Figure 1
Figure 1. Tat-mediated gene modulation in KG-iDC and THP-Mac and its association with cellular promoters. (A) mRNA levels of Tat in KG-1 cells infected with Ad-TatSF2 24 hours after infection at multiplicities of infection (MOI) of 0.1, 1, and 10, analyzed by qRT-PCR. Results are normalized to actin and reported as fold induction relative to Ad-Tat samples infected at MOI of 0.1. The means ± standard error of the mean (SEM) derived from 3 independent experiments are reported. (B) mRNA levels of selected ISGs 24 hours after infection with adenoviral vectors Ad-TatSF2 and negative control Ad-TatSF2G48R57A. Results are reported as fold induction relative to Ad-TatSF2G48R57A samples. (C) Tat association with the MAP2K6 promoter in KG-iDC, detected by ChIP and qPCR. DNA from input (90, 30, and 10 ng of DNA) and immunoprecipitated samples (10 ng of DNA) was amplified by standard PCR using the P1, P2, and P3 set of primers. DNA from cells infected with Ad-TatSF2G48-R57A and Ad-tTA was used as a negative control. One representative experiment is shown. (D) Average fold enrichments of the indicated promoters in the immunoprecipitated DNA relative to input DNA ± SEM from 3 independent experiments are reported. Cycle threshold (Ct) values obtained with 10 ng of immunoprecipitated DNA were compared with the Ct values obtained with 10 ng of the corresponding input DNA. (E) MAP2K6 and Tat mRNA levels in KG-1 cells infected with Ad-TatSF2 and Ad-TatSF2G48-R57A, analyzed by RT-qPCR. Results are normalized to actin and reported as fold induction relative to samples infected with Ad-tTA alone. The means ± SEM of 3 experiments are shown. (F) Luciferase activity in lysates from cells expressing wild-type TatSF2 or TatSF2 G48R57A and transfected with an IRF7- or a MAP2K6-luciferase reporter vector. Firefly luciferase activity was normalized to Renilla luciferase activity. Results are reported as fold induction relative to those in the cells infected with Ad- tTA. (G) mRNA levels of selected ISGs 48 hours after infection with Ad-TatSF2 and Ad-TatSF2G48R57A. Results are reported as fold induction relative to Ad-TatSF2G48R57A samples. (H) Tat association with the IRF7, MAP2K3, and MAP2K6 promoters in THP-Mac expressing TatSF2. DNA from input (90, 30, and 10 ng of DNA) and immunoprecipitated samples (10 ng of DNA) was amplified by qPCR using the P2 and P3 set of primers. DNA from cells infected with Ad-TatSF2G48-R57A and Ad-tTA was used as a negative control. One representative experiment is shown. (I) Average fold enrichments of IRF7, MAP2K3, and MAP2K6 promoters using ChIP-qPCR in THP-Mac expressing TatSF2 relative to input DNA + SEM from 3 independent experiments. (J) Luciferase activity of lysates from THP-Mac expressing the wild-type TatSF2 or TatSF2 G48R57A and transfected with IRF7-, MAP2K6-, or MAP2K3-luciferase reporter vectors.

Tat-mediated gene modulation in KG-iDC and THP-Mac and its association with cellular promoters. (A) mRNA levels of Tat in KG-1 cells infected with Ad-TatSF2 24 hours after infection at multiplicities of infection (MOI) of 0.1, 1, and 10, analyzed by qRT-PCR. Results are normalized to actin and reported as fold induction relative to Ad-Tat samples infected at MOI of 0.1. The means ± standard error of the mean (SEM) derived from 3 independent experiments are reported. (B) mRNA levels of selected ISGs 24 hours after infection with adenoviral vectors Ad-TatSF2 and negative control Ad-TatSF2G48R57A. Results are reported as fold induction relative to Ad-TatSF2G48R57A samples. (C) Tat association with the MAP2K6 promoter in KG-iDC, detected by ChIP and qPCR. DNA from input (90, 30, and 10 ng of DNA) and immunoprecipitated samples (10 ng of DNA) was amplified by standard PCR using the P1, P2, and P3 set of primers. DNA from cells infected with Ad-TatSF2G48-R57A and Ad-tTA was used as a negative control. One representative experiment is shown. (D) Average fold enrichments of the indicated promoters in the immunoprecipitated DNA relative to input DNA ± SEM from 3 independent experiments are reported. Cycle threshold (Ct) values obtained with 10 ng of immunoprecipitated DNA were compared with the Ct values obtained with 10 ng of the corresponding input DNA. (E) MAP2K6 and Tat mRNA levels in KG-1 cells infected with Ad-TatSF2 and Ad-TatSF2G48-R57A, analyzed by RT-qPCR. Results are normalized to actin and reported as fold induction relative to samples infected with Ad-tTA alone. The means ± SEM of 3 experiments are shown. (F) Luciferase activity in lysates from cells expressing wild-type TatSF2 or TatSF2 G48R57A and transfected with an IRF7- or a MAP2K6-luciferase reporter vector. Firefly luciferase activity was normalized to Renilla luciferase activity. Results are reported as fold induction relative to those in the cells infected with Ad- tTA. (G) mRNA levels of selected ISGs 48 hours after infection with Ad-TatSF2 and Ad-TatSF2G48R57A. Results are reported as fold induction relative to Ad-TatSF2G48R57A samples. (H) Tat association with the IRF7, MAP2K3, and MAP2K6 promoters in THP-Mac expressing TatSF2. DNA from input (90, 30, and 10 ng of DNA) and immunoprecipitated samples (10 ng of DNA) was amplified by qPCR using the P2 and P3 set of primers. DNA from cells infected with Ad-TatSF2G48-R57A and Ad-tTA was used as a negative control. One representative experiment is shown. (I) Average fold enrichments of IRF7, MAP2K3, and MAP2K6 promoters using ChIP-qPCR in THP-Mac expressing TatSF2 relative to input DNA + SEM from 3 independent experiments. (J) Luciferase activity of lysates from THP-Mac expressing the wild-type TatSF2 or TatSF2 G48R57A and transfected with IRF7-, MAP2K6-, or MAP2K3-luciferase reporter vectors.

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