (3) after reintroduction of atmospheric oxygen (about 21%) for 1, 2, or 3 h subsequent to the 3 h hypoxic exposure. Total cell lysates were analyzed with immunoblots using 25 µg of protein per lane. Anti-actin was used to normalize the bands to ensure equivalent lane loading. Three experiments, using independent cell populations, were quantified using GraphPad Prism 5. (top panel, left) The phosphorylation of c-Jun, as well as that of Bcl-2, was blocked by treatment with UO126 under all conditions as determined with western blot analysis. Interestingly, Bcl-2 levels were significantly diminished with treatment by UO126 but only upon reintroduction to atmospheric oxygen. (top panel, right) No change in relative density of the ratio of BAX/actin was evident by treatment with UO126 under any condition. Error bars represent standard error. (middle panel, left) A significant drop in Bcl-2 levels was noted but only upon reintroduction of atmospheric oxygen for the 1, 2, and 3 h incubation periods. The asterisk (*) indicates p<0.05, Student t test. (middle panel, right) A significant loss of pBcl-2 was noted under all conditions (continuous atmospheric oxygen, continuous hypoxia, and reintroduction from hypoxia to atmospheric oxygen). The asterisks (***) indicate p<0.001, Student t test. (bottom panel) Unlike with SB216763 (refer to Figure 4), treatment with UO126 resulted in a marked decrease in the phosphorylation of c-Jun, indicating that UO126 adversely affects JNK activity. The asterisks (**) indicate p<0.01, Student t test.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.
9.
Figure 3

Figure 3. From: Lenticular mitoprotection. Part B: GSK-3? and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

Western blot analysis of GSK-3β and GS phosphorylation in HLE-B3 cells treated with UO126 inhibitor. HLE-B3 cell cultures were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either 10 µM UO126 or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h. At the end of the hypoxic incubation period, the hypoxic media were removed, and fresh, oxygenated serum-free MEM with UO126 or DMSO vehicle were added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after (1) continuous normoxic exposure (about 21% oxygen), (2) after the 3 h hypoxic exposure (about 1% oxygen), or (3) after reintroduction of atmospheric oxygen (about 21%) for 1, 2, or 3 h subsequent to the 3 h hypoxic exposure. Total cell lysates were analyzed with immunoblots using 25 µg of protein per lane. Anti actin was used to normalize the bands to ensure equivalent lane loading. The loss of phosphorylation of ERK by UO126 treatment was noted (top, left panel), as was the loss of phosphorylated glycogen synthase by western blot analysis (top, right panel). Three experiments, using independent cell populations, were quantified using GraphPad Prism 5, and the relative densities were plotted for pGSK-3β/GSK-3β and pGS/GS. No change was evident in the ratio of pGSK-3β/GSK-3β while significant inhibition of the phosphorylation of glycogen synthase by UO126 was indicated (middle panel). Error bars represent standard error. The asterisks (***) indicate p<0.001, Student t test. (bottom panel, left) HLE-B3 cells were incubated for 90 min with serum-free MEM, under atmospheric condition, containing 10 µM UO126 or 0.05% DMSO vehicle. Cells were switched to hypoxia for 3 h in the continued presence of UO126 or DMSO vehicle. At the end of the hypoxic exposure, the cells had their media removed, and fresh, oxygenated serum-free MEM containing 5 µg/ml JC-1 and either UO126 or DMSO added for 30 min in atmospheric oxygen. At the end of the 30 min incubation period, the media were again switched with fresh serum-free MEM containing UO126 or DMSO in the absence of the JC-1 dye. The same field of cells was imaged every 150 s for 60 min. Serial confocal imaging of mitochondrial depolarization in HLE-B3 cells in the presence of UO126 demonstrated significant depolarization compared to control cells. (bottom panel, left) Images of the red and green intensity for the UO126- and DMSO-treated cells at t=60 min (bar=20 µm). Note the marked intensity of the green channel with UO126-treated cells relative to DMSO mock treatment at the completion of the 60 min analysis (bottom panel, right) indicating mitochondrial depolarization.

Morgan M. Brooks, et al. Mol Vis. 2013;19:2451-2467.

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