Bdata_apt

Concatenated HTS sequencing data file, containing unique SELEX-enriched sequences after filtering to remove any sequences of size not matching N, where N = 30 nts (size of the random region in the naive RNA library). Bdata_apt contains the sequences enriched in the presence of NSP2

Monitoring oligonucleotide degradation using FCS, FCCS and FRET.

<p>The stability of various RNAs was measured as a function of incubation time in cell extracts. The main changes and parameters corresponding to RNA degradation are shown exemplary for construct 2, representing: (A) the diffusion time from the autocorrelation function (FCS), (B) the amplitude of the cross-correlation function (FCCS), (C) an apparent FRET efficiency determined from the fluorescence intensity and (D) the donor fluorescence lifetime based FRET using a phasor analysis. The colored crosses represent the center of mass in the phasor plot of measurements after 1 min (blue), 60 min (green), 120 min (orange) and 180 min (magenta). The grey arrows indicate the direction of the main changes.</p

Fluorescence intensities of HeLa cells in culture after transfection with oligomer <i>278</i>.

<p>(A) A U-shaped, sequence defined cationizable lipo-oligomer <b><i>278</i></b> for complexation of the dual-labeled RNAs (C: cysteine, K: lysine, Stp: succinoyl-tetraethylene pentamine, linA: linoleic acid). (B) Fluorescence intensity images of the HeLa cells, 15 min, 1 h, 6 h and 24 h after transfection of the four different modifications patterns. The contrast level is equal for all images. The scale bar represents 200 μm. (C) Average fluorescence count rate of the cells at the different conditions shown in (B). The error bars represent the standard deviation of three independent measurements.</p

Design of the dual-labeled RNA oligonucleotide.

<p>(A) 23 nucleotide RNA oligonucleotide conjugated to tetramethylrhodamine (TMR) at its 5’ end <i>via</i> a thioether bond and to Atto488 at its 3’ end <i>via</i> an amide bond. Upon exposure to the cellular environment, the oligonucleotide can be degraded by various RNases. (B) Modification patterns selected to monitor intracellular localization and integrity of the oligonucleotide. RNA backbone modifications to modulate stability towards nucleolytic degradation: 2’-F, 2’-O-Me and phosphorothioate.</p

Quantification of the fluorescence lifetime measurements.

<p>(A-D) Distribution of the pixels along the line connecting the mono-exponential decays at 4.1 ns and at 1.25 ns in the phasor plot for the four modification patterns. (E) Summary of the average fluorescence lifetimes of the cell populations shown in panels A-D using a Gaussian fit to the distribution. The error bars represent the standard deviations of three independent measurements.</p

Lateral mobility of 18.5-kDa MBP in OLN-93 cells.

<p><i>z</i>-scan FCS and <i>z</i>-scan RICS measurements were performed on OLN-P, OLN-G and OLN-GS cells cultured on PLL and transiently transfected with 18.5-kDa MBP-eGFP. Experiments were performed 24 hours after transfection. <b>A</b>) A schematic of a cell showing <i>z</i>-scanning at the basal plasma membrane. <b>B</b>) The total intensity as a function of <i>z</i>-position for a typical z-scanning measurement is shown. <b>C</b>) The averaged autocorrelation curves from 10 cells were fitted with a 2D one component diffusion model. The diffusion coefficients from <i>z</i>-scan FCS are shown as a bar graph. <b>D</b>) A representative autocorrelation curve and corresponding 2D1C fit model is shown from a <i>z</i>-scan RICS measurement at the ventral plasma membrane. <b>E</b>) The diffusion coefficients for the <i>z</i>-scan RICS experiments are shown as a bar graph and represent the average of at least 10 cell measurements. Bars (<b>C,E</b>) represent the mean+SEM. The statistical analysis was performed using GraphPad Prism 5 (one-way ANOVA followed by the Newman-Keuls posttest, * p<0.05).</p

Co-localization of galactolipids with PLP at the plasma membrane of OLN-93 cells and its microdomain association.

<p>OLN-P, OLN-G and OLN-GS cells were cultured on PLL and transiently transfected with 18.5-kDa MBP-eGFP. The experiments were performed 24 hours after transfection. <b>A,B</b>) Representative images of transfected cells stained for either cell surface GalC or sulfatide with O1 and O4 antibodies (red), respectively. Scale bar is 6 µm. Insets show higher power magnifications of which brightness-contrast and median filtering were performed by Image J. <b>C</b>) The fractional co-localization between detection channels was determined by the Manders Correlation Coefficient Calculator (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101834#s2" target="_blank">Materials and Methods</a>) and displayed as a bar graph. Bars represent the mean+SEM of at least 15 cells. The statistical analysis was performed using GraphPad Prism 5 (one-way ANOVA followed by the Newman-Keuls posttest, *p<0.05). <b>D,E</b>) OptiPrep density gradient centrifugation analysis after CHAPS extraction. PLP-eGFP expression was determined using an anti-GFP polyclonal antibody (<b>D</b>). Representative Western blots are shown. The percentages of the PLP-eGFP bands were quantified as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101834#pone-0101834-g002" target="_blank">Figure 2</a> for MBP-eGFP bands (<b>E</b>). A bar of the distribution of PLP-eGFP is shown. The raft fractions are plotted in black and the non-raft fractions are shown as grey bars. Bars represent the mean+SEM. The statistical analysis was performed using GraphPad Prism 5 (n = 3, one-way ANOVA followed by the Newman-Keuls posttest, **p<0.01).</p

Co-localization of galactolipids with PLP at the plasma membrane of OLN-93 cells on Ln2 and Fn.

<p><b>A</b>) OLN-G and OLN-GS cells were cultured on either Ln2 (left panel) or Fn (right panel), transiently transfected with PLP-eGFP, and 24 hours after transfection stained for either cell surface GalC or sulfatide with O1 and O4 antibodies (red), respectively. Representative images are shown. Scale bar is 6 µm. Insets show higher power magnifications of which brightness-contrast and median filtering were performed by Image J. <b>B,C</b>) The fractional co-localization between detection channels was determined by the Manders Correlation Coefficient (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101834#s2" target="_blank">Materials and Methods</a>) and displayed as a bar graph. Bars represent the mean+SEM of at least 15 cells. The statistical analysis was performed using GraphPad Prism 5 [student′s t-test, ** p<0.01, *** p<0.001 (OLN-G and OLN-GS on the same ECM substrate)].</p

Lateral mobility of PLP in the presence of GalC and sulfatide as determined by s-FCS.

<p>OLN-P, OLN-G and OLN-GS cells were transiently transfected with PLP-eGFP on PLL and 24 hours after transfection the mobility was measured using circular scanning-FCS. <b>A</b>) The scanned circle was divided into bins and each individual bin (e.g. highlighted by a red spot) was correlated. For analysis, 10 bins were averaged (dark red area). <b>B</b>) An averaged intensity and correlation carpet for PLP-eGFP diffusion in an OLN-P cell are shown. Inhomogeneous intensity traces were discarded and the remaining FCS curves averaged (arrows). <b>C</b>) The autocorrelation function for all bins (dotted line) and selected bins (black line) where intensity heterogeneities that disturb the measurements have been removed. <b>D</b>) The diffusion coefficients are presented as a bar graph. The bars represent mean+SEM of at least 7 cell measurements. The statistical analysis was performed using GraphPad Prism 5 (one-way ANOVA followed by the Newman-Keuls posttest *p<0.05).</p

Expression and localization of galactolipids in OLN-93 cells.

<p>A) The expression of GalC and sulfatide was characterized in OLN-P, OLN-G, OLN-GS and mock-transduced cells by TLC. <b>B</b>) OLN-G and OLN-GS cells were cultured on PLL and stained for cell surface GalC or sulfatide with O1 and O4 antibodies, respectively. Scale bar is 5 µm.</p