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. 2018 Feb 16;41(5):2535–2544. doi: 10.3892/ijmm.2018.3498

Figure 4.

Figure 4

AMPK activation enhances differentiation and mineralization of MC3T3-E1 cells via the induction of autophagy. (A) Cells were treated with AICAR (10 μM) in the presence or absence of 3-MA (5 mM) or CQ (10 μM) for 24 h, after which western blot analysis was conducted. (B) Semi-quantitative results of western blot analysis. (C and D) Cells were cotreated with AICAR (10 μM) and 3-MA (5 mM) or CQ (10 μM) for 8 days. (C) 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining was used to detect ALP activity. (D) ALP, OCN and Runx2 mRNA expression was detected using reverse transcription-polymerase chain reaction. (E) Cells were cultured for 21 days; representative images (magnification, ×100) of Alizarin red staining are presented. (F) Quantification of Alizarin red staining. *P<0.05, **P<0.01 compared with the control group; #P<0.05, ##P<0.01 compared with the AICAR(A++) group. 3-MA, 3-methyladenine; AICAR, 5-aminoimdazole-4-carboxamide-1-β-D-ribofuranoside; ALP, alkaline phosphatase; AMPK, adenosine monophosphate-activated protein kinase; CQ, chloroquine diphosphatase; OCN, osteocalcin; Runx2, runt-related transcription factor 2.