Figure 7.
Figure 7. Effects of siRNA knockdown of endogenous JAZ expression. Several 21-nt JAZ-siRNAs that targeted different regions of the JAZ sequence and a control siRNA that is based on a JAZ-irrelevant 21-nt sequence in pCDNA3's ampicillin ORF were synthesized. The JAZ-siRNA that targets the central region of JAZ mRNA (5′-AAGAGGCTAGACTCAGATCAG-3′) was selected, since it could effectively reduce expression of JAZ-GFP (but not GFP-only) in NIH3T3 cells (by ∼ 70%, data not shown). (A) siRNA-mediated knockdown of endogenous JAZ expression inhibits p53 transcriptional activity in p53+/+ but not p53–/– MEFs. The JAZ-siRNA (or the control siRNA) was transiently transfected into p53+/+ and p53–/– MEFs. After 48 hours, cells were lysed followed by Western blot analysis using antibodies against JAZ (JAZ111), p53, p21, and tubulin. It should be noted that in order to accurately evaluate the effectiveness of JAZ-siRNA (compared with the control siRNA), a longer exposure of the film (∼ 3 hours) following the enhanced chemiluminescence (ECL) reaction was performed to facilitate detection of the markedly “decreased” endogenous JAZ level in JAZ-siRNA–transfected cells. Endogenous JAZ expression was knocked down by approximately 70%, as determined by densitometry. The JAZ-siRNA was also cotransfected into the MEFs with p53-TA (containing the wt p53 response elements)–luciferase reporter vector and a carrier vector pEGFPN1 (GFP). After 48 hours, cells were lysed and assayed for luciferase activity. p53 transcriptional activity (luciferase reporter) is displayed relative to the control siRNA–expressing p53+/+ MEFs. Con indicates control siRNA. In addition, the control experiment indicates that the JAZ-siRNA had no effect on the activity of the pTA-luciferase control vector (data not shown). (B) The JAZ-siRNA or the control siRNA expression plasmid was transiently cotransfected into p53+/+ and p53–/– MEFs along with a carrier vector pEGFPN1. After 72 hours, cells were harvested for flow cytometry analysis as described in “Materials and methods.” The GFP-positive cells were gated and analyzed for their cell-cycle distribution. (C) NFS/N1.H7 or M1 hematopoietic cells (as described in Figure 1) were stably transfected with the JAZ-siRNA (or the control siRNA) expression plasmid. After IL-3 and/or serum withdrawal for various periods of time, cells were harvested for the viability measurement by trypan blue exclusion method. siR-C indicates the control siRNA; siR-J, the JAZ-siRNA. Bottom panel shows Western blot analysis of siRNA knockdown of endogenous JAZ in H7 or M1 cells. IL-3/serum was present (+) or removed (–) for 2 hours. Error bars represent standard deviations (n = 3).

Effects of siRNA knockdown of endogenous JAZ expression. Several 21-nt JAZ-siRNAs that targeted different regions of the JAZ sequence and a control siRNA that is based on a JAZ-irrelevant 21-nt sequence in pCDNA3's ampicillin ORF were synthesized. The JAZ-siRNA that targets the central region of JAZ mRNA (5′-AAGAGGCTAGACTCAGATCAG-3′) was selected, since it could effectively reduce expression of JAZ-GFP (but not GFP-only) in NIH3T3 cells (by ∼ 70%, data not shown). (A) siRNA-mediated knockdown of endogenous JAZ expression inhibits p53 transcriptional activity in p53+/+ but not p53–/– MEFs. The JAZ-siRNA (or the control siRNA) was transiently transfected into p53+/+ and p53–/– MEFs. After 48 hours, cells were lysed followed by Western blot analysis using antibodies against JAZ (JAZ111), p53, p21, and tubulin. It should be noted that in order to accurately evaluate the effectiveness of JAZ-siRNA (compared with the control siRNA), a longer exposure of the film (∼ 3 hours) following the enhanced chemiluminescence (ECL) reaction was performed to facilitate detection of the markedly “decreased” endogenous JAZ level in JAZ-siRNA–transfected cells. Endogenous JAZ expression was knocked down by approximately 70%, as determined by densitometry. The JAZ-siRNA was also cotransfected into the MEFs with p53-TA (containing the wt p53 response elements)–luciferase reporter vector and a carrier vector pEGFPN1 (GFP). After 48 hours, cells were lysed and assayed for luciferase activity. p53 transcriptional activity (luciferase reporter) is displayed relative to the control siRNA–expressing p53+/+ MEFs. Con indicates control siRNA. In addition, the control experiment indicates that the JAZ-siRNA had no effect on the activity of the pTA-luciferase control vector (data not shown). (B) The JAZ-siRNA or the control siRNA expression plasmid was transiently cotransfected into p53+/+ and p53–/– MEFs along with a carrier vector pEGFPN1. After 72 hours, cells were harvested for flow cytometry analysis as described in “Materials and methods.” The GFP-positive cells were gated and analyzed for their cell-cycle distribution. (C) NFS/N1.H7 or M1 hematopoietic cells (as described in Figure 1) were stably transfected with the JAZ-siRNA (or the control siRNA) expression plasmid. After IL-3 and/or serum withdrawal for various periods of time, cells were harvested for the viability measurement by trypan blue exclusion method. siR-C indicates the control siRNA; siR-J, the JAZ-siRNA. Bottom panel shows Western blot analysis of siRNA knockdown of endogenous JAZ in H7 or M1 cells. IL-3/serum was present (+) or removed (–) for 2 hours. Error bars represent standard deviations (n = 3).

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