Fig 9.

DHX33 is overexpressed in Ras-mutated cancer cell lines and is required for their efficient growth and proliferative properties. (A) A panel of K-Ras mutated or wild-type cancer cell lines (mutation status is shown at the bottom) were screened for total DHX33 protein expression, p14ARF status is also shown at the bottom. (B) 47S rRNA was measured by RT-PCR and normalized to total RNA levels. Error bars represent the standard deviation from three separate experiments. *, P < 0.001. (C) A total of 5 × 10⁴ cells were plated onto six-well culture plates. Cell numbers were counted daily and graphed. The doubling time was calculated based on growth curves and is shown in the table. (D) The indicated cell lines were infected with lentiviruses encoding shScrambled (shSCR) or shDHX33. Whole-cell lysates were extracted 4 days postinfection and subjected to Western blot analysis with antibodies recognizing DHX33 and tubulin. (E) shSCR or shDHX33-infected cells (10⁴) from indicated cancer cell lines were plated onto 100-mm culture dishes. The cells were fixed 10 or 20 days later with 100% methanol and incubated with Giemsa stain for 1 h. Stained colonies were air dried and photographed. (F) shSCR or shDHX33-infected cells (10⁴) from Miapaca-2 and A549 cancer cells were subjected to cell cycle analysis by flow cytometry after propidium iodide staining.