Reduction of SAMHD1 phosphorylation in SCD PBMCs and hemin-treated THP-1 cells. (A) Reduced SAMHD1 Thr-529 phosphorylation in SCD PBMCs. PBMCs obtained from 4 SCD patients and 4 controls were activated with PHA and IL-2 and treated with 75 μM hemin for 24 hours. Cell lysates were resolved on 4% to 12% Bis-Tris gel and probed with antibodies against SAMHD1 and Thr-592 phosphorylated SAMHD1 (SAMHD1-(P)) and β-actin as loading control. Lower panels show quantification for 4 individual samples. Shown are SAMHD1 normalized to β-actin (upper panel), SAMHD1-(P) normalized to β-actin (middle panel), and SAMHD1-(P) normalized to SAMHD1 expression (bottom panel). The means ± SD and P values calculated using Student t test are shown. (B) Increased SAMHD1 expression and reduced Thr-592 phosphorylation in hemin-treated THP-1 cells. THP-1 cells were treated with 75 μM hemin for 24 hours. Cell lysates were analyzed and quantified as in panel A for 4 independent samples. (C) Restoration of hemin-mediated HIV-1 replication by knockdowns of SAMHD1. THP-1 cells were stably transduced with lentiviruses expressing SAMHD1-targeting shRNA and infected with HIV-1-LUC-G virus. Where indicated, the cells were treated with 75 μM hemin. Luciferase activity was measured 2 days PI. The mRNAs were measured by RT-PCR with 18S rRNA as an internal reference for ΔΔCt analysis. The means ± SD and P values calculated using Student t test are shown for 3 independent samples. (D) Hemin treatment increased SAMHD1 mRNA expression. THP-1 cells were treated with hemin for 24 hours. RNA was extracted, reverse transcribed, and analyzed by RT-PCR using primers for SAMHD1 and 18S rRNA as an internal reference for ΔΔCt analysis. The means ± SD and P values calculated using Student t test are shown for 3 independent samples.