FIGURE 2:

Net1 knockdown results in aberrant nuclear morphology. HeLa cells were transfected with isoform-specific shRNA sequences in plasmids coexpressing either hMGFP or puromycin resistance genes. At 48 or 72 h later cells were fixed for immunofluorescence analysis or collected for real-time qPCR and Western blot analysis. (A) Immunofluorescence images of cells expressing nontargeting control, Net1, Net1A, or Net1/Net1A shRNAs. shRNA-transfected cells expressed hMGFP and were stained for α-tubulin (red) and DNA (blue). Bar, 10 μm. (B) Quantification of aberrant nuclear morphology in shRNA-transfected cells selected with puromycin. Average of at least three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001. (C) Real-time qPCR analysis of Net1 and Net1A transcript expression. Transcript levels are normalized to GAPDH. Errors are SEM. (D) Representative Western blot analysis of whole-cell lysates from shRNA-transfected cells. Upper band, Net1; lower band, Net1A.