Fig. 3: LEDGIN CX014442 inhibits HIV-1 integration and hampers HIV-1 transcription and reactivation in SupT1 cells.

a SupT1 cells were transduced with HIV-1 FLuc virus and treated with CX014442 for 3 days. The IC₅₀, 4.05 ± 0.44 μM, was determined using a four-parameter logistic regression of a dose-response curve of the relative luciferase reporter expression (%). Mean ± SD of biological duplicates of 1 independent experiments out of 2 are presented (n = 2). b SupT1 cells were transduced with HIV-1 FLuc virus and treated with CX014442. On day 3, cells were washed and assessed for toxicity. On day 10, cells were reactivated with TNF-α (10 ng/mL) and treated with JQ1/ZL0580. After 24 h, luciferase assays and bDNA imaging were performed. c Cell viability was analyzed via propidium iodide staining on day 3, normalized to non-infected untreated controls, and compared to HIV- (NC) via a One-Way ANOVA test (two-sided test; Dunnett’s multiple comparison test; ns, non-significant). Each dot represents one biological duplicate. Mean ± SD of 2 biological duplicates of 2 independent experiments out of 2 are presented (n = 4). d Luciferase counts (normalized to protein content; BCA assay) are presented for cells harvested on day 11. Each dot represents one biological duplicate. Mean ± SD of 2 biological duplicates of 1 independent experiments out of 2 are presented (n = 2). Statistics were not performed due to a limited number of data points. e, f The vDNA spots per cell (e) and the vRNA spots per infected cell (f) were determined with bDNA imaging. vDNA spots of non-reactivated and reactivated cells and cells treated with different concentrations of JQ1 were pooled if treated with the same concentration of CX014442. VRNA spots were not pooled. Each dot represents a single cell, with a dark blue bar indicating the median. Table S5 shows the number of cells imaged per condition. For vDNA detection, 900 cells were imaged in each condition (n = 900). For vRNA detection, 100 cells were imaged for each condition (n = 100). Except for HIV-, 37 cells were imaged for vDNA and vRNA detection. Results from 1 representative experiment out of 2 are shown. A Kruskal-Wallis test (two-sided test; Dunnett’s multiple comparison test) compared the vDNA/ vRNA spots of the CX014442-treated cells to those of the control (ns non-significant; * p < 0.05; ** p < 0.01; *** p < 0.0001; **** p < 0.0001). Source data are provided in the source data file. HIV-, non-transduced negative control; IC₅₀, 50% inhibitory concentration; NC, negative control; ns, non-significant; TNF-α, tumor necrosis factor α; vDNA, viral DNA; vRNA, viral RNA.