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. 2016 Mar 29;7:11150. doi: 10.1038/ncomms11150

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(a) Transcriptome profiling of SKOV3 cells transfected with miR21 precursor or control miR. A heatmap shows the top 10 miR21 regulated genes related to chemoresistance and metastasis. (b) Pathway analysis showing chemoresistance-related genes that were upregulated (red) or downregulated (green) in miR21-transfected SKOV3 cells. (c) SKOV3 cells incubated with CAA- and CAF-derived exosomes showed lower APAF1 expression than did controls. The results were the average from at least three independent experiments. Mean±s.d.; ***P<0.001; two-tailed Student's t-test. (d) Immunolocalization of APAF1 on ovarian tumour tissue sections (n=5) demonstrated lower APAF1 level in the stromal–epithelial interface compared with that in the centre of the tumour tissues. A representative serial section of the tumour tissue was stained with haematoxylin and eosin (right panel). Dotted line and arrowheads indicate the stromal–epithelial interface between CAF and tumour (T). Scale bar, 10 μm. (e,f) Ovarian cancer cells transfected with miR21 precursor had lower APAF1 mRNA and protein levels than did controls. The results were the average from at least three independent experiments. Mean±s.d.; ***P<0.001 and **P<0.01; two-tailed Student's t-test. (g) The correlation between miR21 and APAF1 mRNA levels in microdissected ovarian cancer tissues was determined using a Spearman's correlation analysis. (h) Recurrent ovarian cancer PEA2 cells expressed lower APAF1 expression than did primary PEA1 cells. The results were the average from at least three independent experiments. Mean±s.d.; **P<0.01; two-tailed Student's t-test. (i) A consensus miR21-binding site was identified within the coding sequence of APAF1. (j) Co-transfection of pre-miR21 and luciferase vector with APAF1 coding sequence decreased the luciferase expression in SKOV3 cells in a dose-dependent manner. The results were the average from at least three independent experiments. Mean±s.d.; ***P<0.001, **P<0.01 and *P<0.05; two-tailed Student's t-test. (k) The luciferase activity was measured following co-transfection with either wild-type (WT) or mutated (Mut) APAF1 coding sequence vectors (mutated sequence shown in red in upper panel). The results were the average from at least three independent experiments. Mean±s.d.; ***P<0.001 and **P<0.01; two-tailed Student's t-test.