Article Figures & Data
Figures
- Figure 1.
Effect of cisplatin on DU145 human prostate cancer cells. A, Cells were treated with indicated concentrations of cisplatin at four different time points. Cell viability was monitored by MTT assay after each time point. Columns, means of triplicate values (n = 3); bars, SE. *, P < 0.05, cisplatin versus untreated cells at 48 hours. ***, P < 0.001, cisplatin versus untreated cells at 96 hours. B, Immunoblot analysis of cell lysates treated with cisplatin at 24-hour and 96-hour time points with monoclonal antibodies against XIAP, caspase-7, and caspase-3. Respective membranes were stripped and probed for β-actin as loading control. Protein bands were quantified by densitometric analysis and expressed as percentage of control. Arrows, 57-kDa XIAP; 43-kDa β-actin; 35-, 32-, and 20-kDa forms of caspase-7; and 17-kDa active caspase-3 bands. C, Akt and phospho-Akt immunoblot analysis of lysates from cells treated with cisplatin at 24-hour and 96-hour time points. The phospho-Akt membrane was stripped and probed for Akt and β-actin as loading control. Protein levels were quantified by densitometric analysis and expressed as percentage of control. Arrows, 60-kDa Akt and phospho-Akt bands.
- Figure 2.
Effect of TRAIL on cell viability and protein expression in DU145 human prostate cancer cells. A, Cells were treated with indicated concentrations of TRAIL at four different time points. Cell viability was examined by MTT assay after each time point. Values obtained were compared with the respective controls. Columns, means of triplicate values; bars, SE. ***, P < 0.001, TRAIL treated versus untreated cells at each time point. B, Immunoblot analysis of lysates from DU145 cells treated with TRAIL for 6 hours. Blots were probed with XIAP and caspase-3 antibodies. Respective membranes were stripped and probed for 43-kDa β-actin as loading control. Numbers below each blot were obtained from densitometric analysis and expressed as percentage of control. Arrows, 57-kDa XIAP, 17-kDa caspase-3, and 43-kDa β-actin bands. C, Immunoblot analysis of lysates from DU145 cells treated with TRAIL for 6 hours. Phospho-Akt and Akt were probed with anti-Akt and phospho-Akt polyclonal antibodies. Respective membranes were stripped and probed for 43-kDa β-actin as loading control. Numbers below each blot were obtained from densitometric analysis and expressed as percentage of control. Arrows, 60-kDa Akt and phospho-Akt bands.
- Figure 3.
Effect of XIAP antisense PMO on XIAP expression and cell proliferation in DU145 cells. A, Immunoblot analysis of lysates from cells treated with XIAP antisense and scrambled PMOs by scrape loading for 24 hours. The same membrane was stripped and probed for β-actin as loading control. Protein levels were determined by densitometric analysis and expressed as percentage of control. Arrows, 57-kDa XIAP and 43-kDa β-actin bands. B, Effect of XIAP antisense PMO on viability of DU145 cells. The number of viable cells was determined by trypan blue exclusion using a hemocytometer. Points, means of triplicate values (n = 3); bars, SE. **, P < 0.01, ***, P < 0.001, XIAP antisense versus scrambled PMO and vehicle at the indicated PMO concentrations. C, Representative phase-contrast photomicrographs of DU145 cells scrape loaded from different treatments.
- Figure 4.
Lysates from the HeLa cells transiently transfected with pCiNeoXIAP-lucΔA and scrape loaded with vehicle, antisense XIAP PMO, or scrambled PMO were also probed for endogenous XIAP expression. Protein bands were quantified by densitometric analysis and expressed as percentage of untreated.
- Figure 5.
A. Caspase-3 immunoblot from cell lysates treated with XIAP antisense and scrambled PMOs by scrape loading for 24 hours. The same membrane was stripped and probed for β-actin as loading control. Numbers below each blot were obtained from densitometric analysis and expressed as percentage of control. Arrows, 17-kDa form of caspase-3 and 43-kDa β-actin bands. B. Levels of M30 antigen in cell lysates as determined by ELISA-based method. Levels of M30 antigen were normalized to total protein content in the cell extracts. Points, means of triplicate values from a single experiment; bars, SE. *, P < 0.05, XIAP antisense PMO versus untreated and scrambled PMO.
- Figure 6.
Role of combined XIAP antisense PMO and cisplatin treatment on protein expression and cell proliferation in DU145 human prostate cancer cells. Cells were treated with cisplatin for 24 hours followed by 24-hour XIAP antisense PMO and scrambled PMO treatments. A. Cell viability was monitored by MTT assay after 24 hours. Columns, means of triplicate values; bars, SE. *, P < 0.05, XIAP antisense PMO versus vehicle and scrambled PMO. **, P < 0.01, cisplatin + XIAP antisense PMO versus cisplatin and cisplatin + scrambled PMO. B. Immunoblot analysis of XIAP expression. Protein levels were quantified by densitometric analysis and expressed as percentage of control. Arrows, 57-kDa XIAP and 43-kDa β-actin bands.
- Figure 7.
Effect of combined TRAIL and XIAP antisense PMO on cell proliferation and protein expression in DU145 cells. Cells were treated with XIAP antisense and scrambled PMOs for 24 hours followed by a 24-hour TRAIL treatment. A. Cell viability was monitored by MTT assay after 24 hours. Columns, means of triplicate values; bars, SE. *, P < 0.05, XIAP antisense PMO versus vehicle and scrambled PMO. **, P < 0.05, TRAIL + XIAP antisense PMO versus TRAIL and TRAIL + scrambled PMO). B. Immunoblot analysis of XIAP expression. Protein levels were quantified by densitometric analysis and expressed as percentage of control. Arrows, 57-kDa XIAP and 43-kDa β-actin bands.
Tables
- Table 1.
Plasmid-based system for screening PMO sequence specificity and antisense activity
Transfection Treatments % Luciferase Activity (Relative Luminescence Units/μg Protein) % Cell Viability pCiNeoXIAP-lucΔA Vehicle 100.00 ± 1.93 100.00 ± 2.72 Scrambled PMO 115.00 ± 4.23 93.78 ± 4.15 XIAP antisense 53.78 ± 9.53 69.30 ± 5.85 Vector control Vehicle 100.00 ± 8.35 100.00 ± 15.38 Scrambled PMO 97.21 ± 3.91 84.89 ± 9.50 LipofectAMINE alone Scrambled PMO 0.006 ± 0.01 83.00 ± 7.57 NOTE: HeLa cells transiently transfected with pCiNeoXIAP-lucΔA, vector alone (pCiNeo-lucΔA), or delivery agent (Lipofectamine) were scrape loaded with different agents as indicated. Luciferase activity was measured 24 hours later. Data are expressed as luciferase activity in lysates normalized for protein content and represent means ± SE of triplicate values (n = 2). Cell viability in each case was assessed by trypan blue staining and expressed as mean ± SE of triplicate values. P < 0.01, vehicle versus XIAP antisense PMO treated cells. P < 0.001, scrambled PMO versus XIAP antisense PMO treated cells transiently transfected with pCiNeoXIAP-lucΔA.
Volume 3, Issue 6
