Supplemental Data
Files in this Data Supplement:
- Supplemental Fig 7 (.pdf, 225 KB) - Supplementary Figure 7. The expression of BI-1 induces autophagy formation. A picture of cell morphology was taken when Neo cells and various clones of BI-1 cells were cultured with thapsigargin for a period of 24 hours (X 200) (A). Neo and BI-1 cells (M1 cells) were treated with 5 ���M thapsigargin and then harvested after the indicated time periods (12, 24, 36, or 48 hours). Cell lysates were immunoblotted after SDS-PAGE with anti-LC3-I/II or ��-actin antibody (B).
- Supplementary Fig. 3 (.pdf, 184 KB) - Supplementary Figure 3. Expression of the 20S proteasome did not differ between Neo and BI-1 cells under ER stress. Neo and BI-1 cells were subjected to either 5 ��M thapsigargin or 5 ��g/ml tunicamycin during the indicated time periods and western blotting was performed with anti-20S proteasome or ��-actin antibody. Representative blots are shown.
- Supplementary Fig. 5 (.pdf, 93 KB) - Supplementary Figure 5. The bafilomycin-induced ER stress response was more active in BI-1 cells than in Neo cells. Neo and BI-1 cells were subjected to 0.001, 0.01, 0.1, 1, and 10 ��M bafilomycin for 4 hours, and then western blotting was performed with anti-GRP78, CHOP, or ��-actin antibody. Representative blots are shown.
- Fig. S6 (.pdf, 170 KB) - Supplementary Figure 6. 100 nM bafilomycin reverses the BI-1-regulated ER stress response. Neo and BI-1 cells were subjected to either 5 ��M thapsigargin or 5 ��g/ml tunicamycin with or without 100 nM bafilomycin for 0, 1, 2, 4, 8, or 12 hours, and then western blotting was performed with anti-GRP78, CHOP, or ��-actin antibody. Representative blots for thapsigargin and tunicamycin are shown in the upper and lower panels, respectively.
- Supplementary Fig. 4 (.pdf, 25 KB) - Supplementary Figure 4. After isolation of the lysosome fraction, the indicated concentrations of bafilomycin were incubated. V-ATPase activity was measured to assess lysosomal activity with the lysosomal fractions described in the Materials and Methods section.
- Supplementary Fig. 2 (.pdf, 57 KB) - Supplementary Figure 2. Proteasome activity did not differ between Neo and BI-1 cells under ER stress. Neo and BI-1 cells were subjected to either 5 ��M thapsigargin or 5 ��g/ml tunicamycin during the indicated time periods. Proteasome activity was calculated by measuring the peptidolytic activity (chymotrypsin-like (A), trypsin-like (B), and caspase-like peptidases (C)). Fluorogenic substrates were incubated with cell extracts for 30 minutes in the presence of 2 mM ATP and proteasome activity was determined by measuring the fluorescence. Each value is expressed as the mean �� SEM of three independent experiments.
- Supplementary Fig. 1 (.pdf, 14 KB) - Supplementary Figure 1. Protein synthesis did not differ between Neo and BI-1 cells. Protein synthesis is similar in Neo and BI-1 cells. 35S was labeled in serum-starved Neo and BI-1 cells. The radioactivity was measured by scintillation counting using Ecoscint A scintillation fluid (National Diagnostics, Atlanta, GA) and detection as counts per minute (cpm) on a Packard 1500 Tri-Carb Scintillation Counter (Packard Instrument Co., Downers Grove, IL).