Article Figures & Data
Figures
- Figure 1.
Modulatory effects of SHetA2 on c-FLIP, survivin, XIAP, Bcl-2, Bcl-XL, Bim, and Bax in human NSCLC cell lines. The indicated cell lines were treated with the indicated concentrations of SHetA2 for 16 h (A) or with 5 μmol/L SHetA2 for the given time as indicated (B). The cells were then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis.
- Figure 2.
Enforced expression of ectopic c-FLIP protects cells from SHetA2-induced cell number decrease (A and B) and apoptosis (C and D). A and B, indicated transfectants were seeded in 96-well plates and treated with the indicated concentrations of SHetA2 ranging from 2.5 to 10 μmol/L. After 48 h, the cells were subjected to the sulforhodamine B assay for measurement of cell number. Mean of four replicate determinations. Bars, SD. C and D, indicated transfectants were treated with 10 μmol/L SHetA2 for 48 h and then harvested for detection of apoptotic cells using Annexin V staining. The percent positive cells in the top right and bottom right quadrants were added to yield the total of apoptotic cells.
- Figure 3.
Enforced expression of ectopic c-FLIP confers resistance to induction of apoptosis by the combination of SHetA2 and TRAIL. A and B, indicated transfectants were seeded in 96-well plates and treated with the indicated concentrations of SHetA2 combined with 10 ng/mL TRAIL (H460) or 20 ng/mL TRAIL (A549). After 24 h, cells were subjected to the sulforhodamine B assay for measurement of cell survival. Mean of four replicate determinations. Bars, SD. C and D, indicated transfectants were treated with DMSO, 5 μmol/L SHetA2 alone, 10 ng/mL (H460) or 20 ng/mL (A549) TRAIL alone, or SHetA2 plus TRAIL for 24 h and then subjected to detection of apoptotic cells using Annexin V staining. The percent positive cells in the top right and bottom right quadrants were added to yield the total of apoptotic cells.
- Figure 4.
Enforced expression of ectopic survivin (A) does not confer resistance to ShetA2 (B and C) or the combination of SHetA2 and TRAIL (D). A, confirmation of ectopic survivin expression in H157 survivin transfectants by Western blotting using survivin antibody. P, pool. B and D, indicated transfectants from H157 cells were seeded in 96-well plates and treated with the given concentrations of SHetA2 (B) or the individual combination of SHetA2 and TRAIL as indicated (D) the next day. After 48 h (B) or 24 h (D), the cells were subjected to the sulforhodamine B assay for measurement of cell survival. Mean of four replicate determinations. Bars, SD. C, indicated transfectants were treated with 10 μmol/L SHetA2 for 48 h and then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for detection of cleaved poly(ADP-ribose) polymerase (cPARP). N.S., nonspecific.
- Figure 5.
Effects of siRNA-mediated reduction of endogenous survivin on SHetA2-induced apoptosis (A and B) and comparison of the potencies of siRNA-mediated down-regulation of c-FLIP and survivin on triggering apoptosis (C). A549 (A) or H1299 (B) cells were seeded in 6-well plates and the next day transfected with 60 nmol/L control (Ctrl) and survivin siRNAs, respectively. Twenty-four hours later, the cells were treated with 10 μmol/L SHetA2. After an additional 48 h, the cells were harvested for evaluation of survivin knockdown efficiency by Western blotting (top) and apoptosis using Annexin V staining/flow cytometry (bottom), respectively. C, A549 cells were seeded in 6-well plates and the next day subjected to transfection with 60 nmol/L control, c-FLIP, and survivin siRNAs, respectively. After 48 h, the cells were harvested for evaluation of c-FLIP or survivin knockdown efficiency by Western blot analysis (left) and for detection of apoptotic cells using Annexin V staining/flow cytometry (right). In addition, the morphologic changes of the transfected cells were also documented (right).
- Figure 6.
SHetA2 down-regulates c-FLIP levels through ubiquitin/proteasome-mediated protein degradation (A and B) independent of JNK (C and D). A, given cell lines were pretreated with 10 or 20 μmol/L MG132 for 30 min before the addition of 10 μmol/L SHetA2. After cotreatment for 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. B, A549-FLIPL-2 cells that stably express ectopic flag-FLIPL were transfected with HA-ubiquitin plasmid using FuGENE 6 transfection reagent for 24 h. The cells were then pretreated with 20 μmol/L MG132 for 30 min and then cotreated with 10 μmol/L SHetA2 for 4 h. Whole-cell protein lysates were then prepared for immunoprecipitation using anti-Flag antibody followed by Western blotting (WB) using anti-HA antibody for detection of ubiquitinated FLIPL (Ub-FLIPL) and anti-Flag antibody for detection of ectopic FLIPL. C, indicated cell lines were treated with 10 μmol/L SHetA2 for the given times. D, A549 cells were pretreated with 20 μmol/L SP600125 for 30 min and then cotreated with 10 μmol/L SHetA2 for another 12 h. After the aforementioned treatments (C and D), the cells were then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for detection of the indicated proteins.
Volume 7, Issue 11
