Fig. 1

miR-132 induction following Tat expression and HIV-1 infection. a U373.MG, SH-SY5Y, and U138.MG were transfected with Tat.Myc plasmid (Tat) or the cloning vehicle pcDNA3 (C3) and cultured for 48 h and harvested for RNA extraction, followed by qRT-PCR for miR-132 level (upper panels), or cell lysates, followed by Western blotting for Tat expression using an anti-C-MYC antibody (lower panels). β-actin was included as a loading control for Western blotting. b Primary astrocytes were isolated from wild-type (WT) mice and doxycycline (Dox)-inducible and astrocyte-specific HIV Tat-transgenic mice (iTat), cultured in the presence of 5 mg/ml Dox (+) or in the absence of Dox (−) for 48 h and harvested for RNA isolation, followed by qRT-PCR for miR-132 level (upper panel) or semi-quantitative RT-PCR for Tat (lower panel). GAPDH was included as a loading control for Tat RT-PCR. c U373.MG and primary human astrocytes (PHA) were infected with VSV-G-pseudotyped HIV (+), cultured for 3 days and harvested for RNA extraction, followed by qRT-PCR for miR-132 level or cell lysates, followed by Western blotting for p24 expression using an anti-p24 antibody. Pseudotyped HIV containing no envelope (−) was included as an infection control (mock). snRNA U6 level was also determined by qRT-PCR. miR-132 level was normalized to snRNA U6 level and expressed as fold changes compared to the control. The data were mean ± SD of triplicates and/or representative of three independent experiments. The data were analyzed using one-tailed Student’s t test, and the comparisons were made between the C3 control and Tat