Effect of M-cadherin RNAi on apoptosis in confluent C2C12 myoblasts.

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Effect of M-cadherin RNAi on apoptosis in confluent C2C12 myoblasts. Effect of M-cadherin RNAi on apoptosis in confluent C2C12 myoblasts. (A) Representative phase-contrast images of C2C12 myoblasts obtained 48 hours after seeding at low density (2.0×103/cm2) or high density (2.1×104/cm2) (top). Scale bar: 200 μm. Immunoblots of cleaved caspase-3 and PARP obtained 48 hours after plating (middle). Cytosolic nucleosomes of low- or high-density cells were measured as an indication of apoptotic DNA fragmentation (bottom). Each data point represents the mean ± s.e.m. of three independent experiments. *P<0.05 vs the low-density group. (B) Expression pattern and protein abundance of M-cadherin at low and high cell densities. Representative confocal images of M-cadherin (red) and DAPI (blue) staining in C2C12 myoblasts at low or high cell density as described in A (top). Scale bar: 10 μm. Immunoblotting analysis of protein abundance of M-cadherin. β-tubulin was probed as a loading control (bottom). (C–H) 80% confluent C2C12 myoblasts were transfected with M-cadherin-targeted siRNA (M-) or non-targeted scramble siRNA (SiCON) as a control. Non-transfected cells with identical culture conditions were used as a normal control (NC) cells. The cells were harvested 48 hours after transfection. Each data point is the mean ± s.e.m. of three independent experiments. *P<0.05 vs the control groups. (C) Phase-contrast images of transfected cells. Scale bar: 100 μm. (D) Immunoblot analysis of transfected cells. (E) The apoptotic DNA fragmentation of transfected cells is shown as mean ± s.e.m. of three independent experiments. *P<0.05 vs both NC and SiCON control groups. (F) Proteins associated with apoptotic signaling (and the relevant control proteins) measured in transfected and control cells (left). The integrity and purity of protein subcellular fractions was verified by immunoblotting with appropriate control antibodies (right). (G) Digital images from control or transfected C2C12 cells stained with NAO (top). Scale bar: 20 μm. The median fluorescence intensity of NAO per cell was determined (bottom). The data are mean ± s.e.m. of three experiments. (H) Mitochondria were isolated from control and transfected cells and stained with JC-1. The data represent FACS analyses of the ratio of orange (intact mitochondria) to green (compromised Δψm) mitochondria. Yan Wang et al. J Cell Sci 2011;124:3835-3847 © 2011.