Article Figures & Data
Figures
- Figure 1.
Thalidomide regulates bFGF expression and cellular distribution in glioma cells. bFGF mRNA expression relative to GAPDH in U-87 MG cells treated with freshly prepared thalidomide or thalidomide with a 9-h preincubation with culture medium for 3 h (A) or treated with liposome-encapsulated thalidomide for 12 or 24 h (B) was analyzed by real-time PCR. Data were collected from at least three independent experiments. Columns, relative index of untreated or empty liposome–treated control; bars, SE. *, P < 0.05; **, P < 0.01, Student's t test. C, immunofluorescence detection of bFGF in U-87 MG cells treated with liposome-encapsulated thalidomide for 12 h. Cellular distribution of bFGF was studied by fluorescence microscopy. DNA was stained with H33258 as a nuclear marker (see also Supplementary Fig. S2). Magnification, ×400. D, protein extracts from U-87 MG cells treated liposome-encapsulated thalidomide for 12 h were subjected to Western blot analysis of bFGF expression. Left, GAPDH was used as an internal control. U-87 MG, C6, and GBM 8401 cells were treated with liposome-encapsulated thalidomide for 24 h. Protein extracts (1 μg) were used to determinate cellular bFGF levels by ELISA. Right, data were normalized with empty liposome–treated control.
- Figure 2.
Effects of thalidomide on cell proliferation and anchorage-independent growth. A, cell proliferation ability for U-87 MG, C6, and GBM 8401 cells treated with indicated concentrations (0, 0.1, 1, 10, and 100 μg/mL) of liposome-encapsulated thalidomide for 72 h was assayed using a resazurin assay. Points, relative index versus empty liposome–treated cells; bars, SE. *, P < 0.05, Student's t test. B, colony-forming ability of U-87 MG, C6, and GBM 8401 cells seeded in culture medium containing 10% FCS and varied concentrations of liposome-encapsulated thalidomide plus 0.3% agar (size, >0.1 mm) were counted 14 d after treatment. Data were collected from three independent experiments, with each experiment repeated six times. *, P < 0.05; **, P < 0.01, Student's t test. C, aggregation ability of U-87 MG, C6, and GBM 8401 cell suspension cultured in 20 μL culture medium contained various concentrations of thalidomide (0, 0.1, 1, and 10 μg/mL). Cells were imaged using phase-contrast microscopy and the aggregation percentage was counted by calculating 20 droplets of each experiment. *, P < 0.05; **, P < 0.01, Student's t test.
- Figure 3.
Down-regulating bFGF expression level is sufficient to decrease the anchorage-independent growth of U-87 MG cells, and exogenous bFGF can partially rescue the effect. A, cellular bFGF protein expression level in bFGF knockdown and control cells was assayed by Western blot. B, cell proliferation ability of each clone. *, P < 0.05; **, P < 0.01, Student's t test. C, colony-forming ability of each knockdown clones in the presence or absence of exogenous bFGF protein. Colony-forming ability (size, >0.1 mm) was measured 14 d later. Data were collected from three independent experiments, with each experiment repeated six times. #, P < 0.01 versus control clone; **, P < 0.01 versus without exogenous bFGF condition, Student's t test. D, tumor growth of control and bFGF knockdown clones. Tumor size was determined by the following formula: 1/2 (length × width2). Data were represented as mean values from three to five mice.
- Figure 4.
Down-regulating bFGF expression level in U-87 MG cells affects the growth rate in a three-dimensional spheroid culture and exogenous bFGF restores their proliferation ability. A, spheroid morphology of bFGF knockdown clones. B, the diameter of each spheroid was measured using Image-Pro Plus software on day 4. Scale bar, 0.1 mm. C, methylene blue uptake by each of the bFGF knockdown clones as an index of spheroid cell numbers. *, P < 0.05; **, P < 0.01, Student's t test.
- Figure 5.
Thalidomide down-regulates bFGF transcription and translation by targeting its G-rich promoter and IRES region. A, EGFP mRNA expression relative to GAPDH in pbFGF-EGFP stably transfected U-87 MG cells treated with thalidomide for 3 h was analyzed by real-time PCR. Data represent at least three independent experiments. Columns, relative index of the untreated control; bars, SE. *, P < 0.05; **, P < 0.01, Student's t test. B, schematic representation of LMW-IRES and HMW-IRES plasmids. C, IRES activity of U-87-LMW-IRES and U-87-HMW-IRES cells treated with indicated concentrations of liposome-encapsulated thalidomide for additional 12 h. Cells were lysed, and luciferase activity was assayed using a Dual Luciferase Assay kit. IRES activity was determined by calculating the Renilla luciferase (LucR) to firefly luciferase (LucF) ratio. Columns, index of the ratio of Renilla luciferase to firefly luciferase normalized to the empty liposome–treated control; bars, SD. *, P < 0.05; **, P < 0.01, Student's t test. D, UV-VIS spectrum of thalidomide incubation with non–G-rich control DNA (top) or G-rich DNA (bottom).
Additional Files
Suplementary Material, Mei and Wu
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Volume 7, Issue 8
