Fig. 2
SULT1A1 knockdown is associated with decreased viral gene expression following infection of MDMs with VSV-G pseudotyped HIV-1 and SIV vectors. a Schematic showing experimental timeline. Briefly, CD14+ monocytes were isolated from human donor PBMCs using positive selection with magnetic beads. Monocytes were differentiated into MDMs and on day 7 were electroporated with siRNA and plated at 1.5 × 104 MDMs per well in a 48 well plate. After 96 h, protein knockdown was confirmed by immunoblot and cells were infected with 100 μl (corresponding to 164 ng p24 HIV-1 and 234 ng p27 SIV viral vectors) of the indicated virus. Luciferase and cell viability measurements were determined at 24 h post-infection. b Representative immunoblot showing SULT1A1 knockdown 96 h post transfection with SULT1A1 siRNAs (1–3) and AllStars Negative siRNA Control (ASN), Qiagen. SULT1A1 expression is compared to endogenous Ku86 used as a loading control. Results from one representative donor are shown. SULT1A1 expression was generally decreased by 70–80 % with siRNA treatment compared to the control ASN siRNA (as shown in Fig. 2c, middle panel). c HIV-1 luciferase reporter expression (left panel), SULT1A1 protein expression (middle panel), and cell viability of MDMs (right panel) were measured 24 h after infection with the VSV-G pseudotyped NL43-Luc HIV-1 vector. Results shown are from 6 donors assayed twice. All values were compared to ASN control. d SIV-1 luciferase reporter expression (left panel), SULT1A1 protein expression (middle panel), and cell viability (right panel) for MDMs 24 h post infection with VSV-G-pseudotyped SIVagm-Luc. Mean and SD shown, *** p < 0.0005 ** p < 0.005 * p < 0.05 one sample t test. Results shown are from 6 donors assayed twice. Samples with <60 % SULT1A1 knockdown and/or <65 % cell viability were not used in the analysis. All values were compared to ASN control