Article Figures & Data
Figures
- Figure 1.
RITA effects on cell growth. RITA reduces cell viability in LAP-35 (A), WE68 (B), TC-71 (C), IOR-BRZ71 (D), 6647 (E), and SK-N-MC (F). Cells were treated with RITA at the indicated concentrations for 24 or 48 hours and then stained with trypan blue for immediate count analysis. Cell growth was calculated as number of trypan blue negative cells. Percentage of cell viability was obtained by setting 100% the cell growth of control vehicle–treated cells (CTR) at each time point and calculating the relative cell growth of treated cells. Data are representative of at least 3 different experiments. G, cells were plated in 96-well plate and treated in octuplicate with increasing concentration of RITA (0.5-1-2-3-6 μmol/L) for 24 or 48 hours (*). Cell viability was assessed by Cell Titer Blue. The dose reported in the graph is the dose able to reduce cell viability to 50% in comparison with untreated cells.
- Figure 2.
RITA causes G2 arrest and cell apoptosis. FACS analysis of LAP-35 (A), TC-71 (B), and SK-N-MC (C) carried out at 12, 24, or 30 hours after treatment with 3 μmol/L RITA. Cells were grown in the presence or absence of RITA and then stained with PI for the immediate cell-cycle analysis. Each panel indicates the percentage of cells in each cell-cycle phase. Sub-G1 region (as indicated) has been independently evaluated relative to the total population. These analyses are representative of at least 2 different experiments. D, TUNEL analysis of indicated cells at 16 or 24 hours after treatment with 3 μmol/L RITA. The percentage of apoptotic cells was measured as TUNEL-positive cells relative to total cells. The data report the mean of 3 independent counts.
- Figure 3.
RITA inhibits anchorage-independent growth. 100,000 cells per 60-mm dish were plated in soft agar and 48 hours after plating, RITA was added at the indicated concentrations. Photos are representative of 2 independent experiments conducted in duplicate.
- Figure 4.
RITA downregulates IGF-1R protein levels. SK-N-MC (A) and TC-71 (B) cells were incubated with 1 or 3 μmol/L RITA for 16 or 24 hours. Whole cells extracts were prepared and analyzed by Western blot for the indicated proteins. Actin was used as loading control. C and D, qReal-time PCR of IGF-1R in SK-N-MC and TC-71 cells, respectively, treated as in A and B. E, SKNMC were incubated with 3 μmol/L RITA and after 14 hours 10 μmol/L MG132 was added. After additional 4 hours, cells were collected for Western blot analysis. F, TC71 were incubated with 3 μmol/L RITA and after 20 hours, 15 μmol/L MG132 was added. After additional 4 hours, cells were collected for Western blot analysis.
- Figure 5.
MDM2 is involved in RITA-mediated IGF-1R degradation. A, SK-N-MC and TC-71 cells were transiently transfected with siMDM2 or CTR oligos (siCTR). After 10 hours, interfered cells were treated with 3 μmol/L RITA and after additional 24 hours collected. Whole cell extracts were prepared and analyzed by Western blot for the indicated proteins. B, cell death was assessed by FACS analysis of Annexin-positive cells (top). Bottom panel, Western blot analysis of a fraction of cells subjected to FACS analysis. SK-N-MC (C) and TC-71 (D) cells were treated with 3 μmol/L RITA for 24 hours or with 10 μg/mL α-amanitin for 27 hours or both. Whole cell extracts were prepared and analyzed by Western blot for the indicated proteins. SK-N-MC (E) and TC-71 (F) cells were treated with 3 μmol/L RITA or treated with vehicle (CTR) and after 4 or 8 hours whole cell extracts collected. MDM2 coimmunocomplexes were analyzed after immunoprecipitation of 800 μg of WCE with α-MDM2 2A10-Ab1 antibodies. WCEs indicate analysis of 1/25 of whole-cell extracts.
- Figure 6.
RITA sensitivity is affected by MDM2 function. A, Western blot analysis of SKNMC cells treated with vehicle, 3 μmol/L RITA for 22 hours, or 3 μmol/L RITA plus 0.5 μg/mL sempervirine added 4 hours before cell lysate collection. B, percentage of cell death after treatment of indicated cells as in A. C, Western blot analysis of TC-71 cells treated with vehicle, 3 μmol/L RITA for 24 hours, or 3 μmol/L RITA plus 0.5 μg/mL sempervirine added 8 hours before cell lysate collection. D, percentage of cell death after treatment of indicated cells as in C. The percentage of cell death was calculated as number of dead/total cell number. Columns, average of 3 independent experiments. Bars, SD. E, Western blot analysis of SKNMC stably transfected with pcDNA 3.1 (CTR), wt-MDM2, or MDM2C438L. Cells were treated with 3 μmol/L RITA and after 15 hours collected for Western blot analysis of indicated proteins. F, cell death of previously indicated SKNMC populations treated as in E. Cell death was calculated as fold of increase of cell death of RITA treated cells/untreated cells. Columns, average of 3 independent experiments. Bars, SD
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file, 44KB.
- Supplementary Figure 1 - PDF file, 53KB, FACS analysis of IOR-BRZ71, WE-68 and 6647 cells upon RITA treatment.
- Supplementary Figure 2 - PDF file, 93KB, IGF-1R and MDM2 protein levels upon RITA treatment in WE-68 and IOR-BRZ71 cells.
- Supplementary Figure 3 - PDF file, 31KB, IGF-1R and MDM2 protein levels upon RITA treatment in LAP-35 and 6647 cells.
- Supplementary Figure 4 - PDF file, 81KB, Levels of EWS-FLI-1 and its targets upon RITA treatment.
Volume 11, Issue 6
