Fig. 5

miR-100-5p and miR-298-5p induce neuronal injury in vitro. a Representative images of C57BL/6 (wild-type, WT) cortical neurons co-cultured with WT microglia. Cells were incubated with 5 μg/ml of indicated miRNAs for 5 d. Cell cultures were subsequently immunostained with NeuN antibody (green) and stained with TUNEL apoptosis assay (red) and DAPI (blue). LPS (100 ng/ml) and loxoribine (1 mM) served as positive control. Mutant control oligonucleotide and unstimulated cells were used as negative control. Scale bar, 20 μM. b, c Quantification of NeuN- and TUNEL-positive neurons in co-cultures containing neurons and microglia. Data are expressed as mean ± SD, n = 4. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 compared to control, two-tailed Student’s t-test. d Representative images of enriched WT cortical neurons, which were incubated with 5 μg/ml of indicated miRNAs for 5 d. Cell cultures were subsequently immunostained with NeuN antibody (green) and stained with DAPI (blue). LPS (100 ng/ml) was used to test for potential relevant contamination with microglia. Mutant control oligonucleotide and unstimulated cells were used as negative control. Scale bar, 20 μM. e Both WT and Tlr7^−/− neurons were incubated with 5 μg/ml of indicated miRNAs for 5 d. LPS (100 ng/ml) was used to test for potential relevant contamination of the enriched neuron cultures with microglia. Mutant control oligonucleotide and unstimulated cells were used as negative control. Subsequently, NeuN-positive cells in WT and Tlr7^−/− cortical neuron cultures were quantified. Data are depicted as relative neuronal viability determined in cell cultures treated with the indicated miRNA compared to control. Results are shown as mean ± SD (n = 4 for WT, n = 3 for Tlr7^−/− neurons). **P < 0.01 WT vs. Tlr7^−/−. P values for relevant groups as determined using the two-tailed Student’s t-test are shown. f Enriched WT cortical neurons were incubated with indicated concentrations of synthetic miRNAs for 5 d. Cultures were immunostained as described above, and NeuN-positive cells were quantified. LPS was used to test for potential relevant contamination with microglia. Mutant control oligonucleotide and unstimulated cells were used as negative control. Data are depicted as relative neuronal viability determined in cell cultures treated with the indicated miRNA. Results are shown as mean ± SD, n = 4