Fig. 1. Structural formula of JPL-32.

Fig. 2. Inhibitory effect of JPL-32 with or without NAC supplementation on TNF-α-induced expression of HIV-1 in OM-10.1 and U1 cells. (A) The latently HIV-infected OM-10.1 and U1 cells were incubated with various concentrations of JPL-32 in the presence of 0, 1 and 4 mM NAC for 2 h, stimulated with 1 ng/ml TNF-α and further incubated for 48 h. Supernatants were then collected for p24 antigen quantification and the p24 levels are expressed as the percent of control (no compound). Cell cultures were examined for cell viability using the MTT method (♦). All experiments were carried out in quadruplicate, and results are presented as mean values with standard deviations. (B) Time-of-addition experiment with 4 mM NAC in OM-10.1 and U1 cell cultures treated with, respectively, 2.5 and 1.25 μg/ml JPL-32 for antiviral activity determination (■) and, respectively, 20 and 10 μg/ml JPL-32 for cytotoxicity assessment (♦). At t = 0, TNF-α-induced expression of HIV-1 was initiated in both cell lines in the presence of JPL-32. Following TNF-α stimulation, NAC was added at different points in time (0–20 h) and further incubated till 48 h post-stimulation. After this 2 day incubation period, p24 levels were assessed in the supernatants and expressed as the percent of control (■) and cell viability was examined using the MTT method (♦).

Fig. 3. Inhibitory effect of JPL-32 with or without NAC supplementation on GFP transactivation. Constructs using the GFP reporter gene transcribed from the HIV-1 LTR (black bars) and the HCMV IE (white bars) promoter were used to investigate the effect of JPL-32 on the transactivation process. HL_tat and Jurkat_tat cells were transfected with the GFP-expressing constructs and incubated with different concentrations of JPL-32 in the presence or absence of 1 mM NAC for 24 h. Quantification of GFP production was measured with the Fluorocount and expressed as the percent of control. Cell viability was determined in the same cell cultures by the MTT method (♦). All experiments were carried out in quadruplicate, and results are presented as mean values with standard deviation.

Fig. 4. Inhibition of TNF-α-induced DNA binding of nuclear NF-κB by JPL-32 with or without NAC supplementation. OM-10.1 and U1 cells were cultured with various concentrations of JPL-32 in the presence and absence of 4 mM NAC for 1 h. Following a 30 min TNF-α stimulation, nuclear extracts were prepared and analyzed for NF-κB binding by EMSA.

Fig. 5. Effect of JPL-32 on TNF-α-induced release and subsequent degradation of IκBα from NF-κB. OM-10.1 and U1 cells were treated with TNF-α as such or in the presence of JPL-32 with or without NAC for the indicated time periods (in min). The cytoplasmic lysates were prepared and fractionated on 4–12% Novex Bis-Tris gels and analyzed for the presence of IκBα.

Fig. 6. Effect of JPL-32 on TNF-α-induced nuclear translocation of NF-κB. (A) immunofluorescence analysis. HL_tat, COS-7 and OM-10.1 cells were treated with TNF-α for 30 min in the presence or absence of JPL-32. Then, the cells were fixed, permeabilized, and examined by fluorescence microscopy. (B) Western blot analysis of nuclear NF-κB in OM-10.1 cells. Cells were cultured with various concentrations of JPL-32 and stimulated with TNF-α for 30 min. The nuclear fractions were fractionated on 4–12% Novex Bis-Tris gels and analyzed for NF-κB.

Fig. 7. Oxidoreductive modulation of the DNA binding activity of nuclear NF-κB. OM-10.1 cells were cultured for 1 h with 8 μg/ml JPL-32. Following a 30 min. TNF-α stimulation, nuclear extracts were treated with 10 mM DTT or 1% β-mercaptoethanol (β-ME) for 1 h and analyzed for NF-κB binding by EMSA.

Fig. 8. Effect of JPL-32 on the direct DNA binding of NF-κB. Nuclear extracts from TNF-α-stimulated OM-10.1 cells (A) and recombinant NF-κB p50 (B) were treated with various concentrations of JPL-32 in the presence or absence of 1% β-mercaptoethanol (β-ME) and analyzed for binding to the ³²P-labeled NF-κB oligonucleotide in the EMSA assay. In order to exclude the effect of the solvent DMSO, which is present in the JPL-32 stock solution, the first three lanes of (B) contains equal amounts of DMSO compared to the following three lanes, containing JPL-32, and the last three lanes, that contain JPL-32 and 1% β-ME.

Fig. 9. Determination of intracellular oxidative stress in OM-10.1 cells. Cells were treated with 10 μM DCFH-DA for 15 min. Then, various concentrations of JPL-32 were added to the cell suspension for another 15 min followed by the absence (dark grey bars) or presence of 50 μM H₂O₂ (light grey bars) for a final 15 min. After this 45 min incubation period, cells were washed in PBS and seeded in a 96-well plate at a density of 2 × 10⁵ cells/well. DCF fluorescence was imaged by fluorescence microscopy (A) and measured with the Fluorocount (B).

Fig. 10. Induction of apoptosis by JPL-32. (A) Jurkat_tat cells were treated with 10 μg/ml JPL-32 for 4 h. Then, the cells were fixed, permeabilized, stained with DAPI and examined by fluorescence microscopy. (B) Jurkat_tat cells were cultured for 15 h with various concentrations of JPL-32 in the presence and absence of 40 ng/ml FasL. Following incubation, cells were incubated for 10 min. with Annexin-V-Fluos reagent and analyzed with a flow cytometer. (C) Jurkat_tat cells were treated with various concentrations of JPL-32 in the presence () or absence (■) of 40 ng/ml FasL for 5 h and assayed for intracellular active caspase-3 by ELISA.

Fig. 11. Induction of intracellular GSH levels by JPL-32 in OM-10.1 and U1 cells. (A) OM-10.1 (■) and U1 (♦) cells were cultured in the presence of respectively, 2.5 and 1.25 μg/ml JPL-32. After different periods, samples were taken and analyzed for intracellular GSH content by two different methods. (B) OM-10.1 (■) and U1 (♦) cells were treated with various concentrations of JPL-32 for 4 h. Following incubation, samples were subjected for intracellular GSH determination by the two different methods. (C) OM-10.1 (■ and □) and U1 (♦ and ) cells were cultured with respectively, 2.5 and 1.25 μg/ml JPL-32 in the presence (not filled) and absence (filled) of 200 μM buthionine sulfoximine (BSO). After an incubation period of 24 h, samples were taken and analyzed for intracellular GSH content by the Bioxytech GSH-400 method.

Fig. 12. Redox regulation of NF-κB activation. Activation and nuclear translocation of NF-κB has been reported to occur under oxidative conditions and are stimulated by JPL-32, whereas the binding of NF-κB to the DNA promoter sequences has been shown to be dependent on reducing conditions which are blocked by JPL-32.