Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: Cd induced oxidative stress in neuronal cells. ( A ) SH-SY5Y cells were treated with indicated concentrations of Cd for 24 h, and subsequently, ROS levels were analyzed by flow cytometry. ( B ) SH-SY5Y cells were incubated with 5 μM Cd for various time periods. The formation of ROS was measured by flow cytometry. ( C ) SH-SY5Y cells were exposed to 5 μM Cd and 2.5 mM NAC alone or together for 24 h; subsequently, ROS levels were analyzed by flow cytometry. ( D , E ) SH-SY5Y cells were exposed to 5 μM Cd at the indicated times. Western Blot analysis was performed to determine SOD2 protein expression, and a CuZn/Mn-SOD Assay Kit was used to measure SOD2 activity. ( F ) Rat cerebral cortical neurons were incubated with 5 μM Cd for various time periods, and subsequently, ROS levels were analyzed by fluorescence microscopy. All results are representative of three independent experiments. The provided scale bar in the merged image represents 200 μm. * p < 0.05, ** p < 0.01 versus the control group; ## p < 0.01 versus the Cd group.

Article Snippet: Primary rat cerebral cortical neurons were obtained from the cerebral cortex of one-day-old Sprague–Dawley rats, with a typical yield of approximately 3–4 × 10 6 viable neurons per pup.

Techniques: Flow Cytometry, Incubation, Western Blot, Expressing, Activity Assay, Fluorescence, Microscopy, Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: Cd induces the nuclear expression of FoxO3a in neuronal cells. SH-SY5Y cells were treated with 5 μM Cd for various time periods. ( A ) The cell lysates were subjected to Western Blot analysis to measure FoxO3a protein expression; ( B ) nuclear and cytoplasmic extracts were subjected to Western Blot analysis to measure FoxO3a expression. ( C , D ) SH-SY5Y cells and rat cerebral cortical neurons were exposed to 5 μM Cd for 6 h, and confocal laser scanning microscopy was performed to determine FoxO3a localization. FoxO3a is shown in green, and nuclei were counterstained with DAPI (blue). All results are representative of three independent experiments. The provided scale bar in the merged image represents 20 μm. * p < 0.05, ** p < 0.01 versus the control group.

Article Snippet: Primary rat cerebral cortical neurons were obtained from the cerebral cortex of one-day-old Sprague–Dawley rats, with a typical yield of approximately 3–4 × 10 6 viable neurons per pup.

Techniques: Expressing, Western Blot, Confocal Laser Scanning Microscopy, Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: p38 regulates the Cd-induced nuclear expression of FoxO3a. ( A ) SH-SY5Y cells were treated with 5 μM Cd for various time periods, and phosphorylated and total MAPK proteins were detected by Western Blot analysis. ( B ) SH-SY5Y cells were treated with 5 μM Cd for 6 h after preincubating with 10 μM SB203580 for 1 h. Western Blot analysis was performed to determine FoxO3a expression. ( C , D ) SH-SY5Y cells and rat cerebral cortical neurons were exposed to 5 μM Cd for 6 h after preincubation with 10 μM SB203580 for 1 h. Confocal laser scanning microscopy was performed to determine FoxO3a expression. FoxO3a is shown in green, and nuclei were counterstained with DAPI (blue). ( E ) SH-SY5Y cells were treated with 5 μM Cd for various time periods. Western Blot analysis was performed to determine phosphorylation of FoxO3a-Ser7. ( F – H ) SH-SY5Y cells and rat cerebral cortical neurons were incubated with 5 μM Cd for 6 h after preincubation with 10 μM SB203580 for 1 h. Western Blot analysis and confocal laser scanning microscopy were performed to determine phosphorylation of FoxO3a-Ser7 expression. The provided scale bar in the merged image represents 20 μm. ( G – H ) p-FoxO3a is shown in red, and nuclei were counterstained with DAPI (blue). All results are representative of three independent experiments. * p < 0.05, ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the Cd group.

Article Snippet: Primary rat cerebral cortical neurons were obtained from the cerebral cortex of one-day-old Sprague–Dawley rats, with a typical yield of approximately 3–4 × 10 6 viable neurons per pup.

Techniques: Expressing, Western Blot, Confocal Laser Scanning Microscopy, Phospho-proteomics, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: The effects of the p38/FoxO3a pathway on the Cd-induced oxidative stress. ( A ) SH-SY5Y cells were treated with 5 μM Cd for various time periods after being pretreated with 10 μM SB203580 for 1 h. Western Blot analysis was performed to determine SOD2 protein expression. ( B ) SH-SY5Y cells were treated with 5 μM Cd for 6 h after preincubating with 10 μM SB203580 for 1 h. A CuZn/Mn-SOD Assay Kit was used to measure SOD2 activity. SH-SY5Y cells and cerebral cortical neurons were preincubated with 10 μM SB203580 for 1 h, followed by treatment with 5 μM Cd for various time periods. ROS levels were analyzed by flow cytometry or fluorescence microscopy ( C , D ); cells were exposed to 5 μM Cd for 24 h after preincubation with SB203580, and subsequently, cell viability of SH-SY5Y cells and rat cerebral cortical neurons was analyzed by CCK8 ( E , F ). All results are representative of three independent experiments. The provided scale bar in the merged image represents 200 μm. * p < 0.05, ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the Cd group.

Article Snippet: Primary rat cerebral cortical neurons were obtained from the cerebral cortex of one-day-old Sprague–Dawley rats, with a typical yield of approximately 3–4 × 10 6 viable neurons per pup.

Techniques: Western Blot, Expressing, Activity Assay, Flow Cytometry, Fluorescence, Microscopy, Control