Figure 1. NH043-1 improved MSG-induced cell death by using MTT assay in SH-SY5Y cells.
(A) Cell death was measured after treating MSG with various concentrations in 2 x 104 SH-SY5Y cells. (B) Cell viabilities were measured after treating with 100 mM MSG for 24 h in the absence or presence of NH043-1 at indicated doses, or NMDA receptor antagonist, MK801, in SH-SY5Y cells. Data were the mean values of three
Figure 2. NH043-1 inhibited MSG-induced cell apoptosis by Annexin-V staining.
Apoptosis detection was measured after treating with 100 mM MSG and 15 uM NH043-1 or 10 uM MK801 for 24 h in 1 x 106 SH-SY5Y cells.
Cells were stained with Annexin-V/PI and measured with a flow cytometry. (A) Control (B) 100 mM MSG (C) 100 mM MSG with 15 uM NH043-1 or (D) 10 uM MK801.
(A)
(D) (C)
(B)
Figure 3. NH043-1 decreased Calpain-2 and SBDPs expressions by MSG in SH-SY5Y cells.
(A) Calpain-2 and SBDPs expressions were measured by western blot analysis after treating with 100 mM MSG and 15 uM NH043-1 or 10 uM MK801 for 6 h in 1 x 106 SH-SY5Y cells. (B) and (C) Quantification of the intensities of Calpain-2 and SBDPs protein bands was analyzed by ImageJ software and actin was used as loading control.
Calpain2
Figure 4. Effects of NH043-1 on the changes of Bcl-2 and Bax expressions by MSG in SH-SY5Y cells
(A) Bcl-2 and Bax expressions were measured by western blot analysis after treating with 100 mM MSG and 15 uM NH043-1 or 10 uM MK801 for 6 hours in 1 x 106 SH-SY5Y cells. (B) and (C) Quantification of the intensities of Bcl-2 and Bax protein bands was detected by ImageJ software and actin was used as loading control. The results were shown as mean ± SEM, n=3, *p < 0.05, comparing to MSG group.
(A)
Figure 5. Effects of NH043-1 on cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP expressions by MSG induction in SH-SY5Y cells (A) 1 x 106 SH-SY5Y cells were treated with 100 mM and 15 uM NH043-1 or 10 uM MK801 for 24 h. The expressions of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP were measured by Western blot analysis. (B), (C), and (D) Quantification of the intensities of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP protein bands was detected by ImageJ software and actin was used as loading control. The results were shown as mean ± SEM, n=3, *p < 0.05, comparing to MSG group.
Figure 6. NH043-1 decreased the ROS production in SH-SY5Y cells by MSG.
(A) ROS productions were measured by CL analysis after treating with 100 mM MSG for 24 h in the absence or presence of 15 uM NH043-1 or NMDA receptor antagonist, 10 uM MK801, in 1 x 106 SH-SY5Y cells with a luminol-enhanced chemiluminescence detector. (B) Total counts of ROS production.
Relative of CL (% of contr ol)
Ctrl MSG NH043-1 MK
Relative of all counts (% of control) +MSG (B)
Figure 7. NH043-1 rescued MSG-induced loss of MMP by using JC-1 staining in SH-SY5Y cells.
(A) Loss of MMP were measured after treating with 100 mM MSG for 12 hours in the absence or presence of 15 uM NH043-1 or NMDA receptor antagonist, 10 uM MK801 in 1 x 106 SH-SY5Y cells, with JC-1 staining and analyzed with a flow cytometry. The treatments included control, 100 mM MSG, 100 mM MSG with 15 uM NH043-1, 100 mM MSG with 10 uM MK801, and 5 uM CCCP (disruptor of electron transport chain). (B) The ratio of JC-1 oligomer/monomer was determined. The results were shown as mean ± SEM, n=3, *p < 0.05, comparing to MSG group.
Ctrl MSGNH043-1 MK CCCP
0 50 100 150
+ MSG
Relative of MMP (% of control)
(B) (A)
*
(A)
Figure 8. NH043-1 improved cell viability and inhibited cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP expressions after doxycycline induction in nTBP/Q79 cells.
(A) 1.0 x 106 nTBP/Q36 and nTBP/Q79 cells were induced with 10 ug/mL doxycycline for 1, 3, and 5 d after pretreatment of 15 uM NH043-1 for 1 h. Cell viabilities were measured using MTT assay. (B) 1.0 x 106 nTBP/Q36 and nTBP/Q79 cells were induced with 10 ug/mL doxycycline for 5 d after pretreatment of 15 uM NH043-1 for 1 h. The expressions of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP were detected by western blot. (C) Quantitative analysis of the intensities of cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP protein executed in nTBP/Q79 cells by ImageJ software and actin was used as loading control. The results were shown as mean ± SD, n=3, *p < 0.05, comparing to Dox group.
(A)
(B)
TBP (N12)
Ctrl Dox Dox
0 1 2 3 4
Relative fold change
+ NH043-1
(C)
*
Figure 9. Effects of NH043-1 on protein aggregation after doxycycline induction of nTBP/Q36 and nTBP/Q79 by using dot-blot assay
1.0 x 106 nTBP/Q36 or nTBP/Q79 cells were treated with 10 ug/mL doxycycline for 5 days after pretreatment of 15 uM NH043-1 for 1 hour.
Cell lysates were analyzed using anti-TBP (N12) antibody on (A) western blot and (C) dot-blot assay. (B) Quantitative analysis of the intensities of TBP (N12) protein executed in nTBP/Q79 cells by ImageJ software. The values represent means ± SD, n=3. *p< 0.05, comparing to Dox group.
Figure 10. Effects of NH043-1 on body weight and motor performance in SCA17 transgenic mice
Mice were grouped into WT_Saline, TG_Saline, and TG_NH043-1 groups. (A) Mice body weight was measured from 8- to 20-week-old. (B) The performance of motor coordination was determined by rotarod assay.
The values represent means ± SD, n=6. *p< 0.05, comparing to TG_Saline group. Mice were intraperitoneal-injected with saline or NH043-1 (4.5 mg/kg) at 10-week by three times a week.
(A)
WT_Saline TG_Saline TG_NH043-‐1
Figure 11. NH043-1 treatment improved gait abnormalities in SAC17 mice.
Footprint analysis of WT_Saline, TG_Saline, and TG_NH043-1 (4.5 mg/kg) mice were performed at 20-week. Mice were intraperitoneal-injected with saline or NH043-1 from 10-week by three times a week. (A) The footprint patterns of WT_Saline, TG_Saline, and TG_NH043-1 were shown. (B) The front/hind footprint overlaps (cm), (C) the parallel length (cm), and (D and E) the front/hind stride lengths (cm).
The results were shown as mean ± SEM, n=6, *p < 0.05, comparing to TG_Saline group.
TBP(N12) cleaved-caspase-3 expression in SCA17 transgenic mice.
(A) The extent of TBP aggregation [detected using TBP (N12) antibody], and the expression of cleaved-caspase-3 in the cerebella of mice were