Fig 4. Proliferation, migration and endosomal recycling in MYO5B-mutants.

Proliferation with CyQuant NF assay in SK-N-AS clones (A) shown as fold change at 24h, 48h, and 72h compared to mean MYO5B wild type (WT). Graphs show three independent experiments, each run in sextuplicates. Grey = empty vector, black = MYO5B^WT, red = p.L587P, blue = p.G1611S, green = p.R1641C. Mean fold change (FC) and significance (p-value) compared to MYO5B^WT within each time point are presented (lower panel). * p<0.05, ** p<0.01, paired t-test. Migration (Oris) in HEK293 clones (B) shown as representable photos from light microscope (5X) of cell area remaining in MYO5B-mutants (p.L587P, p.G1611S, and p.R1641C), MYO5B^WT, and empty vector at 24 h compared to the starting wound area. Scale bar shown is 100μm. Graph show percent wound area left after 24 h (mean±SEM, n = 2) in each construct. Transferrin uptake assay in SK-N-AS clones (C), with transferrin stained in red and nuclei stained in blue (DAPI). Pictures are taken with 40X Zeiss Axioscope 2 Plus fluorescence microscope, and small inserts show representative pictures taken with 63X, LSM700 confocal microscope. Scale bars shown are 50 μm and 100μm, respectively. Graphs plot fold change of corrected total cell fluorescence (CTCF) of transferrin (Tf) uptake (from four fields of view taken with 40X), and Western Blot analysis of the Transferrin receptor (TfR) expression (from two independent experiments) in mutants and empty vector compared to MYO5B^WT (n = 2). GAPDH was included as a loading control.