Article Figures & Data
Figures
- Figure 1.
VES induces cell death and growth arrest in PC-3 cells. A, PC-3 cells were plated on a six-well plate and each group (three wells) was treated with various concentrations of VES for 48 h. After VES treatment, cells were trypsinized and combined with floating cells. The number of trypan blue–stained cells was counted. B, PC-3 cells were plated on a six-well plate and each group (three wells) was treated with various concentrations of VES for 48 h. After treatment, attached cells were harvested by trypsinization and counted under the microsccope.
- Figure 2.
VES induces NAG-1 protein/mRNA expression in a concentration- and time-dependent manner. A, PC-3 cells were treated with the indicated concentrations of VES for 48 h and total lysates were subjected to Western blot analysis for NAG-1. For Western analysis, 39-kDa form of NAG-1 protein (proform) was shown. The membrane was stripped and reprobed for β-actin to confirm equal loading. B, PC-3 cells were treated with 20 μmol/L VES for the indicated amount of time and total cell lysates were subjected to Western blot analysis for NAG-1. For Western analysis, 39-kDa form of NAG-1 protein (proform) was shown. The membrane was stripped and reprobed for β-actin to confirm equal loading. C, PC-3 cells were treated with the indicated concentrations of VES for 48 h and mRNA was subjected to Northern blot analysis for NAG-1. The membrane was stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to confirm equal loading of RNA. D, PC-3 cells were treated with 20 μmol/L VES for indicated amount of time and mRNA was subjected to Northern blot analysis for NAG-1. The membrane was stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase to confirm equal loading of RNA. E, PC-3 cells were transfected with NAG-1 promoter reporter plasmid (-966/70) and phRL-null plasmid. Twenty-four hours after transfection, cells were treated with 20 μmol/L VES for 48 h. NAG-1 promoter reporter activity was normalized to Renilla luciferase activity.
- Figure 3.
VES induces NAG-1 protein/mRNA expression in a p38 kinase-dependent mechanism. A, PC-3 cells were treated with 20 μmol/L VES for the indicated amount of time, and total cell lysates were subjected to Western blot analysis for NAG-1. For Western analysis, 39-kDa form of NAG-1 protein (proform) was shown. Membranes was stripped and reprobed with indicated antibody. B, PC-3 cells were pretreated with 2.5 μmol/L Go6983, 20 μmol/L PD980509, 20 μmol/L Ly294002, 20 μmol/L SP600125, or 20 μmol/L SB203580 for 50 min and treated with 20 μmol/L VES for 48 h. Total cell lysates were subjected to Western blot analysis for NAG-1. For Western analysis, 39-kDa form of NAG-1 protein (proform) was shown. C, PC-3 cells were pretreated with 20 μmol/L SB203580 or SB202190 for 50 min and treated with 20 μmol/L VES for 48 h. Total cell lysates were subjected to Western blot analysis for NAG-1. For Western analysis, 39-kDa form of NAG-1 protein (proform) was shown. CTL, vehicle treated. D, PC-3 cells were pretreated with 20 μmol/L SB203580 for 50 min and treated with 20 μmol/L VES for 48 h. Total RNA was purified and 5 μg total RNA was reverse transcribed. The level of NAG-1 cDNA was analyzed with real-time PCR analysis. CTL, vehicle treated; SB203580, treated with SB203580; SB203580/VES, pretreated with SB203580 before VES treatment; VES, VES treated.
- Figure 4.
VES induces p38 kinase activation and nuclear translocation. A, PC-3 cells were pretreated with 20 μmol/L SB203580 for 50 min and treated with 20 μmol/L VES for 48 h. Total cell lysates were subjected to Western blot analysis for phospho-Hsp27. Membrane was stripped and reprobed for total Hsp27. CTL, vehicle treated; SB203580, treated with SB203580; SB203580/VES, pretreated with SB203580 before VES treatment; VES, VES treated. B, PC-3 cells were plated on coverslip and treated with indicated concentration of VES for 24 h. Cells are stained by p38 antibody followed by FITC-conjugated anti-rabbit antibody.
- Figure 5.
MKK6 activates NAG-1 promoter in a p38 kinase-dependent mechanism. A, PC-3 cells were cotransfected with NAG-1 promoter reporter plasmid (-966/70) and pcDNA3.1-lacZ or pcDNA3-MKK6b(E). As an internal control, the phRL-null vector was used to correct for transfection efficiency. Twenty-four hours after transfection, medium was changed and cells were incubated for another 24 h in the presence or absence of 20 μmol/L SB203580. Luciferase activity was measured 48 h after transfection and firefly luciferase activity was normalized to Renilla luciferase. B, PC-3 cells were cotransfected with indicated length of NAG-1 promoter reporter plasmid and pcDNA3.1-lacZ or pcDNA3-MKK6b(E). As an internal control, the phRL-null vector was used to correct for transfection efficiency. Luciferase activity was measured 48 h after transfection and firefly luciferase activity was normalized to Renilla luciferase.
- Figure 6.
NAG-1 mRNA is stabilized by VES treatment in a p38 kinase-dependent mechanism. A, PC-3 cells were transfected with pGL3 promoter or pGL3 promoter plasmid with 3′-UTR of NAG-1. As an internal control, the phRL-null vector was used to correct for transfection efficiency. Twenty-four hours after transfection, cells were treated with or without VES for 48 h. Luciferase activity was measured and firefly luciferase activity was normalized to Renilla luciferase. B, PC-3 cells were cotransfected with pGL3 promoter or pGL3 promoter plasmid with 3′-UTR of NAG-1 and pcDNA3.1-lacZ or pcDNA3-MKK6b(E). As an internal control, the phRL-null vector was used to correct for transfection efficiency. Luciferase activity was measured 48 h after transfection and firefly luciferase activity was normalized to Renilla luciferase. C, PC-3 cells were treated with or without VES in the presence or absence of SB203580. Forty-eight hours after VES treatment, cells were treated with 5 μg/mL actinomycin D for the times indicated and NAG-1 mRNA levels adjusted to actin were measured by real-time reverse transcription-PCR analysis. The level of NAG-1 mRNA was expressed as the percentage of the NAG-1 mRNA at the 0 h of actinomycin D treatment in each group.
- Figure 7.
NAG-1 siRNA-expressing PC-3 cells are resistant to VES-induced apoptosis. Empty vector or NAG-1 siRNA-expressing PC-3 cells were treated with 20 μmol/L VES for 48 h. Total lysates were subjected to Western blot analysis for PARP and NAG-1. For Western analysis, 39-kDa form of NAG-1 protein (proform) was shown. The membrane was stripped and reprobed for β-actin to confirm equal loading.
Volume 7, Issue 4
