Fig. 3: Plasma membrane molecules affect the mobility of CD47.

CD47 was detected on viable HeLa cells with an anti-CD47) antibody(B6H12) labeled with A647 and imaged by TIRF microscopy during 60 s (a,b) or 120 s (c) at 33 Hz. a Viable cells were stained to detect CD47 and then fixed: with respectively 4% formaldehyde, 3% formaldehyde and 0.1% glutaraldehyde or 4% formaldehyde followed by 0.05% saponin. Mean Jump Distances (MJDs) was were calculated and each CD47 trajectory was colored according to its MJD value. A range of 250 frames of CD47 tracking was represented. Scale bar, 5 µm. b MJD distributions were obtained after merging the MJDs from several cells (4% formaldehyde: n = 20 cells, 3% formaldehyde + 0.1% glutaraldehyde: n = 14 cells, 4% formaldehyde + 0.05% saponin: n = 13 cells) measured in two independent experiments. The dotted red line corresponds to the mean value of the low-mobility (111.9 nm) and high-mobility (172.7 nm) MJDs extracted from viable cell MJD distribution (represented by the gray dotted line). c Cells were treated by UVB irradiation (n = 45), Cytochalasin D (CytD, n = 22), EDTA (n = 22), Mn²⁺ (n = 18) and MβCD (n = 22) and compared to non-treated viable (control) cells (n = 84). MJDs were calculated for each cell. Representative individual MJD histograms are shown in Figure S7. The Gaussian distribution of low- and high-mobility fractions was fitted, and the fraction of the high-mobility MJDs relative to the total number of measured MJDs is represented (R-value). Each dot corresponds to one cell and each experiment was performed at least two times independently. The mean and the standard deviation for each treatment are represented. *** denotes p < 0.0005, ** denotes p < 0.005, and n.s. denotes p > 0.05 as determined by a two-sided Student’s t test in comparison to the control.