Supplementary Figure 4: Cells containing CYRI-B inducible vector but treated with the vehicle solution show similar phenotype to the control cells.

a-b - Western blot of COS-7 cells transiently transfected with a mVenus-TetON inducible CYRI-B construct probed with anti-GFP (detects mVenus co-expression). Cells were collected and analysed 48h after vehicle or doxycycline treatment (a). Percentage of overexpression relative to the EV (standardized at 100% - Red dotted line) (b) (n = 3 independent western blot). See also Supplementary Fig. 7. c-f - Immunofluorescence of vehicle-treated negative control (EV) or CYRI-B COS-7 cells, fixed after 4h of spreading and stained for WAVE2 (magenta), nuclei (blue) and GFP (green). Scale bar = 50 μm. Inset panels are a magnified view of the white dashed field. Scale bar = 10 μm (c). WAVE2 ratio and circularity were measured and reported in (d) and (e) respectively. Cell area quantification was based on the phalloidin staining (f). Two-tailed Mann-Whitney test was applied. n.s. p > 0.05. (Veh/EV n = 81 cells ; Veh/CYRI-B n = 78 cells). All data presented are representative of at least 3 biologically independent experiments. Bar and scatter plots show data points with mean and s.e.m.