Figure 2.

PA200 affects growth in glutamine-limiting conditions. A, after transfection of HeLa cells and plating in 2 mmol/L glutamine, the glutamine remaining in the media was measured using a Nova Flex device at each time point. B, clonogenic survival assay using untransfected HeLa cells in media supplemented with indicated concentrations of glutamine. Colony formation was assessed 13 days after plating without media replacement. C, HeLa cells transfected with the PA200-specific siRNA (white bars) or nonspecific siRNA (Ctrl si, black bars) were plated (0.33 × 10⁴/cm²) in media with 0.05 or 0.2 mmol/L L-glutamine for 72 hours. Cell growth was assessed using Promega cell proliferation kit (MTS assay). D and E, untransfected HeLa cells were plated at 1 × 10⁶ cells per 10-cm diameter dish in complete growth media in the presence of az-NC-001 at 4 µmol/L or dimethyl sulfoxide (DMSO, vehicle) for 48 hours. Cells were harvested and replated at 10,000 cells per well of a 96-well dish in fresh media containing inhibitor az-NC-001 or DMSO for 2 days. Pictures were taken (E) and cell growth was measured by Promega Proliferation Kit (MTS; D). *, P < 0.005. A representative figure of at least 3 independent experiments is shown for each panel.