Fig 2: Molecules acting in concert with RAB35 to promote Dorsal Ruffles formation. a Schematic representation of the signalling pathway leading to CDR formation. b RAB5 and RAC are required for RAB35-dependent CDR formation but PDGFRB is dispensable. pSLIK-HA-RAB35-human-MEFs were silenced for Pdgfrb, or Rab5 or Rac1 and treated or not with doxycycline to induce the transgene expression. CDR formation was monitored by time-lapse phase contrast microscopy. Still phase contrast images from time-lapse sequence (Supplementary Movie 13) are shown. Red arrows indicate CDRs. Scale bar, 100 µm. The number of CDRs/cell/h was reported as mean ± SEM (>30 cells/condition, three independent experiments). ****p < 0.0001; ns not significant, paired Student’s t-test. The silencing of genes was verified by qRTPCR. c Silencing of Rab35 GEFs or effectors. MEF cells were interfered for the indicated Rab35 GEFs or effectors and tested for their ability in forming CDRs upon PDGF stimulation. Representative Phalloidin-stained images are reported for each experimental condition. Scale bar, 50 µm. CDRs were manually counted and normalized against the scrambled sample. Data are the mean ± SD (n > 200 cells/condition in three independent experiments). ****p < 0.0001; **p < 0.01, paired Student’s t-test. The silencing of the targeted genes was verified by qRTPCR. In the case of p85, double p85a and ß KO MEFs47 were employed and the loss of p85 isoforms was confirmed by western blotting. In the latter case, as control we used both p85a+/-p85ß-/- MEFs (p85-/+, derived from sibling mice) as well as p85a-/-p85ß-/- MEF in which p85a was re-expressed

Fig 3: siRNA screening for RAB GTPases involved in CDRs formation. a siRNAs targeting RAB GTPases were ranked according to their CDR score (Methods and Supplementary Figure 1). Representative images of positive (siPdgfrb) and negative (siEGFP) controls are, respectively, in red and green. siRNAs best inhibiting CDR formation (more than 60% reduction relative to scrambled siEGFP) are enclosed into the light grey box. The oligo s95003 targeting Rab35, indicated in blue, is reported as a representative example. Red arrows indicate CDRs. Scale bar, 50 µm. b The siRNAs that inhibited CDRs formation more efficiently were validated in a completely independent experiment. Left: MEF cells interfered for Rab35 (ID: s95002, s95003), or Rab8a (ID: s69778, s69779), or Rab8b (ID: s108154, s108155, s108156), Pdgfrb (positive control), or Scrambled oligo (negative control). Upon PDGF stimulation, cells were fixed and stained with phalloidin. Representative images are shown for each experimental condition. Red arrows indicate CDRs. Scale bar, 50 µm. Right: CDRs were manually counted and normalized against scrambled-transfected, control samples. Data are the mean ± SD (n > 200 cells/condition in three independent experiments). The silencing of the targeted genes was verified by qRTPCR