Figure 1.
Temperature increase exacerbated sodium azide/2-DG-induced cell death.
(A) Sodium azide caused dose-dependent cell death. Neuronal death was induced with different concentrations of sodium azide plus 0.5 mM 2-DG for a period of 1 hour. The time point of cell collection for the analysis was decided based on the findings from our preliminary studies. Cell survival was detected with trypan blue exclusion assay. Error bars represent mean ± SEM. n = 4. * denotes significant difference from 37°C control cell (0 mM) (p<0.01). (B) Increased temperature resulted in more cell death. Cells were treated as indicated for a period of 1 hour and cell survival was detected with trypan blue exclusion assay. * denotes significant difference from 37°C control (p<0.01). † denotes significant difference from 37°C with sodium azide/2-DG (p<0.01). ‡ denotes significant difference from 41°C without sodium azide/2-DG (p<0.05).
Figure 2.
Temperature increase worsened sodium azide/2-DG induced reduction of cellular volume.
Cells received different treatments for 1 hour, and then cell volume was measured by BD FACScan flow cytometer with an aid of Cell Quest Pro. The data were acquired using FCS with voltage E00. A total of 50,000 cells were analyzed per sample. Flow cytometry analysis showed smaller forward scatter value following the treatment with sodium azide/2-DG or increased temperature alone. Combination treatment of sodium azide/2-DG and increased temperature produced addictive actions in the decrease of forward scatter value. Data were representative of one of three independent experiments. w/: with; w/o: without.
Figure 3.
Apoptosis was detected with Annexin V-FITC and PI staining.
Cells were double stained with Annexin V-FITC and PI after the treatments for 1 hour. Apoptosis with Annexin V-FITC positive staining (green fluorescence) was detected in cells treated with high temperature alone, sodium azide/2-DG alone or the combination of sodium azide/2-DG plus high temperature. Representative images were shown in the figure. w/: with; w/o: without.
Figure 4.
Intracellular and extracellular ATP levels changed following treatments.
Neuronal death was induced by treating the cells with 0.5 mM 2-DG and 5 mM sodium azide for the indicated time. Control cells were incubated with fresh regular medium for 10 minutes. (A) Intracellular ATP was measured. Error bars represent mean ± SEM. n = 4. * denotes significant difference from 37°C controls (p<0.01); † denotes significant difference from 41°C control (p<0.01); ‡ denotes significant difference from the cells received the treatment at 37°C (p<0.05). (B) Extracellular ATP was measured in the culture medium following the treatments. Error bars represent mean ± SEM. n = 4. *denotes significant difference from 37°C controls (p<0.01); † denotes significant difference from the group received the treatment with sodium azide/2-DG at 37°C (p<0.01).
Figure 5.
Temperature increase worsened sodium azide/2-DG induced ER stress.
(A) Cells were subjected to treatments with sodium azide/2-DG, increased temperature or combination of sodium azide/2-DG plus increased temperature for a period of 30 minute or 1 hour. Control cells received no treatment. The cells were then collected and protein expressions were analyzed with western blot. β-actin was used as a loading control. (B) CHOP shRNA transfection significantly inhibited CHOP expression. SH-SY5Y cells were transfected with lentiviral particles encoding copGFP (mock) or CHOP shRNA. CHOP protein levels were analyzed by western blot. β-actin was used as a loading control. (C) CHOP shRNA transfection significantly reduced sodium azide/2-DG-induced cell death. Mock or CHOP shRNA transfected SH-SY5Y cells were treated with sodium azide/2-DG for 1 hour at 37°C and 41°C. Cell death was detected with trypan blue exclusion assay. * (p<0.05) and † (p<0.01) denote significant difference from the group received mock transfection.
Figure 6.
Caspase-3 was activated following the treatments.
Cells were subjected to treatments with sodium azide/2-DG, increased temperature or combination of sodium azide/2-DG plus increased temperature for a period of 30 minute or 1 hour. Control cells received no treatment. Cleavage of caspase-3 and PARP-1 were analyzed with western blot. β-actin was used as a loading control.
Figure 7.
Caspase-3 activity was increased following the treatments.
Sodium azide/2-DG, increased temperature or combination of sodium azide/2-DG plus increased temperature resulted in increased caspase-3 activity. Cells were pre-incubated with DMSO or caspase-3 inhibitor (z-DEVD-FMK) for 2 hours, and then cells were subjected to the indicated treatments for 1 hour. * denotes significant difference from the 37°C DMSO cell (p<0.01); † denotes significant difference from the 37°C plus sodium azide/2-DG and 41°C alone cells (p<0.05); ‡ (p<0.05) and § (p<0.01) denote significant difference from the DMSO groups, respectively. n = 3.
Figure 8.
Inhibition of caspase-3 reduced neuronal death following the treatments.
Cells were pre-incubated with DMSO or caspase-3 inhibitor (z-DEVD-FMK) for 2 hours, and then cells were subjected to the indicated treatments for 1 hour. Cell death was detected with trypan blue exclusion assay. * (p<0.05) and † (p<0.01) denote significant difference from the DMSO groups, respectively. n = 4.