Small-Molecule Inhibition of HIV pre-mRNA Splicing as a Novel Antiretroviral Therapy to Overcome Drug Resistance (original) (raw)
Figure 6
Comparison of HIV-1 Inhibition in MT2 Cells Treated with AZT or IDC16 and Analysis of Splicing Profiles of Apoptotic Genes in PBMC-Treated Cells
(A and B) MT2 cells cultured in a 96-well plate were infected with pNL4.3 at 100 TCID50 in the absence or presence of IDC16 (A) or AZT (B) for 18 h. Cells were then washed and changed to fresh medium with/without IDC16 or AZT. Half of the culture medium was refreshed each 2 or 4 d in the presence of drugs. The formation of syncytia was scored at the indicated time points.
(C) Four ug of total RNA from triplicate of PBMCs untreated (lane Ctl) or treated with IDC16 (lane IDC16) or AZT (lane AZT), was reverse transcribed with Omniscript reverse transcriptase (QIAGEN) using random hexamers and oligo dT and. The mixture was aliquoted in a 96-well plate and subjected to PCR amplification using 0.375U/15 μl of hotStarTaq DNA Polymerase with specific primers (0.3–0.6 μM) using the buffer provided by the manufacturer (QIAGEN). The PCR reaction was carried out in a GeneAmp 9700 PCR system. Following an incubation of 15 min at 95 °C, and 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C, the reaction was ended with an extension step of 10 min at 72 °C. PCR products were fractionated on a LabChip HT DNA assay station (Caliper) for quantitation and sizing. The full data can be accessed through http://www.lgfus.ca/Tazi/, username = Tazi, password = sc35. Three examples of genes altered by AZT treatment (HDM2), (BRCA1), and (DATF1) are shown.