Fig. 5

BP-CM could enhance the osteo/odontogenic capability of BMMSCs through autophagy. a BMMSCs of each group were cultured for an hour simultaneously, and culture medium was replaced with BP-CM at different time points (15 min, 30 min, and 60 min). Western blot was conducted to detect the autophagy-related markers (LC3, Beclin1, and p62). Quantification was done by ImageJ, *P < 0.05 and ***P < 0.001. b BMMSCs were divided into four groups and treated with different medium for 30 min. The normalized expression of p62 and Beclin1, and the ratio of LC3II/LC3I represented the relative expression. ***P < 0.001. c TEM pictures of autophagosomes showed that BP-CM-induced BMMSCs had more autophagic vacuoles as compared with cells in the control group. Autophagosomes are indicated by black arrows. Scale bar = 2 μm. d The images of LC3 (red) and DAPI (blue) in BMMSCs cultured with different media for 30 min were observed by immunofluorescence staining. Scale bar = 50 μm. Quantification was done by ImageJ. ***P < 0 001. e, f The effects of autophagy inhibitor 3-MA on osteo/odontoblastic-associated markers of BP-CM-induced BMMSCs were investigated by western blot and real-time RT-PCR. *P < 0.05, **P < 0.01, and ***P < 0.001. g, h Suppression of autophagy decreased the expression of ALP and mineralization of BMMSCs, while complete culture medium was served as a blank control and BP-CM was served as a positive control. Scale bar = 100 μm, ***P < 0.001