Fig. 6.
Fig. 6. Modulation of CD11c promoter activity in hairy and nonhairy cells. / (A) Effect of the inhibition of AP-1 and Ras on CD11cpromoter activity in Mo hairy cells. The expression construct p11Wt containing nucleotides − 128 to + 36 of the CD11c gene promoter was transfected in parallel with the empty vector pATLuc into untreated Mo hairy cells mixed with pCMV, pTAM67, or pRasN17. The expression constructs pTAM67 and pRasN17 were generated by insertion into pCMV of, respectively, TAM67, encoding a dominant-negative mutant of c-Jun,34 orRasN17,36 encoding a dominant-negative mutant of Ras. After correction for transfection efficiency, the level ofluciferase reporter gene activity directed by p11Wt above that conferred by pATLuc in the presence of pCMV was assigned a value of 100% (UT). The level of activity directed by p11Wt in parallel transfections in the presence of pTAM67 (TAM67) or pRasN17 (RasN17) is expressed as a percentage of this value. Each bar represents the mean ± the standard deviation of 3 independent experiments. Using the Student t test, the probability values for the reduction in CD11c promoter activity caused by pTAM67 and pRasN17 were calculated as P = .001 andP = .012, respectively. (B) Effect of exogenous expression of JunD and RasV12 on CD11c promoter activity in Jurkat nonhairy cells. The expression construct p11Wt was transfected into the cell line Jurkat in the presence of pRSV, RSV-hjD, pCMV, or pRasV12. The expression construct RSV-hjD contains the coding region of humanjunD inserted downstream of the Rous Sarcoma virus promoter.37 The plasmid pRSV was generated by removal of the junD coding region from RSV-hjD. The expression construct pRasV12 was generated by insertion ofRasV12,38 encoding a dominant-positive mutant of Ras, into pCMV. Transfected cells were left untreated for 16 hours prior to harvesting. The level of luciferase reporter gene activity directed by p11Wt above that conferred by pATLuc in the presence of pRSV and after correction for transfection efficiency is assigned a value of 100% (UT). The level of activity directed by p11Wt in parallel transfections in the presence of RSV-hjD is expressed as a percentage of this value (JunD). Similarly, the level ofluciferase reporter gene activity directed by p11Wt above that conferred by pATLuc in the presence of pCMV is assigned a value of 100% (UT) and the activity directed by p11Wt in parallel transfections in the presence of pRasV12 expressed relative to this value (RasV12). Each bar represents the mean ± the standard deviation of 3 independent experiments.

Modulation of CD11c promoter activity in hairy and nonhairy cells.

(A) Effect of the inhibition of AP-1 and Ras on CD11cpromoter activity in Mo hairy cells. The expression construct p11Wt containing nucleotides − 128 to + 36 of the CD11c gene promoter was transfected in parallel with the empty vector pATLuc into untreated Mo hairy cells mixed with pCMV, pTAM67, or pRasN17. The expression constructs pTAM67 and pRasN17 were generated by insertion into pCMV of, respectively, TAM67, encoding a dominant-negative mutant of c-Jun,34 orRasN17,36 encoding a dominant-negative mutant of Ras. After correction for transfection efficiency, the level ofluciferase reporter gene activity directed by p11Wt above that conferred by pATLuc in the presence of pCMV was assigned a value of 100% (UT). The level of activity directed by p11Wt in parallel transfections in the presence of pTAM67 (TAM67) or pRasN17 (RasN17) is expressed as a percentage of this value. Each bar represents the mean ± the standard deviation of 3 independent experiments. Using the Student t test, the probability values for the reduction in CD11c promoter activity caused by pTAM67 and pRasN17 were calculated as P = .001 andP = .012, respectively. (B) Effect of exogenous expression of JunD and RasV12 on CD11c promoter activity in Jurkat nonhairy cells. The expression construct p11Wt was transfected into the cell line Jurkat in the presence of pRSV, RSV-hjD, pCMV, or pRasV12. The expression construct RSV-hjD contains the coding region of humanjunD inserted downstream of the Rous Sarcoma virus promoter.37 The plasmid pRSV was generated by removal of the junD coding region from RSV-hjD. The expression construct pRasV12 was generated by insertion ofRasV12,38 encoding a dominant-positive mutant of Ras, into pCMV. Transfected cells were left untreated for 16 hours prior to harvesting. The level of luciferase reporter gene activity directed by p11Wt above that conferred by pATLuc in the presence of pRSV and after correction for transfection efficiency is assigned a value of 100% (UT). The level of activity directed by p11Wt in parallel transfections in the presence of RSV-hjD is expressed as a percentage of this value (JunD). Similarly, the level ofluciferase reporter gene activity directed by p11Wt above that conferred by pATLuc in the presence of pCMV is assigned a value of 100% (UT) and the activity directed by p11Wt in parallel transfections in the presence of pRasV12 expressed relative to this value (RasV12). Each bar represents the mean ± the standard deviation of 3 independent experiments.

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