Fig. 7: Sprouty4 acts through a non-canonical pathway, by regulating RhoA levels, to induce the effects of Mdm2 knockdown in cell migration.

A, B Effect of Spry4 modulation in the phosphorylation of ERKs in HT1080 p53KO cells. Protein levels of phospho-ERK and total ERK levels (A) after Spry4 silencing using a pool of siRNAs and (B) in response to silencing of Mdm2 alone or Mdm2 and Spry4 together. C, D RhoA modulation in HT1080 p53KO cells transfected with siRNAs against Mdm2 or treated with MEL23. C Protein levels and (D) mRNA levels of RhoA after transfection with indicated siRNAs (n = 3 samples) or treatment with MEL23 (n = 3 samples) for 24 h. β-actin was used as a loading control for immunoblots. RPL32 was used as a housekeeping control for qPCR. E Immunoprecipitation of Mdm2 in the presence of MG132. Lysates were probed for the presence of RhoA, and MdmX was used as a positive control. Mdm2 was pulled down by using a mix of antibodies against Mdm2 that recognize different domains within the protein. F RhoA protein levels in HT1080 p53KO cells transfected with siRNAs against Mdm2 alone or against Mdm2 and Spry4. α-tubulin was used as a loading control. G Quantification of wound scratch migration assay comparing cells transfected with siRNAs against Mdm2 alone or against Mdm2 and a pool of Spry4 siRNAs in the presence or absence of the RhoA inhibitor Rhosin (50 µM) for 24 h. The graph represents mean ± SD, n = 3 technical replicates. The graph for the other two independent experimental replicates can be found in the supplementary material. H Immunoblot of levels of total and phospho-cofilin-1(Ser3) in HT1080 p53KO cells silenced for Mdm2 alone or with double KD of Mdm2 and Spry4. I Immunofluorescence showing F- (red) and G-actin (green) staining. Nuclei (blue) as detected by DAPI staining, n = 3 groups. Representative images (left) and quantification of F/G ratio (right). Graph represents mean ± SD of independent experimental replicates, in each replicate the F/G-actin ratio was quantified in at least 30 events/condition for a total of at least 90 events/condition. More details about the statistical tests used can be found in the Source Data file.