Fig. 5
From: Mutant p53s generate pro-invasive niches by influencing exosome podocalyxin levels
mutp53 exosomes promote integrin recycling in fibroblasts to influence ECM architecture. a, b mutp53-expressing R175H or R273H H1299 cells, or cells generated by CRISPR from the latter (PODXL-CRSP; Rab35-CRSP), were transfected with GFP or PODXL-GFP or were left untransfected. Exosomes collected from H1299-p53−/− and the transfected and untransfected mutant p53-expressing H1299 cells were used to treat TIFs and receptor recycling (a) and migratory characteristics of these (b) were determined as for Fig. 2a, b. In a R59022 (10 μM) or DMSO was added to TIFs as indicated. Mean ± SEM, n = 6. In a ***red versus blue, and ***green versus black are p < 0.001, ANOVA. In b, n = >52; ***red versus blue,***purple versus yellow, ***green versus black, ***light blue versus black are p < 0.001, Mann–Whitney. In the right panel of b, *** is p < 0.001, Mann–Whitney. c TIFs were incubated with exosomes from H1299 (p53−/−, p53R273H, p53R175H) cells or left untreated and allowed to deposit ECM in the presence and absence of R59022 (10 μM) or DMSO. ECM was then de-cellularised, stained with antibodies recognising fibronectin and image stacks were collected using confocal microscopy. Extended focus projections of these stacks are displayed in the left panel of c, bar, 50 μm. The organisation of the ECM fibres in these was determined using GLCM. The decay curves and the weighted means of the decay distances derived from these are presented in the centre and right panels of c respectively. Weighted mean ± SEM, n = 8, * is p < 0.05, Mann–Whitney. d TIFs were treated with exosomes from H1299 (p53−/− or p53R273H) cells or were left untreated and allowed to deposit ECM. De-cellularised ECM was analysed using AFM. The left and right panels indicate ECM stiffness and stickiness respectively. Mean ± SEM, n > 6 ECM preparations from two individual experiments *** is p < 0.001, Mann–Whitney. e MDA-MB-231 cells were seeded onto de-cellularised ECM deposited by exosome-treated TIFs as indicated. Cells were fixed and cell:ECM adhesions visualised by immunofluorescence. Top panel shows representative images (bar, 20 μm). Total area of paxillin per cell and average area of paxillin-positive particles are plotted in the bottom left (n > 6) and right panels (n > 16). Mean ± SEM, * is p < 0.05, Mann–Whitney
