Fig. 5: The mut-p53/miR-30d secretome induces a supportive TME.

a GO enrichment analysis of differentially secreted proteins (Fig. 2g data), using DAVID database. All terms were significant (p < 0.05 Benjamini–Hochberg correction). b Analysis of intracellular (cell lysates) and secreted (CM) proteins in MDA-MB-231 cells transfected with mut-p53 siRNA (sip53) alone or with miR-30d mimic. c AFM measurement of the stiffness of ECM deposited by MDA-MB-231 cells transfected with mut-p53 siRNA alone or with miR-30d mimic. Boxplot represents median and whiskers (Tukey method). d H1299 cells were treated with H1299-conditioned DMEM or conditioned DMEM (CM) from MDA-MB-231 cells transfected as in (b); cell migration was analyzed by transwelling (Boyden chamber) assays (16 h). Right: cell amounts and mut-p53 levels were compared by western blot. Bottom: Representative light microscope images of migrated cells. e H1299 cell migration analysis performed as in (d), using CM from MDA-MB-231 cells transduced with control (CTRL) or dy-30d. Control of decoy vector was GFP. f Levels of intracellular and secreted VEGFA analyzed as in (b). Right: relative VEGFA secretion quantified by densitometry. g Analysis of endothelial network formation by HUVEC cells on Matrigel upon addition of CM from MDA-MB-231 cells transfected as in (b). The graph shows numbers of endothelial closed loops (n = 4). VEGF (20 ng/ml for 24 h) was used as control. h Dextran permeability assay of HUVEC monolayers was performed upon addition in the upper chamber of MDA-MB-231 CM as in (b). FITC-Dextran fluorescence in the lower chamber was measured after 30′. TNFα (100 ng/ml for 30 min) was used as control. i Representative light microscopy images of wound healing assays of WI-38 fibroblasts treated with their medium (CTRL) or with MDA-MB-231 CM as in (d). The graph shows percentage of wound closure. j Immunoblot analysis of fibronectin1 in WI-38 cells treated as in (i). The blot is representative of n = 3 biological replicates. Bottom: densitometry quantification of FN1 expression relative to GAPDH. Graphs represent individual data points and mean +/− SEM; p value (*p < 0.05, **p < 0.01. ***p < 0.001, ****p < 0.0001) calculated by two-tailed unpaired Student’s t-test. Blots are representative of n = 3 biological replicates. Source data are provided as Source data file.