Fig. 2: Loss of Mdm2 function decreases individual and collective invasion in 3D and metastatic burden in vivo.

A, B Tumor spheroid invasion in collagen matrix after (A) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or (B) treatment with MEL23. Representative images above and quantification below of the area invaded and the number of cells invading 24 h after implantation. C, D Tumor spheroid invasion in the collagen-BME matrix after (C) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or (D) treatment with MEL23. Representative images and quantification of the area invaded 24 h after implantation. Graphs show three pooled independent experimental replicates, the total number of spheroids in each condition is: siCtrl = 10, siMdm2#1 = 15, siMdm2#2 = 12 in panel (A); DMSO = 25, MEL23 = 24 in panel (B); siCtrl = 12, siMdm2#1 = 14, siMdm2#2 = 15 in panel (C), and DMSO = 8, MEL23 = 9 in panel (D). E Representative images of cell morphology of HT1080 p53KO cells transfected with siRNAs against Mdm2 or siCtrl in collagen matrix, 6 h after implantation. F Quantification of more circular cells (circularity of 0.75 or higher) in each condition shown in (E), n = 3 groups. The graph represents the average fold change of more circular morphology in Mdm2 silenced cells compared to control in three independent experimental replicates. More details about the quantification can be found in the method’s session. G–I HT1080 p53KO cells stably expressing shRNA scramble (shScramble) or a pool of Mdm2 shRNAs (shMdm2) were used to analyze metastatic burden using mouse models. G Protein levels of Mdm2 and MdmX in HT1080 shScramble and shMdm2 stable cell lines. β-actin was used as a loading control. H, I Representative images above and quantification below of metastatic foci in the lungs after implantation of shScramble or shMdm2 cells using (H) orthotropic model, n = 4 mice/group or (I) tail-vein model, n = 8 mice/group. In all box and whisker plots the boxes extend from 25 to 75 percentiles and whiskers show min and max values, The line in the center of each box represents the median. Graphs shown in panels (F, H, I) represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.