Fig. 2
CircSIPA1L1 stimulates DPSC osteogenesis. a Western blot results revealed that the protein levels of OSX, RUNX2, and ALP significantly increased in the circSIPA1L1 group compared with the NC group. b Western blot assay showed higher protein levels of ALP, RUNX2, and OSX in the Si-NC group than the Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group, respectively. GAPDH was the internal control. c Images of alkaline phosphatase (ALP) staining in the different groups, and Si-NC treatment led to the highest ALP activity. Cells were cultured for 7 days. After 14 days of co-culture, the formation of mineralized nodules in DPSCs in the circSIPA1L1 group generated more calcified nodules than the NC group; meanwhile, the Si-NC group has more calcified nodules than Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group. d Immunofluorescence staining showed positive expressions of ALP and OSX in DPSCs transfected with si-NC, si-circSIPA1L1-1 or si-circSIPA1L1-3, respectively. *P < 0.05, **P < 0.01. e The mRNA levels of OSX, RUNX2, and ALP in DPSCs measured by RT-PCR following the 3-day osteogenesis. The results showed higher levels of ALP, RUNX2, and OSX in the circSIPA1L1 group than the NC group. f Histograms showed quantification of Alizarin red staining by spectrophotometry of the NC group and circSIPA1L1 group. g H&E staining and Masson staining Si-NC, si-circSIPA1L1-1, and si-circSIPA1L1-3 groups. H&E and Masson staining showed less bone-like structures and collagen deposits in DPSCs of the circSIPA1L1-downexpressing group than the control group. Bone/dentin-like tissues (arrow), S around the scaffold, scale bar = 100 μm. h The results of RT-PCR showed that the expression of ALP, RUNX2, and OSX were decreased in the Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group compared with the Si-NC group. n = 3. **2-ΔΔCt > 2, P < 0.01; *1 < 2-ΔΔCt < 2, P < 0.05. i The quantification of Alizarin red staining by spectrophotometry revealed that si-circSIPA1L1-1 or si-circSIPA1L1-3 showed less calcified nodules than si-NC group