Figure 8
Knockdown of CFL1 reduces total CFL1 and pCFL1 expression in PBMCs and reverts E2-mediated anti-HIV-1BaL activity in PBMCs. (a) PBMCs (106 cells/ml) were transfected with CFL1 siRNA, Trilencer-27 Universal scrambled negative control siRNA duplex and Trilencer-27 HPRT Positive control siRNA duplex using Viromer Green transfection reagent. 48 h post-transfection, PBMCs were split in two groups and either incubated with E2 10,000 pg/ml for additional 48 h or left untreated. 96 h post-transfection, PBMCs were challenged with 1000 TCID50/106 cells HIV-1BaL and cultured for 3 days. WB analysis was done at 48 h, 96 h and 168 h post-transfection (2 experiments). WCEs (120 µg) were prepared at indicated time points and probed for CFL1 and pCFL1. β-actin (120 µg) was used as internal control. Uncropped blots are presented in Supplementary Fig. S8. (b) Transfected PBMCs were incubated with or without E2 10,000 pg/ml for 48 h followed by HIV-1BaL challenge as described in (a) and 14 days culture. E2 was added on days 3, 7 and 11 of culture. Infection was monitored by HIV-1 gag qRT-PCR. Shown are the viral growth kinetics (HIV-1 gag copy numbers/ml) over 14 days of culture in individual experiments (Mean ± SEM of replicates) and Log10-transformed SOFT and CUM analyses (Mean ± SEM, D3-14) of 2 experiments. NC siRNA negative control/Trilencer-27 Universal scrambled negative control siRNA duplex, PC siRNA positive control/trilencer-27 HPRT Positive control siRNA duplex.
