Data are based on the analysis of rearrangement of membrane caveolar rafts and were obtained by pooling data from each single cell. Treatments operated on ECFCs are shown in the first column. The targets of the statistical analysis are indicated in the top row. Control data (CTRL) are presented as raw data obtained by ImageJ analysis of a wide pool of control cells. Other data are presented as percentage changes compared with control. The second column reports the percentage change in the area of labeled contiguous structures that is taken as a measure of raft clusterization. These data are in agreement with results obtained by the biochemical analysis performed with membrane fractionation. Quantitative analysis and colocalization of antigens were performed on acquired images analyzed as individual channels. The Manders overlap coefficients (M1 and M2) were determined using JACoP plugin and also calculated using the intensity correlation analysis plugin of the open-source software WCIF-ImageJ as previously described.²⁶  We used the same operating mode of image analysis: Manders overlap coefficients indicate an overlap of the signals and thus represent the degree of colocalization between the red and green pixels: their values range from 0 (no overlap) to 1 (complete overlap). Briefly, the background signal on each image was initially corrected using the ImageJ background subtraction function; and whenever possible, single cells on the images were selected using the lasso tool (which defines a so-called region of interest). Colocalization was then calculated, after choosing the threshold values for the green and red channels, with the aforementioned plugin on the regions of interest previously defined. Zero/zero pixel was excluded. The Manders coefficient relative to the uPAR fraction that overlap Cav1 was reported. Quantization of caveolin-1 fluorescence and uPAR fluorescence was conducted on z stacks of 70 sections corresponding to a thickness of approximately 0.8 μm passing through the middle of the cells. Regions of interest were manually drawn around each cell, and the integrated intensity after background correction was measured in caveolin-1 and uPAR channels. To account for differences in cell size, the integrated intensity in each region of interest was divided by its measured perimeter. ImageJ software was used for fluorescence quantization, and Origin 6.1, Version 6.1052 (B232) was used for statistical analysis. Quantitative colocalization analysis of antigens was performed on acquired images analyzed as individual channels. Results are expressed as mean ± SD or SE. Multiple comparisons were performed by 1-way ANOVA with Bonferroni correction. A P value < .05 was considered statistically significant.

This data indicates that almost all uPAR molecules remaining after aODN treatment, although reduced, colocalized with caveolin-1.