Figure 2: Effects of metformin on HIV-1 transcription in memory CD4⁺ T-cells of ART-treated PWH.

(A) Shown is the experimental flow chart. Briefly, memory CD4⁺ T-cells from ART-treated PWH were stimulated by CD3/CD28 Abs in presence of ARVs (Saquinavir 5 μM, Raltegravir 200 nM) and in the presence or the absence of metformin (1 mM) or INK128 (50 nM) for 3 days. Cells were collected for dual extraction of cell-associated (CA) RNA and DNA. (B) Levels of integrated HIV-DNA Alu/LTR primers were quantified by nested real-time PCR and normalized per number of CD3 copies. (C) Levels of CA unspliced (US) HIV-RNA (Gag primers) were quantified by nested real-time RT-PCR and normalized to the number of HIV-DNA copies per 10⁶ cells. (D) The HIV RNA/DNA ratio was used as a surrogate marker of HIV-1 transcription. Each symbol represents one donor (n=7; median ± interquartile range). Friedman p-values are indicated on the graphs. Uncorrected Dunn’s multiple comparison p-values did not reach statistical significance and are not shown.