![Figure 4. Effects of BB-1 on TRAIL-R2 expression, TRAIL sensitivity, and p53 expression. (A) KOB cells (5 × 105/mL) were incubated with or without 12.5 μg/mL BB-1 for 24 hours, and the cells were stained with antibodies to TRAIL receptors or with an isotype-matched control antibody. Cells (104) were counted and analyzed by FCM. Shaded peaks, solid lines, and dotted lines correspond to treated, untreated, and control staining, respectively. RFI was determined as the ratio of mean fluorescence intensity for specific staining to that for control staining. (B) Effects of TRAIL-R2-Fc on TRAIL-induced apoptosis. KOB cells (5 × 105/mL) and Jurkat cells (106/mL) were cultured for 48 hours in the presence of the indicated concentrations of TRAIL (□, ○). KOB cells were incubated with 12.5 μg/mL BB-1 for 24 hours, followed by the indicated concentrations of TRAIL for 24 hours (▪). Inhibition of TRAIL-mediated cell death was examined by adding 2.5 μg/mL TRAIL-R2-Fc fusion protein against each of the above treatments (▪, • with dotted lines). Cell proliferation was assessed by MTS assay, and results are expressed relative to the value for the control cells cultured without TRAIL or BB-1. Data are the mean ± SD from 3 independent experiments. (C) Effect of siRNA determined by RT-PCR. At 24 hours after transfection, cells were harvested and subjected to RT-PCR for the number of cycles indicated. mRNA expression of TRAIL-R1 and -R2 in Mock-, GFP siRNA (siGFP)–, TRAIL-R1 siRNA (siTRAIL-R1)–, and TRAIL-R2 siRNA (siTRAIL-R2)–treated cells is shown with that of GAPDH. (D) Effect of siRNA on DR expression evaluated by FCM. At 24 hours after transfection, cells were incubated with or without 12.5 μg/mL BB-1 for 24 hours and were examined by FCM. Shaded and unshaded areas correspond to specific and control staining, respectively. RFI was determined as the ratio of mean fluorescence intensity for specific staining to that for control staining and is indicated in each panel. (E) Effect of siRNA on cell proliferation assay. At 24 hours after transfection, cells were treated for 48 hours with 0.5 μg/mL TRAIL [T], 12.5 μg/mL BB-1 [B], or BB-1+TRAIL (0.5 μg/mL TRAIL was added after incubation with 12.5 μg/mL BB-1 for 24 hours [B→T]) or without reagents [C]. Cell proliferation was assessed by MTS assay, and results are expressed relative to the value for the control cells. Data are the mean ± SD from 3 independent experiments. *P < .01. (F) p53 expression. KOB cells (5 × 105/mL) were incubated with or without 12.5 μg/mL BB-1 for 24 hours, and cells were harvested at the indicated time points. Immunoprecipitation (IP) was performed as described in “Materials and methods” using p53 monoclonal antibodies.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/107/2/10.1182_blood-2005-05-1982/4/zh80020689690004.jpeg?Expires=1722763129&Signature=WndGyUKoZLxnFB894T2h2h6Mt2tDMPyOTWBhRhAYHq-uhBrOSVHrE6HR5SFdGsJk5E4eufszZHu0tG-mp14-14M4HCSzGsqTtI6GwpmYK~SY~qSfmPcZDl~DmIXM4L4X~8--93yH120rmAUK6Dqo01-WL7vZLHwVqXoHfefIBAj4NZjUQr7YLh6E6aRzSuwMfU7sr4q9yOKBUL1S976Um-0MeBsWRBH5DiUnBblrezSMgXwRArZwk4UUfb8ybbu9gab9UglDvWF8uerPOoKWttmqNrAXniVBDxd1fB5GwetUOkxnREqsN6gveSdMHo2uKEmUtZovAa1Q516jcGedCA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effects of BB-1 on TRAIL-R2 expression, TRAIL sensitivity, and p53 expression. (A) KOB cells (5 × 105/mL) were incubated with or without 12.5 μg/mL BB-1 for 24 hours, and the cells were stained with antibodies to TRAIL receptors or with an isotype-matched control antibody. Cells (104) were counted and analyzed by FCM. Shaded peaks, solid lines, and dotted lines correspond to treated, untreated, and control staining, respectively. RFI was determined as the ratio of mean fluorescence intensity for specific staining to that for control staining. (B) Effects of TRAIL-R2-Fc on TRAIL-induced apoptosis. KOB cells (5 × 105/mL) and Jurkat cells (106/mL) were cultured for 48 hours in the presence of the indicated concentrations of TRAIL (□, ○). KOB cells were incubated with 12.5 μg/mL BB-1 for 24 hours, followed by the indicated concentrations of TRAIL for 24 hours (▪). Inhibition of TRAIL-mediated cell death was examined by adding 2.5 μg/mL TRAIL-R2-Fc fusion protein against each of the above treatments (▪, • with dotted lines). Cell proliferation was assessed by MTS assay, and results are expressed relative to the value for the control cells cultured without TRAIL or BB-1. Data are the mean ± SD from 3 independent experiments. (C) Effect of siRNA determined by RT-PCR. At 24 hours after transfection, cells were harvested and subjected to RT-PCR for the number of cycles indicated. mRNA expression of TRAIL-R1 and -R2 in Mock-, GFP siRNA (siGFP)–, TRAIL-R1 siRNA (siTRAIL-R1)–, and TRAIL-R2 siRNA (siTRAIL-R2)–treated cells is shown with that of GAPDH. (D) Effect of siRNA on DR expression evaluated by FCM. At 24 hours after transfection, cells were incubated with or without 12.5 μg/mL BB-1 for 24 hours and were examined by FCM. Shaded and unshaded areas correspond to specific and control staining, respectively. RFI was determined as the ratio of mean fluorescence intensity for specific staining to that for control staining and is indicated in each panel. (E) Effect of siRNA on cell proliferation assay. At 24 hours after transfection, cells were treated for 48 hours with 0.5 μg/mL TRAIL [T], 12.5 μg/mL BB-1 [B], or BB-1+TRAIL (0.5 μg/mL TRAIL was added after incubation with 12.5 μg/mL BB-1 for 24 hours [B→T]) or without reagents [C]. Cell proliferation was assessed by MTS assay, and results are expressed relative to the value for the control cells. Data are the mean ± SD from 3 independent experiments. *P < .01. (F) p53 expression. KOB cells (5 × 105/mL) were incubated with or without 12.5 μg/mL BB-1 for 24 hours, and cells were harvested at the indicated time points. Immunoprecipitation (IP) was performed as described in “Materials and methods” using p53 monoclonal antibodies.
