Fig. 4: MiR-27b regulated PAN-induced apoptosis in NHP podocytes.

a miR-27b was significantly suppressed upon PAN treatment. Cells were exposed to 10 µg/ml PAN for 3–5 days before collected for RT-qPCR. Relative miR expression was calculated using U6 as normalization control. Error bar represents data from two independent experiments. b PAN-induced miR-27 change was also observed in primary rat podocytes. Cells were treated with PAN for 3 and 5 days before collected for miR-qPCR analysis. Error bar represents data from two independent experiments. c miR-27b was efficiently transfected into NHP podocytes. 50 nM miRNA mimic and siControl were transfected into NHP podocytes using Lipofectamine RNAiMax. Cells were collected at day 3 and day 5 post transfection and miRNA expression was analysis using miR-X kit (Clontech). Error bar represents data from two independent experiments. Data were normalized to U6. d Endogenous miR-27b was efficiently knocked-down by transfecting miR-27b hairpin inhibitor into NHP podocytes. miR inhibitor control and miR-27b inhibitor were transfected into NHP podocytes at the final concentration of 50 nM. Cells were collected for miRNA qPCR at day 3 and day 5 post transfection. Data were normalized to U6. Error bar represents data from two independent experiments. e miR-27b overexpression enhanced PAN-induced cell death while miRNA inhibition led to increased cell viability. Cells were transfected with 50 nM miRNA mimic or inhibitor and treated with 10 µg/ml PAN for 5 days before collected for cell viability test using CellTiter 96 kit (Promega). Relative cell viability was calculated by normalizing absorbance readings from PAN-treated samples to non-treated controls. Error bar represents data from three independent experiments with eight replicates. **p < 0.01 by Student's t-test. f miR-27b overexpression led to increased caspase3 activation. Cells were transfected with 50 nM miRNA mimic and treated with 50 µg/ml PAN for 36 h before fixation and immunostaining. Scale bar: 200 µm. g Cleaved caspase-3 expression was increased by miR-27b overexpression in NHP podocytes. Cells were treated with 50 µg/ml PAN for 48 h before collected for active caspase3 by western blotting. Actin was used as the loading control. h miR-27b overexpression led to increased caspase3/7 activation. Cells were treated with 50 µg/ml PAN for 36 h before collected for /7 glo assay (Promega). Error bars represent data from eight replicates. *p < 0.05 by Student's t-test. i miR-27b overexpression led to increased caspase3/7 activation. Cells were treated with 10 µg/ml PAN for 36 h before collected for /7 glo assay (Promega). Error bars represent data from eight replicates. *p < 0.05 by Student's t-test