Skip to main content
. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: FEBS J. 2017 Nov 8;284(23):4096–4114. doi: 10.1111/febs.14293

Figure 5. Inhibiting proviral activity in HIV-NanoLuc clone E9.

Figure 5

A gRNA designed to mutate the TLR-4 receptor using wildtype Cas9 were developed and transfected into clone E9. (A) T7E1 assay confirms mutagenesis at the TLR-4 genomic sequence in E9 cell populations. (B) TLR-4 mutated cell populations were stimulated with LPS (100 ng/ml) with untreated E9 cells and E9 cells receiving TH gRNAs were used as a comparison. ***p<0.0001 vs untreated, *p<0.05 vs untreated. Average fold changed listed above graph. #p<0.05 vs fold change of HIV-NanoLuc CHME-5 E9. (C) Cells were treated with TNF-α (50 ng/ml) to show that the LPS effect is specific to TLR-4 mutation. ***p<0.0001 vs untreated. Average fold changes listed above graphs. No significant difference vs. fold change of HIV-NanoLuc CHME-5. (D) Inhibition of proviral activity using NF-κB inhibitor sulfasalazine. Sulfasalazine (0.1mM, 0.5 mM or 1 mM) was added to cells one hour before LPS (100 ng/ml) stimulation. Luciferase was measured after 24 hours. 1mM sulfasalazine prevented LPS mediated proviral activty. p<0.0001 vs untreated, n.s. vs untreated. (E) Cell viability assay shows no significant difference among treatment groups. (F) Photomicrographs of cells density. Data are expressed as the mean ± SEM. N=3 independent cell passages.