Cell viability assays were represented as mean ± S.E. (n=3). Comparisons between the

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1 Supplemental Data 1 Online supplemental information Statistical analysis Cell viability assays were represented as mean ± S.E. (n=3). Comparisons between the groups were performed using repeated measures analysis of variance (ANOVA). Whereas differences between means were inspected with Dunnett s multiple comparison post hoc tests. A p value < 0.01 ( ) and p<0.05( ) was considered statistically significant. The data was analyzed using KaleidaGraph version 4.0 (Synergy software, Reading, PA). Figure legends: Figure S1. This data supports experimental data presented in Fig. 2. LDH activity (A) and cell viability (B) assayed at different time points. The data is represented as means ± S.E.M of three independent experiments. The p<0.05 ( ) and p < 0.01 ( ) are the only time points depicted as significant difference between the groups when compared to control for both LDH activity and % cell viability. At these time points there was <2% of cell death. (C). shows a bar graph representation of percent of the total LDH (cells + medium) released into the medium. There is very little change in LDH release over a time period of 180 min. Note that the activity at zero time is high to start with. It is possible that at zero time some serum may still remain behind even after three washes with PBS. (D). Amount of αb in the medium plotted as a percentage of total αb protein. Additionally we have assessed expression of αb by QRTPCR; there is insignificant change in the expression of αb in a four hr time period (unpublished).

A p value < 0.01 ( ) and p<0.05( ) was considered statistically significant. The data was analyzed using KaleidaGraph version 4.0 (Synergy software, Reading, PA). Figure legends: Figure S1.

2 Supplemental Data 2 Figure S2. Effect of various inhibitors on the secretion of αb. (A) Effect of sodium azide on αb secretion at two different concentrations. (B) LDH activity in the sodium azide experiments. There is no significant difference between control and 3 mm sodium azide treatment cells. However, 10 mm sodium azide showed a significant (, p< 0.01) increase in LDH release and decrease in cell viability (, p< 0.01) (C) Data is shown in means ± S.E.M of three independent experiments as shown in (Figure S2 B and C). At 10 mm concentration there was about 8% cell death. (D) Immunoblot showing classical protein transport inhibitors has minimal effect on αb secretion. (E) Effect of four classical protein transport inhibitors on the distribution of Calnexin(1,2), Enolase and Galectin-3 (3) in ARPE cell membranes isolated using Mem-PER Eukaryotic membrane Protein Extraction reagent (Pierce-Thermo Scientific, Inc. Rockford, IL). These proteins were selected in order to assess if indeed the drugs used were effective physiologically. The data shows that all these drugs, which do not impact αb secretion in any significant fashion indeed impact the distribution of various membrane proteins indicating that these inhibitors are active in ARPE cells. (F) Corresponding LDH activity and (G) % cell death. Data is shown in means ± S.E.M; significant difference were determined only for Noc and Tun treated cells with p<0.05 ( ) and p < 0.01( ) versus control as shown in B and C. <5% cell death was observed in Noc and Tun treatments. GS = Golgi stop (Monensin); GP = Golgi plug (Brefeldin A), Noc = Nocodazole and Tun = Tunicamycin. Figure S3. This data is relevant to Fig. 6. Assessment of cell death by measurements of LDH. (A) LDH activity in untreated and MBCD treated samples from three independent experiments were represented as means ± S.E.M. (B) % cell death. Significant LDH activity is seen in cells treated with 10mM MBCD (, p < 0.01) for 6 hr, whereas, 5mM

01) increase in LDH release and decrease in cell viability (, p< 0.01) (C) Data is shown in means ± S.E.M of three independent experiments as shown in (Figure S2 B and C).

3 Supplemental Data 3 MBCD treated cells show minimal LDH release and % cell death as shown by p<0.05 ( ) versus control. At 10 mm concentration there was significant cell death (~15%). A concentration of 5 mm was used in the experiments shown in Fig. 6. Figure S4. This data supports Figs. 8 and 9. Data presented in Fig. 3. Pre-immune controls for immunogold labeling with anti αb and anti Hsp70. (A) Unpermeabilized exosomes. (B) Permeabilized exosomes.

A concentration of 5 mm was used in the experiments shown in Fig. 6. Figure S4. This data supports Figs. 8 and 9.

4 Fig. S1 Supplemental Data 4

5 Supplemental Data 5 Fig. S2 Fig. S3

6 Fig. S4 Supplemental Data 6

7 Supplemental Data 7 Supplemental references: 1. Delom, F., Fessart, D., and Chevet, E. (2007) Apoptosis 12, Fuentes, F., and Arregui, C. O. (2009) Mol Biol Cell 20, Bao, Q., and Hughes, R. C. (1999) Glycobiology 9,

CD3/TCR stimulation and surface detection Determination of specificity of intracellular detection of IL-7Rα by flow cytometry

CD3/TCR stimulation and surface detection Stimulation of HPB-ALL cells with the anti-cd3 monoclonal antibody OKT3 was performed as described 3. In brief, antibody-coated plates were prepared by incubating