Figure 1.

Activation of autophagy following exposure to CoPs in murine calvaria resorption models and osteoblast cells. (A) Immunofluorescence was performed to examine the expression of LC3 in osteoblasts. Sections of murine calvaria are presented for animals from each group. Scale bar: 50 µm. Red, LC3; green, osteoblasts (BGLAP); blue, DAPI nuclear staining. (B) Immunofluorescence was performed to examine the expression of LC3 in osteoblast cells. Cells were treated with 50 µg/ml CoPs for 12 h. Scale bar: 10 µm. Red, LC3; blue, DAPI nuclear staining. (C) Quantification of the percentage of cells with LC3 puncta shown in (B). Data are presented as the means ± S.E.M. from 3 independent experiments. **, P < 0.01 vs. control. (D) Western blots performed after osteoblast cells were treated with various concentrations (0, 10, 50, 100 µg/ml) of CoPs for 12 h. Rap, rapamycin. (E) Western blots performed after osteoblast cells were cultured in CQ for 3 h prior to being treated with 50 µg/ml CoPs for 12 h. (F) Transmission electron microscopy of osteoblasts cultured with or without 10 mM 3-MA prior to being treated with 50 µg/ml CoPs for 12 h. The double-membrane autophagosome (AP) containing CoPs clusters and the rough endoplasmic reticulum are shown in the middle and right images of the upper panel. High-magnification view of an autolysosome containing CoPs clusters, partially degraded ribosomes and rough endoplasmic reticulum in the middle image of the lower panel. AVd, degradative autophagic vacuole.