Fig. 2
MΦ uptake of miR-375 as a non-exosome entity. a, b Primary human MΦ were cocultured with MCF-7 cells for 24 h. a One hour before and during the coculture period, MΦ were treated either with vehicle, cytochalasin B, nocodazole, and b carbenoxolone. MiR-375 abundance was quantified via qPCR and normalized to untreated MΦ or untreated coculture MΦ, respectively (n ≥ 3). c MiR-375 was measured by qPCR in the supernatants of MΦ, viable MCF-7 cells (MCF-7), STS-treated apoptotic MCF-7 cells (ap MCF-7), media, and normalized to untreated MΦ. Synthetic cel-miR-39a was used as spike-in control (n ≥ 2). d VCM and ACM of ER+ (EFM-192A, MCF-7, T47D) and ER− (MDA-MB-468, MDA-MB-231, SKBR3, HCC1937) breast carcinoma cells, mammary epithelial cells (MCF-10A), and primary mammary epithelial cells (HMEC) were analyzed for the abundance of miR-375 (n = 3). e MiR-375 level was measured by qPCR in MΦ cocultured with STS-treated apoptotic MCF-7 cells for 4 h. MCF-7 cells were removed from cocultures and MΦ were further cultivated for 20 h (24 h time point). Data are normalized to control MΦ (n = 5). f MΦ were treated with STS as a control, or 1:1 diluted supernatants of viable (VCM) or apoptotic (ACM) MCF-7 cells for 30 min. Cells were washed and further cultured in MΦ media for 4 and 24 h, and miR-375 abundance was measured and normalized to untreated MΦ (n ≥ 5). g ACM was incubated with control or 50 µg/mL RNase A at 37 °C for indicated time. Before RNA isolation, cel-miR-39a was added as a normalization control. MiR-375 abundance was quantified by qPCR and normalized to control ACM (n ≥ 3). h MΦ were incubated for 30 min with either MCF-7 control ACM or RNase A-treated ACM. Cells were washed and cultured for another 24 h in MΦ media. MiR-375 level was determined and normalized to untreated MΦ (n = 5). Data of a–h are mean ± SEM and p-values were calculated using one-sample t-test. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant
