Dubińska-Magiera M , Chmielewska M , Kozioł K , Machowska M , Hutchison CJ , Goldberg MW , Rzepecki R .

	Fig. 1. XLAP2β localizes in X. laevis XTC cells typically for the inner nuclear membrane protein. Cells were grown on coverslips, fixed with PFA or methanol, and stained for XLAP2β, lamin B2, nucleoporins F/G, XBAF, TPX2, actin, γ-tubulin, β-tubulin, and membranes (DHCC). DNA was visualized with DAPI (blue). Single confocal sections (1.5 μm) through the center of nuclei are shown. Bar: 5 μm. Interphase XLAP2β protein colocalizes with lamin B2, shows apparent colocalization with FG-repeat nucleoporins, and partly colocalizes with BAF proteins at the NE only. LAP2β does not localize to cytoplasm or ER. In XTC cells, centrosomes locate immediately next to NE. XTC cells show a fibroblast-like distribution of microtubules and actin. In interphase XTC cells, TPX2 protein is weakly expressed. It gradually appears in the mid-S phase and is dispersed through the whole nucleoplasm so does not colocalize with XLAP2β
	Fig. 2. The main fraction of XLAP2β does not colocalize with TPX2 in XTC cells during interphase. XTC cells were grown on coverslips, fixed with methanol, and stained for XLAP2β, lamin B2, TPX2, and membranes (DHCC). DNA was visualized with DAPI (blue). Single confocal sections (1.5 μm) through the center of nuclei are shown in a. Bar: 10 μm. They illustrate typical localization for those antigens in XTC cells. For TPX2 localization and all analyses of its colocalization, only cells with high level (easily detectable) of TPX2 were chosen. b Line sections (long red arrows) used for qualitative colocalization analyses (relative fluorescence intensities distribution). c Results of these analyses. Left marker (cyan) indicates point outside the cell (threshold) and right marker (violet) points the nuclear envelope. It is clearly seen that TPX2 does not localize to nuclear envelope where the main signal from XLAP2 locates. b Presents three type of regions of the cell (intranuclear, envelope, and cytoplasm), shown as red circles, which were the basis for quantitative colocalization calculations resulted in the graph displayed in d. Bars of the chart indicate colocalization coefficients of lamin B2 with XLAP2 either TPX2. Red asterisks show statistically significant differences between analyzed colocalizations. Lamin B2 colocalizes with XLAP2 in the nuclear envelope and in the nuclear interior but only partially with TPX2. In the envelope, the difference between colocalization coefficients for XLAP2 and for TPX2 is statistically significant, and inside the nucleus is on the borderline of significance. Hence, TPX2 and XLAP2 do not colocalize in examined regions
	Fig. 3. Analysis of the XLAP2 protein knockdown in tissue cultured X. laevis XTC cells. XTC cells were plated at 7.5 × 104 cells/well, transfected with 100 nM siRNA sequences, and grown for 24 and 72 h. C-XTC, untreated cells; 15, 98, 237xlap, cells treated with different siRNAs. a Schematic diagram of the Xenopus LAP2β protein. The positions of amino acid residues are marked above the diagram. Locations of predicted domains are marked by different patterns: striped box common domain of all potential isoforms, white box LEM domain, hatched box the transmembrane region. cDNA sequences given below the diagram were used to design 25 nt ds siRNAs for XLAP2 protein expression silencing. Numbers denote a position of nucleotides. b Western blotting analysis of the XLAP2 protein silencing in tissue cultured X. laevis XTC cells after 24 and 72 h post- transfection. Equal amounts of the protein were loaded. Numbers denoted above each lane represent the relative amounts of XLAP2 protein. Molecular masses of the proteins (kDa) are marked on the left side of the picture. XLAP2 knockdown was most efficient with 98xlap and 15xlap siRNAs after 3 days post-transfection. c, d Effect of XLAP2 protein knockdown on the morphology of XTC cells was also analyzed by confocal and immunofluorescence microscopy. XTC cells were stained for XLAP2 (red), DNA was visualized with DAPI (blue). c General view of the control, non-treated, and siRNA transfected XTC cells. Note the complete loss of XLAP2 signal after 72 h post-transfection with 15xlap siRNA. d Higher magnification of the control and silenced nuclei imaged in non-confocal (3 left columns) or confocal mode (right column) visualize that the degrading XLAP2 protein is no longer localized in the NE. DNA is amorphous (98xlap). Note the apoptotic nucleus with lobulations (15xlap lower photograph). Images were combined and processed in Adobe Photoshop. Single confocal sections (1.5 μm) through the center of nuclei are shown. Bars, 20 μm (c), 10 μm (d)
	Fig. 4. Microscopic immunofluorescence analyses of XTC cells subjected to knockdown or mock-knockdown procedures. Knockdown of XLAP2 protein in XTC cells results in inhibition of growth, increased cell death and cellular abnormalities. a, b Tissue cultured cells were grown on glass coverslips and transfected either with 15XLAP2 and 98XLAP2 siRNAs or scrambled control siRNA (C-15). Untreated cells (C-XTC) were used as a control. After the indicated period of time, cells were fixed and costained for XLAP2 and α-tubulin and DNA was visualized with DAPI (not shown). a Cells from 10 representative fields were counted. The diagram represents total cell count from 10 fields. Asterisk denotes statistically significant lower cell number in 15 + 98xlap siRNA treated XTC cells versus c15 siRNA control cells (P < 0.05). b Analysis of cellular organization in XTC cells from 10 fields. Normal, well-organized tubular network, average nucleus size; disturbed tubulin, cells showing partially condensed tubulin but attached to the surface, small nucleus; shrinked, condensed tubulin, small nucleus; apoptotic phenotype, weak or no tubulin staining, no NE staining, degraded DNA. XLAP2 knockdown results in phenotype abnormalities in XTC cells, as well as in cell death
	Fig. 5. Knockdown of XLAP2β in tissue cultured X. laevis XTC cells results in cell abnormalities. XTC cells were transfected with plasmid encoding antisense (15) or scrambled (C15) siRNA together with GFP as a transfection marker directed against an N-terminal fragment of XLAP2. For immunofluorescence, cells were grown on glass coverslips for 24 or 48 h after transfection, fixed in PFA, and stained for XLAP2 (red) and DNA (DAPI, blue), GFP (green). Single confocal sections (1.5 μm) through the center of nuclei are shown. Bar: 5 μm. Untreated cells (C) or cells treated with a plasmid encoding scrambled siRNA and GFP (C15) were used as controls. Note that nuclear shape abnormalities are concomitant with loss of XLAP2 protein from NE. Note XLAP2 speckles and discontinuous XLAP2 in NE in nucleus transfected with 15 siRNA plasmid after 24 h and lobular shape of apoptotic nucleus after 48 h
	Fig. 6. XLAP2β knockdown affects cell nuclei morphology and interferes with the proper location of lamin B2 and nucleoporins. XTC cells were transfected with siRNA plasmid, encoding silencing (15) or scrambled (C15) siRNA with GFP as a transfection marker, directed against an N-terminal fragment of XLAP2. For immunofluorescence, cells were grown on coverslips for 48 h after transfection, fixed with PFA or methanol, and stained for XLAP2β (red), DNA (blue), and nucleoporins with F/G repeats, lamin B2, β- and γ-tubulin (yellow). Expressed GFP proteins (green) localize throughout the whole cell volume. For most images, GFP looks precipitated and weakly visible because of methanol fixation method used to obtain the best performance of individual antibodies. Single confocal sections (1.5 μm) through the center of nuclei are shown. Bar: 5 μm. XTC cells with decreased level of XLAP2β protein have abnormally shaped nuclei, and F/G-repeat nucleoporins and lamin B2 are redistributed. The microtubule network is frequently shrunk, but centrosomes remain their normal position directly at the nuclei

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