Fig. 2. mut-p53 and miR-30d regulate protein secretion.
a Gene Ontology (GO) enrichment analysis of genes differentially expressed in MDA-MB-231 cells transduced with miR-30d decoy compared with control, using DAVID database. All terms were significant (p < 0.05) following Benjamini–Hochberg correction. b Gene set enrichment analysis (GSEA) of hallmarks PROTEIN_SECRETION in MDA-MB-231-dy-30d transcriptome compared with control (n = 3). c Analysis of protein secretion in MDA-MB-231-dy-30d cells by [35S]-methionine/cysteine labeling, SDS-PAGE, and autoradiography (top). The inhibitor of ER-GA protein transport Brefeldin A (BFA, 2.5 μM) was used as control. Intracellular proteins are shown in Supplementary Fig. 3c. Right: ratio of secreted vs intracellular labeled proteins quantified by densitometry. d Protein secretion analysis in MDA-MB-231 cells transfected with mut-p53 siRNA, miR-30d mimic or combination, performed as in (c) (intracellular proteins are in Supplementary Fig. 3d). e MDA-MB-231 cells were transduced with construct encoding secretion signal-fused GFP (ssGFP). Forty-eight hours upon silencing mut-p53, overexpressing miR-30d mimic or combination, fresh medium was added and collected after 2 h. Bottom: ratio of secreted versus intracellular ssGFP quantified by densitometry. f Intracellular and medium (CM) ssGFP levels were analyzed as in (e) in MCF10A cells silenced for wt-p53 (shp53) and overexpressing mut-p53R280K, and transduced with dy-30d or control. g Intracellular and CM ssGFP levels were analyzed as in (e) in MDA-MB-231 cells upon silencing HIF1α or culturing in hypoxia (2% pO2) for 16 h, and treated or not with miR-30d inhibitor. h MDA-MB-231 cells were transfected as in (d), and CM was analyzed by LC–MS/MS. Heatmap shows proteins whose secretion was significantly altered. i Venn diagram showing overlap between proteins differentially secreted (h: secretome) upon mut-p53 silencing in MDA-MB-231 cells and genes differentially expressed at protein (proteome) or RNA level (transcriptome) as reported in ref. 30. j Venn diagram showing overlap between differentially secreted proteins (secretome) and differentially expressed transcripts (transcriptome) upon inhibiting miR-30d in MDA-MB-231 cells by decoy construct (dy-30d). Graphs represent the individual data points, the mean +/− SEM of three independent experiments. Blots are representative of n = 3 biological replicates. P value (*p < 0.05, **p < 0.01, ***p < 0.001) was calculated by two-tailed unpaired Student’s t-test. Source data are provided as Source data file.