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cytoa22246-sup-0001-SuppFig1.tif13266KSupporting Information Figure 1. eGFP-S2-ΔN is not enriched in nuclear speckles in living cells. ASF/SF-2 colocalization studies. (A-C) Confocal fluorescence micrographs of living MCF-7 cells overexpressing mCherry-ASF/SF-2 (A) with eGFP-S2-ΔN (B). In the merge (C) ASF/SF-2 is in red and S2-ΔN is in green. Note that eGFP-S2-ΔN is not enriched in nuclear speckles in living cells. (D-F) Confocal fluorescence micrographs of living MCF-7 cells as described in (A-C) but after incubation for 6 hours with 5μg/ml of a-amanitin. Note that the inhibition of transcription by a-amanitin affects the shape of nuclear speckles (compare A to D) and that eGFP-S2-ΔN is not recruited to nuclear speckles after a-amanitin treatment. (G-I) Confocal fluorescence micrographs of MCF-7 cells as described in (A-C) after paraformaldehyde fixation. Note the clear enrichment of eGFP-S2-ΔN in nuclear speckles. (J-L) Confocal fluorescence micrographs of MCF-7 cells as described in (D-F) after paraformaldehyde fixation. Note again the change in shape of nuclear speckles after a-amanitin treatment (compare J with G). Note eGFP-S2-ΔN enrichment in nuclear speckles. Scale bars 10μm.
cytoa22246-sup-0002-SuppFig2.tif13359KSupporting Information Figure 2. eGFP-S2-ΔN is not enriched in nuclear speckles in living cells. ΔSF3b155 colocalization studies. (A-C) Confocal fluorescence micrographs of living MCF-7 cells overexpressing mCherry-ΔSF3b155 (A) with eGFP-S2-ΔN (B). In the merge (C) ΔSF3b155 is in red and S2-ΔN is in green. Note that eGFP-S2-ΔN is not enriched in nuclear speckles in living cells. (D-F) Confocal fluorescence micrographs of living MCF-7 cells as described in (A-C) but after incubation for 6 hours with 5μg/ml of a-amanitin. Note that the inhibition of transcription by a-amanitin affects the shape of nuclear speckles (compare A to D) and that eGFP-S2-ΔN is not recruited to nuclear speckles after a-amanitin treatment. (G-I) Confocal fluorescence micrographs of MCF-7 cells as described in (A-C) after paraformaldehyde fixation. Note the clear enrichment of eGFP-S2-ΔN in nuclear speckles. (J-L) Confocal fluorescence micrographs of MCF-7 cells as described in (D-F) after paraformaldehyde fixation. Note again the change in shape of nuclear speckles after a-amanitin treatment (compare J with G). Note eGFP-S2-ΔN enrichment in nuclear speckles. Scale bars 10μm.
cytoa22246-sup-0003-SuppFig3.tif1837KSupporting Information Figure 3. All ACFs measured in the nucleoplasm of MCF-7 cells transiently transfected with different constructs as indicated are best described by the two-component diffusion model. (A) Averaged residual plots obtained after fitting the ACFs obtained from 19 independent FCS measurements in the nucleoplasm of MCF-7 cells transiently transfected with eYFP. The one-component diffusion, the two-component diffusion and the anomalous diffusion model were used to fit as indicated. Note that the two-component diffusion model described the ACF of eYFP the best. (B) Averaged residual plots, obtained from 27 independent FCS measurements in the nucleoplasm of transiently transfected MCF-7 cells, showing the fit of the measured ACFs of eYFP-S2-ΔN with the anomalous, the two-component and the one-component diffusion model as indicated. Note that the two-component diffusion model described the ACF of eYFP the best. (C) Averaged residual plots showing the fit of the ACFs obtained from 32 independent FCS measurements in the nucleoplasm of MCF-7 cells transiently with eYFP-S2-ΔN and after incubation for 6 hours with 5μg/ml of ?-amanitin. Both the anomalous and the two-component diffusion model were used to fit the obtained ACFs, but the two-component diffusion model describes the data the best. (D-F) Averaged residual plots obtained after fitting the measured ACFs of eYFP-S2-ΔN-δ1,2 (D), eYFP-S2-ΔN-δ1 (E) or eYFP-S2-ΔN-δ2 (F) using the two-component diffusion and the anomalous diffusion model. The averaged residuals were calculated from fitting minimum 20 ACFs obtained in independent FCS measurements in the nucleoplasm of MCF-7 cells. Note that the ACFs of all the eYFP-S2-ΔN?PIP2 mutants are best described by the two-component diffusion model. The deviation from the fits [δG(τ)] is given in function of autocorrelation time (τ in μs).
cytoa22246-sup-0004-SuppFig4.tif964KSupporting Information Figure 4. All ACFs measured in nucleoli of MCF-7 cells transiently transfected with different constructs as indicated are best described by the two-component diffusion model. (A-C) Averaged residual plots obtained after fitting the ACFs of minimum 13 independent nucleolar FCS measurements in MCF-7 cells transiently expressing eYFP-S2-ΔN (A), eYFP-S2-ΔN-δ1 (B) or eYFP-S2-ΔN-δ2 (C). The two-component diffusion and the anomalous diffusion model were used. Note again that the two-component diffusion model describes the ACFs the best. The fit of eYFP-S2-ΔN-δ1,2 is not shown since this mutant is excluded from nucleoli. The deviation from the fits [δG(τ)] is given in function of autocorrelation time (τ in μs).
cytoa22246-sup-0005-SuppFig5.tif22218KSupporting Information Figure 5. S2-ΔN and S2-ΔN-δ1,2 form homo-dimers in the nucleoplasm (A-D) Mean normalized autocorrelation functions (ACF) and cross-correlation functions obtained from minimum 12 independent FCCS experiments measured in the nucleoplasm of MCF-7 cells transiently co-expressing eGFP-S2-ΔN and mCherry (A), eGFP-mCherry (B), eGFP-S2-ΔN and mCherry-S2-ΔN (C) or eGFP-S2-ΔN-δ1,2 and mCherry-S2-ΔN-δ1,2 (D). Cross-correlation between mCherry and eGFP-S2-ΔN was used as a negative control. Fusion of eGFP and mCherry was used as a positive control. Note the co-diffusion of eGFP-S2-ΔN and mCherry-S2-ΔN. Note also that eGFP-S2-ΔN-δ1,2 and mCherry-S2-ΔN-δ1,2 still co-diffuse.
cytoa22246-sup-0006-SuppTab1.doc34KSupporting Information Table 1. P-values as obtained by unpaired two-tailed t-tests listed to identify significant differences between the diffusion coefficients (Dfast, 37°C and Dslow, 37°C) or the percentage of the fast component (Ffast, 37°C) of the different constructs as indicated and measured by FCS. A P-value lower than lower than 0.05 denotes a significant difference, n = degrees of freedom.
cytoa22246-sup-0007-SuppTab2.doc29KSupporting Information Table 2. P-values as obtained by unpaired two-tailed t-tests listed to identify significant differences between the immobile fractions (Fimm) of the different constructs as indicated and measured by FRAP. A P-value lower than lower than 0.05 denotes a significant difference, n = degrees of freedom.

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