Article Figures & Data
Figures
- Fig. 1.
The expression of SAG in human tumors and normal counterparts. A, SAG overexpression in multiple human primary tumor tissues. Tumor tissue arrays containing multiple normal and tumor tissues from different organs were stained with purified SAG mAb on the DAKO AutoStainer using the DakoCytomation EnVision+ System-HRP (DAB) detection kit and counterstained with hematoxylin (Surgipath). The stained slides were observed under a microscope (Olympus 1X71) and images were acquired using software DP controller. B, SAG staining in lung tissues, normal versus cancer. Lung tumor tissue arrays containing normal lung and tumor tissues were stained for SAG expression. Stained normal and tumor tissues were classified into four groups (+ to ++++) according to the staining intensity of each tissue. C, percentage of normal or tumor tissues in each staining group. Tissue samples with different staining intensity were grouped and tabulated.
- Fig. 2.
SAG silencing selectively inhibits the growth of human cancer cells. H1299 human lung cancer cells, U87 human glioblastoma cells, NL20 normal bronchial epithelial cells, and MRC-5 lung fibroblast cells were infected with LT-CONT and LT-SAG for 96 h and then split for assays as follows. A, SAG silencing effects were determined by immunoblotting, with β-actin as the loading control, 96 h after cell splitting. B, ATPlite cell proliferation assay. Cells, after lentivirus-based siRNA silencing, were split and seeded into 96-well plates at 3,000 per well in quadruplicates and subjected to ATPlite cell proliferation assay over periods up to 96 h. *, P < 0.05; **, P < 0.01. C, clonogenic cell survival assay in H1299 (top) and U87 (bottom) cells. Cells, after lentivirus-based siRNA silencing, were split, seeded into six-well plates at 100 cells (H1299) or 300 cells (U87) per well in triplicates, and incubated at 37°C for 9 d, followed by 0.05% methylene blue staining and colony counting. D, soft agar anchorage-independent growth assay in H1299 and U87 cells. Ten thousand cells after lentivirus-based siRNA silencing were seeded in 0.33% agar containing 1× cell culture medium and 10% FBS in 60-mm Petri dish, then grown at 37°C for 14 d, followed by staining with p-iodonitrotetrazolium overnight and colony counting.
- Fig. 3.
SAG silencing sensitizes cancer cells to radiation. Cells, after lentivirus-based siRNA silencing, were seeded in six-well plates at three different cell densities in duplicates. The next day, cells were exposed to different doses of radiation followed by incubation at 37°C for 9 d for colony counting. The surviving fraction was calculated and plotted after comparison with the corresponding controls (0 Gy). The sensitizing enhancement ratio (SER) was calculated as the ratio of the inactivation dose under scrambled siRNA control conditions divided by the inactivation dose after SAG silencing. Points, mean from three independent experiments; bars, SEM.
- Fig. 4.
SAG silencing induces apoptosis with NOXA accumulation. H1299 and U87 cells were infected with LT-SAG, along with LT-CONT, for 96 h, then split and cultured for 72 h, followed by propidium iodide staining and FACS analysis for apoptosis detection, caspase-3 activity assay, cell cycle profile, and Western blotting for the levels of apoptosis-associated proteins. A, induction of apoptosis by SAG silencing. Apoptotic cells were determined by sub-G1 fraction in FACS analysis. B, caspase-3 activation on SAG silencing. Caspase-3 activity in infected cells was determined by caspase-3 activity assay. Columns, mean of three independent experiments; bars, SEM. C, expression of apoptosis-associated proteins. The status of a panel of apoptosis-associated proteins including proapoptosis proteins and antiapoptosis proteins were detected by Western blotting, with β-actin as the loading control.
- Fig. 5.
SAG manipulation changes NOXA level and protein half-life. A, SAG overexpression eliminates endogenous NOXA. U87 cells were transiently transfected with pcDNA3-FLAG-SAG, along with pcDNA3 as a control. Cells were harvested 30 h later and subjected to Western blotting using anti-NOXA antibody, with β-actin as the loading control. B, SAG overexpression reduces the level of ectopically overexpressed NOXA. U87 cells were transiently cotransfected with FLAG-NOXA and FLAG-SAG or FLAG-NOXA and pcDNA3. Cells were harvested 30 h later and subjected to Western blotting using anti-FLAG antibody (for FLAG-NOXA) and anti-SAG (for FLAG-SAG and endogenous SAG), with β-actin as the loading control. C, SAG overexpression shortened the protein half-life of NOXA. U87 cells were transiently cotransfected with FLAG-NOXA and FLAG-SAG or FLAG-NOXA and pcDNA3. Twenty-four hours later, cells were treated with 20 μg/mL cycloheximide for the indicated periods of time, followed by Western blotting using antibodies against FLAG (for FLAG-NOXA) and SAG (for FLAG-SAG), with β-actin as the loading control. The relative NOXA levels were quantified by densitometry analysis using the ImageJ1.410 image processing software. D, SAG knockdown extended the half-life of endogenous NOXA. U87 cells were infected with LT-SAG, along with LT-CONT, for 96 h and then split. Twenty-four hours later, cells were treated with 20 μg/mL cycloheximide for the indicated periods of time, followed by Western blotting using antibody against endogenous NOXA, with β-actin as the loading control. The relative NOXA levels were quantified by densitometry analysis using the ImageJ1.410 image processing software. Endo-NOXA, endogenous NOXA.
- Fig. 6.
SAG silencing inhibits the growth of orthotopic pancreatic tumors. PANC-1 human pancreatic carcinoma cells were infected with LT-CONT and LT-SAG, followed by determination of SAG silencing effect (A), cell survival assay in vitro (B), tumor formation in vivo (C), and Western blotting (D). A, SAG silencing effects. Panc-1 cells stably transfected with luciferase were infected with LT-CONT or LT-SAG, and SAG levels were determined 96 h postinfection by Western blotting, with β-actin as the loading control. B, clonogenic cell survival assay. Cells, after SAG silencing, were split, seeded into six-well plates at 100 per well in triplicates, and incubated at 37°C for 9 d, followed by 0.05% methylene blue staining and colony counting. C, bioluminescence imaging of implanted tumors and measurement of tumor weight. Panc-1 cells stably transfected with luciferase were infected with LT-CONT or LT-SAG and implanted into the pancreata of mice (five mice per group) for the evaluation of tumor growth in vivo. After 5 wks, tumors were bioluminescence imaged using a cryogenically cooled imaging system coupled to a data acquisition computer running Living Image software at the University of Michigan Small Animal Imaging Core. Mice were then sacrificed, and tumors were harvested and weighed. D, NOXA expression in tumors. The levels of Noxa and SAG in tumor tissues were determined by Western blotting using antibodies against NOXA and SAG, with β-actin as the loading control.
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Volume 16, Issue 3
