Positioning effects are not due to changes in nuclear size and shape, distribution of other nuclear proteins, or loss of nuclear permeability. HT1080 cells transiently transfected with tagged NETs were tested for parameters that could theoretically indirectly influence chromosome position. (a) The area of the nuclear midplane (left, distribution plots) and the longest and shortest distance across it (right, scatter plots) were measured and quantified for over 40 transfected cells for each NET. In each case the NET transfected cells were compared to untransfected (UT) cells in the same population. Only very moderate fluctuations in nuclear size or shape were observed. (b) Transfected cells were stained with antibodies for nucleolin to determine if NETs had indirect disruptive effects on nucleoli and for lamins and the NET emerin to determine if NETs generally altered NE organization. No changes in the distribution of these markers were observed. (c) The permeability barrier function of the NE in NET-transfected HT1080 cells was tested by expressing a reporter transport cargo in the same cells. The cargo contains two GFP molecules in tandem with a strong nuclear localization signal and a weak nuclear export signal so that it can shuttle but accumulates predominantly in the nucleus. If nucleocytoplasmic transport or NE permeability is compromised, the reporter accumulates more in the cytoplasm as when the herpesvirus protein ICP27, which interferes with nuclear transport through binding to Nup62, is co-expressed (second panel). None of the NETs affected the distribution of the reporter. For both (b) and (c) scale bar = 10 μm.