Figure 1

Dicer expression in HIV-1 infected and target cell-lines. A) Thirty micrograms of whole cell extract from CD4+ T-cell lines (lane 1 – Hut78, lane 2 – Molt 4, lane 3 – H9, lane 4 – Jurkat, lane 5 – CEM) and HIV-1 infected CD4+ T-cell lines (lane 6 – J1.1, lane 7 – ACH2) was resolved by SDS-PAGE and western blotted for the presence of Dicer and β-actin. Asterisks indicate the position of the 220, 45 and 30 kDa mass standards. Densitometry was performed and the ratio of Dicer to β-actin was determined and is shown below the western blot images. B) Thirty micrograms of whole cell extract from monocytes (lane 1 – THP-1), pro-monocytes (lane 2 – U937), HIV-1 infected pro-monocytes (lane 3 – U1) and HIV-1 infect pro-myelocytes (lane 4 – OM10.1) was resolved by SDS-PAGE and western blotted for the presence of Dicer and β-actin. C) Monocytes and lymphocytes were isolated from three normal donors (designated A, B, C, D and E) as described in materials and methods. Five micrograms of whole cell extract from lymphocytes and 20 micrograms from monocytes was resolved by SDS-PAGE and western blotted for the presence of Dicer. D) Decreasing amounts of CEM extract were western blotted for Dicer Lanes 1 – 20 micrograms, 2 – 10 microgramsm 3 – 5 micrograms. 100 micrograms of U937 and U1 extract were blotted (lanes 4 and 5). Urea buffer was used to extract proteins from U937 and U1. Fifty micrograms of extract was western blotted for Dicer (lanes 6 and 7). U937 and U1 cells were dissolved in Laemlii buffer for 2 minutes at 95° and loaded directly onto the gel (lanes 8 and 9) E) Quantitative RT-PCR analysis was performed to examine the level of Dicer expression in CEM, ACH2, U937 and U1. Relative expression values were obtained by normalization to actin. F) U937 and THP-1 were cultured in the presence of 0, 10, 20 or 100 nM PMA for 48 hours to induce differentiation. Twenty micrograms of whole cell extract from U937, THP-1, CEM, H9 and BJAB were resolved by SDS-PAGE and western blotted for Dicer. Densitometry was performed as in panel A.