Figure 2
SAMHD1 silencing promotes HIV replication in astrocytes. (A) Astrocytes (2 × 105 cells/ml) were transfected with 75 pmols SAMHD1-specific siRNA using Lipofectamine 2000. SAMHD1-knockdown was assessed by immunoblotting with SAMHD1-specific antibodies. One representative blot is shown. (B) SAMHD1-siRNA or control siRNA transfected astrocytes were infected by HIV (100 ng p24). Cells were treated with or without 100 nm AZTTP. After 72 h, p24 was measured in supernatants by ELISA. (C) Total RNA was extracted from corresponding cells from ‘B’ and amplified by RT-PCR for HIV-1 LTR/RU-5 gene. (D) SAMHD1- siRNA transfected astrocytes were infected with the HIV-luciferase pseudovirus (20 ng) for 3 days, harvested and lysed. Luciferase concentration in whole cell lysates was determined. (E) Astrocytes were treated with or without raltegravir (10 μM) for 6 h followed by TSA (50 nM) treatment. Cells were infected with the HIV-luciferase pseudovirus (20 ng) for 3 days. Luciferase concentration in whole cell lysates was determined. (F) Astrocytes (1 × 106 cells/ml) were treated with trichostatin A (TSA; 50 nM) for 24 h or left untreated. Following the treatment, SAMHD1 mRNA levels were quantified by real-time PCR analysis. The SAMHD1 mRNA level in cells without TSA was set to 1. P value ≤0.05 were considered to be significant. (G) Cell lysate was prepared from the corresponding cells in (F), and SAMHD1 protein expression was detected in corresponding cell lysates by immunoblotting with SAMHD1-specific antibodies. Equal amounts of total protein (30 μg) from both cells were loaded. GAPDH was used as a loading control. Results shown are representative of mean ± SEM of three independent of experiments (except A and G). P value ≤0.05 were considered to be significant. SAMHD1, sterile alpha motif and histidine/aspartic acid domain-containing protein 1; LTR, long terminal repeat; AZTTP, azidothymidine triphosphate; TSA, trichostatin A; VSV-G, vesicular stomatitis virus; RLU, relative light units; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.