Fig 2: Expression of LINC complex proteins is increased in patient MD-1 (SUN1 p.G68D/p.G338S) myoblasts.(A) Control and MD-1 myoblasts were fixed in methanol and analysed by immunofluorescence microscopy using SUN1, SUN2, emerin, nesprin-2G and lamin A/C antibodies, as indicated, together with DAPI staining of DNA. Scale bar, 22 µm. (B) Mean fluorescence intensity of SUN1, SUN2, emerin, nesprin 2 and lamin A/C was measured in individual DAPI-stained nuclei using an Olympus Scan?R screening station and analysed using Scan?R analysis software. The results are presented as mean ± S.E. of 1000 cells taken from at least 3 independent experiments. **P=0.05. Significant P-value for SUN1 was P = 0.009. (C) Total protein extracts from control (C) and patient MD-1 myoblasts were Western blotted using antibodies against LINC complex-associated proteins, as indicated. (D) Samples prepared as in A were Western blotted using nesprin-2 (N2–N3) antibodies. (E–F) Protein expression was quantified by densitometric analysis of at least 3 independent experiments. The results are presented as mean ± S.E. *P=0.05 and **P=0.01. Each significant P- values are as follows: SUN1 P = 0.05, a-tubulin P = 0.003, nesprin-2 P = 0.002. P- value for emerin was P = 0.06.

Fig 3: LINC complex localization in EWS cells is rescued by lamin A expression.a Lamin A-GFP (green), SUN1 (red), SUN2 (red), nesprin 2 (red) and emerin (red) localization in TC-71 Empty-GFP clone (pEV #2) and TC-71 lamin A-GFP #84 (#84). DNA was counterstained with 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI). Merge of fluorescence signals are shown (MERGE). Graphs indicate the fluorescence intensity profile along the white arrows. Representative graphs of at least 30 nuclei analyzed for each sample were shown; magnification 100x, scale bar 10 µm; b Western blotting analysis of lamin A, SUN1, SUN2, emerin and nesprin 2 in TC-71 Empty-GFP clone (pEV #2) and TC-71 lamin A-GFP #84 (#84). GAPDH was used as loading control. Densitometric analysis is shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to TC-71 Empty-GFP clone; two-tailed unpaired Student’s t-test, *p < 0.05.

Fig 4: Prelamin A-related decrease of cell migration and motility in EWS cells.a Western blotting analysis of prelamin protein expression in parental TC-71 (CTRL), empty vector cells (E) and prelamin transfected TC-71 cells (OV) performed at 24 and 48 hours. GAPDH was used as loading control; b Wound healing assay of empty vector cells (EMPTY) and prelamin overexpressed TC-71 cells (OV). Representative pictures were taken at 0 and 24 h after scratching. Magnification 10×. Histograms were plotted as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to empty vector cells; two-tailed unpaired Student’s t-test, *p < 0.05; c Migration assay of empty vector cells (EMPTY) and prelamin overexpressed TC-71 cells (OV). Histograms show the percentage of migrated cells respect to empty vector cells, which were considered as 100%. Histograms were plotted as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to empty vector cells; two-tailed unpaired Student’s t-test, *p < 0.05; d Invasion assay of empty vector cells (EMPTY) and prelamin overexpressed TC-71 cells (OV). Histograms show the percentage of migrated cells respect to empty vector cells, which were considered as 100%. Histograms were plotted as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to empty vector cells; two-tailed unpaired Student’s t-test, *p < 0.05. e Western blotting analysis of prelamin A and lamin A/C protein expression in non-treated TC-71 cells (CTRL) and in mevinolin treated EWS cells (2.5 µM or 5µM MEV). GAPDH was used as loading control. Densitometric analyses are shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to CTRL cells; one-way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001; f Wound healing assay of non-treated TC-71 cells (CTRL) and mevinolin treated EWS cells (2.5 µM or 5µM MEV). Representative pictures were taken at 0 and 24 h after scratching. Magnification 10×. Histograms were plotted as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to non-treated TC-71 cells; one-way ANOVA test, **p < 0.01; g Migration assay of non-treated TC-71 cells (CTRL) and mevinolin treated EWS cells (2.5 µM or 5µM MEV). Histograms show the percentage of migrated cells respect to non-treated TC-71 cells, which were considered as 100%. Histograms were plotted as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to non-treated TC-71 cells; one-way ANOVA test, *p < 0.05; h Invasion assay of non-treated TC-71 cells (CTRL) and mevinolin treated EWS cells (2.5 µM or 5µM MEV). Histograms show the percentage of migrated cells respect to non-treated TC-71 cells, which were considered as 100%. Histograms were plotted as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to non-treated TC-71 cells; one-way ANOVA test, *p < 0.05. i Lamin A/C (green), SUN1 (red), SUN2 (red), Nesprin2 (red), and Emerin (red) localization in non-treated TC-71 cells (CTRL) and mevinolin treated (5 µM) EWS cells (MEV). DNA was counterstained with DAPI (DAPI). Merge of fluorescence signals are shown (MERGE). Graphs indicate the fluorescence intensity profile along the white arrows. Representative graphs of at least 30 nuclei analyzed for each sample were shown; magnification 100x, scale bar 10 µm; l Western blotting analysis of SUN1, SUN2, Emerin, and Nesprin 2 in non-treated TC-71 cells (CTRL) and mevinolin treated (5 µM) EWS cells (MEV). GAPDH was used as loading control. Densitometric analysis is shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to CTRL cells; two-tailed unpaired Student’s t-test, *p < 0.05.