Fig. (2).

Tat ameliorates the inhibitory effect of Per-1 on HIV-1 transcription. (A) 293T cells were cotransfected with a construct containing FLAG-tagged Per-1-001, Per-1-002, or Mock along with HIV-1 LTR (LTR-Luc) or CMV (CMV-Luc) promoter-driven luciferase reporter constructs. After 48 h of cotransfection, the cells were lysed to measure the luciferase reporter levels. Western blotting was conducted to detect FLAG-tagged Per-1 isoform or GAPDH expression using anti-FLAG or anti-GAPDH antibodies. (B) 293T cells were cotransfected with a construct of FLAG-tagged short isoform Per-1-002 or Mock and with or without Tat in the form of a pNL4.3.Luc construct. After 48 h of cotransfection, the cells were lysed to measure the luciferase reporter levels. Western blotting was conducted to detect FLAG-tagged Per-1-002 or GAPDH expression using anti-FLAG or anti-GAPDH antibodies. (C) 293T cells were cotransfected with a construct of FLAG-tagged short isoform Per-1-002 or Mock and an LTR-Luc construct in the presence or absence of an HA-Tat expression vector. After 48 h of cotransfection, the cells were lysed to measure the luciferase reporter levels. The inhibition of viral promoter LTR-mediated luciferase expression was accordingly calculated between Per-1-002 and Mock. Western blotting was conducted to detect FLAG-tagged short isoform Per-1-002, HA-Tat, or GAPDH expression using anti-FLAG, anti-HA, or anti-GAPDH antibodies (D). (E) Total RNA was extracted from resting and stimulated CD4+ T-cells, monocytes, MDMs, MDDCs, 239T, Jurkat, HeLa, and THP-1 as well as differentiated THP-1 cells and used for qPCR to assess Per-1 transcripts normalized to GAPDH. RLU, relative luminescence units. **P < 0.01 (t-test). NS, not significant. Data are presented as mean ± S.E.M. from three independent experiments.