Figure 2: Cocaine-induced HIV transcription augments overall HIV replication.

Structure of the lentiviral vector (pHR’-PNL-Luc) carrying the reporter luciferase gene under the HIV LTR promoter (A). Schematic representation of the cocaine (Coc) treatment for the luciferase reporter assay (B). Jurkat-pHR’-P-Luc cells were chronically treated with 5 µM – 20 µM of cocaine. The cells were lysed, and luciferase reporter protein expression levels were assessed using luciferase assays (C). Schematic depiction of the cocaine treatment and subsequent infection of PBMC cells with replication-competent HIV (D). HIV transcripts were quantified by real-time PCR using primer sets that amplify the Envelope (Env) region of the HIV genome (E). The level of Gag/p24 protein was analyzed by immunoblotting with specific antibodies against HIV p24 (F). Actin, a constitutively expressed protein, was used as a loading control in the same blot. Densitometric analysis of protein bands (normalized to actin) confirmed a significant increase in p24 levels compared to untreated cells (Ctrl) (G). Immunoblots are representative of at least three independent experiments. The results are expressed as mean ± SD, analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (C & E) or unpaired t-test (G). Asterisks over the bars indicate significant differences: ∗p < 0.05 for the comparison of cocaine-treated cells vs. untreated cells.