To test for STP0404’s inhibitory effects during the late stage of HIV-1 life cycle such as the maturation step, we employed the “producing cells” setup. In T75 tissue culture flasks, 293T cells were pre-treated with and without STP0404 (0 and 60 nM) for 2 hrs prior to transfection with pD3-HIV (13 μg) and pVSV-g (6 μg), using 0.3 mg/ml polyethylenimine. After 24 hrs incubation at 37°C, the supernatant was removed and replaced with fresh media with and without STP0404 (0 and 60 nM). 48 hrs later, D3-HIV vector from each treatment group was concentrated from the collected supernatant via ultracentrifugation (22,000 rpm for 2 hrs). Following p24 quantification, using 96-well plates, similar amount of respective D3-HIV vector was used to transduce Jurkat cells in triplicates for 48 hrs. The cells were harvested and fixed with 3.7% formaldehyde before being analyzed for GFP expression using FACS (Miltenyi Biotec, VYB). Separately, the “infecting cells” setup was used to investigate the inhibitory effects of STP0404 against the early stages of HIV-1 life cycle such as the integration and transcription steps. D3-HIV vector collected from transfected, non-treated 293T cells were used to transduce LEDGF/p75 +/- Jurkat cells which have been subjected to 2 hrs pre-treatment with and without STP0404 (0 and 60 nM). After 48 hrs, the cells were collected and fixed for FACS analyses as described above. All data were analyzed using GraphPad Prism (Version 9). Unpaired t tests were performed to determine the significance of the readings in respective experimental set up relative to the untreated controls. The data were presented as means ± S.D. of triplicates, whereby p-value < 0.05 is represented as *; p-value < 0.001 is represented as ***; ns indicates not significant.

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