Fig 8.

DHX33 induction is required for Ras^V12-initiated tumor formation. (A) Arf-null ear fibroblasts from 2-month-old mice were infected with retroviruses encoding pBABE-empty vector (EV) or pBABE-Ras^V12. Cells were then infected with lentiviruses encoding shScrambled (shSCR), shLuciferase (shLuc), or shDHX33. Whole-cell lysates were extracted and analyzed by Western blotting with Ras, DHX33, and tubulin antibodies. (B) A total of 5 × 10³ infected cells were plated onto soft agar 60-mm plates in triplicate to measure anchorage-independent cell growth after 14 days. Quantitation of the colony numbers is presented from three representative fields under ×4 magnification. Error bars represent the standard deviation calculated from three different fields of colonies on triplicate plates. (C) Arf-null NIH 3T3 cells were infected with retroviruses encoding Ras^V12, followed by infection with lentiviruses encoding shScrambled (shSCR) or shDHX33. Whole-cell lysates were subjected to Western blot analysis with Ras, DHX33, and tubulin antibodies. (D) A total of 3 × 10⁶ infected NIH 3T3 cells were subjected to cytosolic ribosome profiles. (E) The upper panel shows NIH 3T3 cells infected with retroviruses encoding Ras^V12 that were then infected with shSCR or shDHX33 lentiviruses. A total of 10⁶ infected NIH 3T3 cells were injected into the flanks of nude mice. Tumor formation was visualized and photographed after 14 days. For the lower panel, mice were sacrificed at day 14 postinjection, and tumors were excised and photographed.