TC-PTP knockdown results in reduced proliferation of murine B-cell lymphoma cells. (A) Validation of TC-PTP knockdown. DBL-114 cells were lentivirally transduced with either a control shRNA against firefly luciferase or 1 of 2 shRNAs specific to murine TC-PTP expressed in vector pLL3.7. Cells were sorted for less than 95% transduction and sorted cells were lysed in 1% TX-100. A total of 5 μg of each lysate was subjected to semiquantitative Western blot analysis with 6F3 anti–TC-PTP and anti–β-actin as a loading control. A representative Western blot is shown along with quantitation of 3 different blots. (B) TC-PTP knockdown confers a competitive growth disadvantage to mouse B-cell lymphoma cells relative to cells expressing normal levels of TC-PTP. DBL-114 cells (left panel) or TBL-1 (right panel) were lentivirally transduced with the listed shRNAs, resulting in a mixed population of transduced (GFP⁺) and nontransduced (GFP⁻) cells. The frequency of GFP⁺ cells (relative to an empty vector control) for each construct was then monitored for 2 weeks after infection. (C) TC-PTP knockdown results in an accumulation of cells in the G₁ phase of the cell cycle. DBL-114 cells sorted for control shRNA (shLuc) or TC-PTP–specific shRNA (shTC-PTP.2) were fixed with cold ethanol and stained with propidium iodide to examine total cellular DNA content. Cells were subjected to FACS analysis, and 20 000 events were collected for each shRNA. The cell-cycle data are as follows (mean ± SD): shLuc; apoptotic, 0.73% ± 0.09%; G₁, 40.1% ± 0.46%; S, 13.0% ± 0.25%; G₂/M, 24.8% ± 0.80%; 4N⁺, 16.7% ± 3.40%; shTC-PTP, apoptotic, 0.63% ± 0.22%; G₁, 45.8% ± 1.31%; S, 12.8% ± 0.74%; G₂/M, 23.8% ± 1.46%; 13.4% ± 4.69%; n = 3. (D) TC-PTP knockdown retards cellular proliferation. DBL-114 cells were transduced to express the control shRNA (shLuc) or shTC-PTP.2, and a thy1.1 reporter. Cells were labeled with CFSE 3 days after infection and evaluated at 48 and 72 hours later. Cells were counterstained with OX7 anti–Thy1.1–Alexa 647 to differentiate shRNA expressing cells and analyzed by flow cytometry. The data are shown for both transduced cells (solid line, labeled) and untransduced cells (shaded, labeled) from each population. Shown are representative plots from 3 different datasets.