Fig 4.

DHX33 protein knockdown or overexpression influences ribosome biogenesis and protein translation. (A) Arf/p53/Mdm2^−/− MEFs were infected by lentivirus encoding shRNA-DHX33 or shScrambled (shSCR). At 3 days after infection, the cell lysates were subjected to Western blot analysis with anti-DHX33 and tubulin antibodies. (B) Infected cells from above were pulsed with [³H]uridine and chased for the indicated time points to monitor newly synthesized rRNA. Equal numbers of cells were pelleted for total RNA extraction. RNA was separated and transferred onto nylon membranes for autoradiography. (C) Equal numbers of the above-mentioned cells were subjected to total RNA isolation and then isolated by formaldehyde RNA denaturing gel. 28S and 18S rRNA were visualized by ethidium bromide staining and quantified. Bars were taken from three different experiments. (D) Equal numbers of Arf/p53/Mdm2^−/− MEFs infected by the indicated short-hairpin lentiviruses were subjected to cytosolic ribosome profile analysis at 4 days postinfection. (E and F) Arf/p53/Mdm2^−/− MEFs were infected with lentiviruses encoding empty vector, DHX33 (wild type) or mutant DHX33 (K94R). At 4 days postinfection, infected cells were subjected to cytosolic polysome profile analysis (E) and Western blot analysis with the indicated antibodies (F).