Kinetics of transferrin recycling in ALMS and control fibroblasts.

<p>(A–L) ALMS and control fibroblasts were incubated with unlabelled transferrin for 30 minutes. TfR and nuclei are depicted in red and blue, respectively. Early and recycling endosomes were immunostained with Alexa Fluor 488-labelled EEA1 (A–F) and Rab11 (G–L), respectively. Scale bars = 25 µm. (M–N) Co-localization with pericentrin (PCTN;red) and TfR (green) shows overlay at the pericentrosome. (O–Q) Quantification of TfR and its colocalization with Rab11 at the pericentrosome. Mean fluorescence intensity per 2 µm diameter area (dashed circle) was measured by ImageJ/Fiji software and used as an estimate of the number of TfR positive endosomes. Results are from 40 patient and 47 control cells and error bars indicate ± SEM; Star indicates p<0.0001. Scale bars = 5 µm. (R) Cells were ‘pulsed’ with Tf-Alexa Fluor 647 for 30 min, followed by a ‘cold chase’ of unlabelled holo-transferrin for indicated times. Data was pooled from three fibroblast cell lines from ALMS and control subjects. The graph represents the mean +/− SEM of four independent experiments as a mean percentage of Tf internalization at each time point. The values (MFI) obtained at time 0 following the pulse were set at 100%. Star denotes p<0.0001.</p>