Figure 7.

Gesicle Dosage and Viability

(A–D) HIV-NanoLuc CHME-5 microglia were treated with gesicles containing LTR gRNA 4 at varying concentrations (0.01–100 μg protein/mL). (A) Live-cell images 24 hr post-centrifugation indicated CherryPicker Red fluorescence increases as gesicle dose increases. (B) Viability 24 hr post-gesicle treatment was assayed using the ATP viability assay. No significant differences were detected between the different gesicle concentrations. (C) Long-term viability after cellular expansions and culture was measured by cell yield and % viability using trypan blue. No significant differences were observed between treatment conditions. (D) Cells treated with 10 and 100 μg protein/mL concentrations of LTR gRNA 4 or protamine alone. After expansion, cells were plated and stimulated with pro-inflammatory factors LPS (100 ng/mL) and TNF-α (50 ng/mL), which was repeated for 2 additional weeks. A significant decrease in proviral activity was observed using 100 μg/mL at all conditions and time points versus control samples. Cells treated with 10 μg/mL showed significant proviral inhibition after LPS and TNF-α stimulation at weeks 1 and 2, but no significant differences were observed at week 3 versus control samples. Data are the mean + SEM of three experiments (B), or three stable cultures with three experiments each (C and D), analyzed using a one-way ANOVA with post hoc test (B) or analyzed using a two-way ANOVA with post hoc test for each stimulation group (C and D); *p < 0.05 versus untreated cells: unstimulated; ^#p < 0.05 versus untreated cells: LPS stimulated; ^ˆp < 0.05 versus untreated cells: TNF-α stimulated.