Figure 5.
Figure 5. Effects of JAZ on p53 transcriptional activity. (A) JAZ-GFP/pEGFPN1 or pEGFPN1 (GFP-only) was cotransfected with a p53-luciferase reporter vector (p53-TA-luc) into COS-7, CV-1, and NIH3T3 cells. After 24 or 48 hours, JAZ-GFP– or GFP-positive COS-7, CV-1, and NIH3T3 cells were sorted by fluorescence-activated cell sorting (FACS). The same numbers of such sorted GFP-only and JAZ-GFP–positive cells were lysed and then assayed for luciferase activity. G indicates GFP; J-G, JAZ-GFP. Luciferase activity is displayed relative to that of the GFP control. (B) FLAG-JAZ/pcDNA3 (empty vector pcDNA3 as a control) was cotransfected with PG13 or MG15 luciferase reporter vector into p53-null SAOS-2 cells, in the presence or absence of p53wt/pCMV. pAc-β-GAL internal control vector was also included to normalize transfection efficiencies. After 24 hours, cells were lysed and assayed for luciferase activity; V indicates pcDNA3; F-J, FLAG-JAZ/pcDNA3; and p53, p53wt/pCMV; PG13 and MG15 luciferase vectors contain the wild-type and the mutant p53 DNA-binding site, respectively.51 Luciferase activity is displayed relative to that of the pcDNA3 vector. (C) JAZ-GFP/pEGFPN1 was cotransfected with a p21-promoter luciferase vector (p21-P-luc) into p53+/+ and p53–/– MEFs followed by FACS and luciferase assays. (D) FLAG-JAZ/pcDNA3 was cotransfected with p53wt/pCMV or p53ΔC(30)/pcDNA3 plus PG13 and pAc-β-GAL vectors into p53–/– MEFs following luciferase assays and normalization. (E) Mutational analysis of JAZ stimulation of p53 transcriptional activity in CV-1 cells. To determine the role of JAZ's ZF domains, JAZ-GFP ZF mutants (in pEGFPN1) containing a single or multiple C2H2→C2AH32 mutations were also used. Wt JAZ-GFP and its ZF mutants were cotransfected with the PG13 vector into CV-1 cells. After 24 hours, cells were sorted by FACS for luciferase activity. The ZF mutations H91A, H152A, H203A, and H257A are in JAZ's first, second, third, and fourth ZF motifs, respectively. NF represents the mutant in which all 4 zinc fingers are point mutated. JAZ-GFP deletion mutants were also used. J(1-171) is a JAZ-GFP deletion mutant containing 1 to 171 amino acids of JAZ, while J(167-294) deletion mutant contains 167 to 294 amino acids. Error bars represent standard deviations (n = 3).

Effects of JAZ on p53 transcriptional activity. (A) JAZ-GFP/pEGFPN1 or pEGFPN1 (GFP-only) was cotransfected with a p53-luciferase reporter vector (p53-TA-luc) into COS-7, CV-1, and NIH3T3 cells. After 24 or 48 hours, JAZ-GFP– or GFP-positive COS-7, CV-1, and NIH3T3 cells were sorted by fluorescence-activated cell sorting (FACS). The same numbers of such sorted GFP-only and JAZ-GFP–positive cells were lysed and then assayed for luciferase activity. G indicates GFP; J-G, JAZ-GFP. Luciferase activity is displayed relative to that of the GFP control. (B) FLAG-JAZ/pcDNA3 (empty vector pcDNA3 as a control) was cotransfected with PG13 or MG15 luciferase reporter vector into p53-null SAOS-2 cells, in the presence or absence of p53wt/pCMV. pAc-β-GAL internal control vector was also included to normalize transfection efficiencies. After 24 hours, cells were lysed and assayed for luciferase activity; V indicates pcDNA3; F-J, FLAG-JAZ/pcDNA3; and p53, p53wt/pCMV; PG13 and MG15 luciferase vectors contain the wild-type and the mutant p53 DNA-binding site, respectively.51  Luciferase activity is displayed relative to that of the pcDNA3 vector. (C) JAZ-GFP/pEGFPN1 was cotransfected with a p21-promoter luciferase vector (p21-P-luc) into p53+/+ and p53–/– MEFs followed by FACS and luciferase assays. (D) FLAG-JAZ/pcDNA3 was cotransfected with p53wt/pCMV or p53ΔC(30)/pcDNA3 plus PG13 and pAc-β-GAL vectors into p53–/– MEFs following luciferase assays and normalization. (E) Mutational analysis of JAZ stimulation of p53 transcriptional activity in CV-1 cells. To determine the role of JAZ's ZF domains, JAZ-GFP ZF mutants (in pEGFPN1) containing a single or multiple C2H2→C2AH32  mutations were also used. Wt JAZ-GFP and its ZF mutants were cotransfected with the PG13 vector into CV-1 cells. After 24 hours, cells were sorted by FACS for luciferase activity. The ZF mutations H91A, H152A, H203A, and H257A are in JAZ's first, second, third, and fourth ZF motifs, respectively. NF represents the mutant in which all 4 zinc fingers are point mutated. JAZ-GFP deletion mutants were also used. J(1-171) is a JAZ-GFP deletion mutant containing 1 to 171 amino acids of JAZ, while J(167-294) deletion mutant contains 167 to 294 amino acids. Error bars represent standard deviations (n = 3).

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