Article Figures & Data
Figures
- Figure 1.
Nelfinavir had anti-proliferative activity against renal cancer cells. A: MTS assay. Cells were treated for 48 h with 2-20 μM nelfinavir. Bars represent mean±SD, n=12. B: Colony-formation assay. Cells were treated for 48 h with 5-20 μM nelfinavir, then given fresh media and allowed to grow for 1-2 weeks. Bars represent mean±SD, n=3. *Relative to the untreated control.
- Figure 2.
Nelfinavir induced apoptosis of renal cancer cells. A: Cell-cycle analysis. Cells were treated for 48 h with 5-20 μM nelfinavir. Changes in the cell-cycle distribution were evaluated using flow cytometry; 10,000 cells were counted. The number inset in each graph shows the percentage of cells in the sub-G1 fraction. B: Western blotting for cyclin D1 and cyclin-dependent kinase 4 (CDK4). Cells were treated for 48 h with 5-20 μM nelfinavir. Actin was used as the loading control. Representative blots are shown. C: Annexin-V assay. Cells were treated for 48 h with 10 or 20 μM nelfinavir. Apoptotic cells were detected by annexin-V assay using flow cytometry; 10,000 cells were counted. Bar graphs show the percentages of apoptotic cells. Data are expressed as mean±SD from three independent experiments. FITC, Fluorescein isothiocyanate; 7-AAD, 7-amino-actinomycin D. D: Western blotting for cleaved poly(ADP-ribose) polymerase (PARP), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), and survivin. Cells were treated for 48 h with 5-20 μM nelfinavir. Actin was used for the loading control. Representative blots are shown.
- Figure 3.
Nelfinavir induced endoplasmic reticulum (ER) stress in renal cancer cells. Western blotting for the ER stress markers glucose-regulated protein 78 (GRP78), endoplasmic oxidoreductin-1-like protein alpha (ERO1-Lα), and endoplasmic reticulum resident protein 44 (ERp44). Cells were treated for 48 h with 5-20 μM nelfinavir. Actin was used for the loading control. Representative blots are shown.
- Figure 4.
Nelfinavir caused aggresome formation and induced autophagy in renal cancer cells. A: Aggresome detection after 48 h treatment with 10 or 20 μM nelfinavir. Red, aggresome; blue, nucleus. Original magnification, 100×. B: Western blotting for the autophagy marker light chain 3 (LC3). Cells were treated for 48 h with 5-20 μM nelfinavir. The emergence of LC3-II marks the occurrence of autophagy. Actin was used for the loading control. Representative blots are shown.
- Figure 5.
Nelfinavir increased the expression of death receptor 4 (DR4) and DR5 and sensitized renal cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). A: Western blotting for DR4 and DR5. Cells were treated for 48 h with 5-20 μM nelfinavir. Actin was used as the loading control. Representative blots are shown. B: MTS assay. Cells were cultured for 24 h in medium with or without 20 μM nelfinavir. Then they were given 25 ng/ml TRAIL with or without 1 μg/ml DR4/Fc or DR5/Fc chimeric protein and incubated for another 24 h. Bars represent mean±SD, n=12. Significantly different at *p=0.0001, **p=0.0007, ***p=0.0033, and ****p=0.0021; N.S., not significantly different. C: Photomicrographs after 48-h treatment. Cells were cultured for 24 h in medium with or without 20 μM nelfinavir. Then they were given 25 ng/ml TRAIL with or without 1 μg/ml DR4/Fc or DR5/Fc chimeric protein and incubated for another 24 h. Note that TRAIL exerted very strong cytotoxicity only when it was preceded by nelfinavir treatment, and this cytotoxicity was blocked by DR4/Fc and DR5/Fc chimeric protein. Original magnification, 100×. D: Annexin-V assay. Cells were cultured for 24 h in medium with or without 20 μM nelfinavir. Then they were given 25 ng/ml TRAIL with or without 1 μg/ml DR4/Fc or DR5/Fc chimeric protein and incubated for another 24 h. Apoptotic cells were detected by annexin-V assay using flow cytometry; 10,000 cells were counted. Bar graphs show the percentages of apoptotic cells. Data are expressed as mean±SD from three independent experiments. FITC, Fluorescein isothiocyanate; 7-AAD, 7-amino-actinomycin D. *Significantly different at p=0.0495; N.S., not significantly different.
