Fig 2: Orai1 downregulation reduces the proliferation of MC3T3-E1 cells and causes their cell cycle arrest. Control siRNA and Orai1 siRNA transfected into MC3T3-E1 cells for an appropriate period. The 2-APB stock solution was diluted to a concentration of 50 μM in the cell culture medium. DMSO alone was dissolved as negative (vehicle) control. After that, cell proliferation and cell cycle assays were performed at an appropriate period. a The cell proliferation was evaluated by the MTT assay of MC3T3-E1 cells transfected with either control siRNA, Orai1 siRNA, DMSO or 2-APB. b Apoptosis was evaluated by Tunel staining of the MC3T3-E1 cells transfected with either control siRNA or Orai1 siRNA (Scale =100 μm). c The cell cycle distribution was investigated using PI staining of the MC3T3-E1 cells transfected with either control siRNA, Orai1 siRNA, DMSO or 2-APB. (Data are presented as means ± SD from three independent experiments. *p < 0.05 as compared with the control

Fig 3: Orai1 downregulation lowers the expression of cyclin D1 in MC3T3-E1 cells. a Western blotting was performed to assess the expression levels of cyclin D1, cyclin E, CDK4, and CDK6 in the MC3T3-E1 cells transfected with either control siRNA or Orai1 siRNA. b Real-time PCR was carried out to measure mRNA levels of cyclin D1, cyclin E, CDK4, and CDK6 in the MC3T3-E1 cells transfected with either control siRNA or Orai1 siRNA. Data are presented as means ± SD from three independent experiments. *p < 0.05 as compared with the control

Fig 4: Orai1 downregulation reduces the proliferation of osteoblasts by inactivating the Ras-NF-κB signaling pathway. a The amounts of Ras-GRF and p-p65-NF-κB were measured in the MC3T3-E1 cells transfected with either control siRNA or Orai1 siRNA. ARS-853 was dissolved in DMSO to prepare a stock solution. After MC3T3-E1 cells were transfected with either the control siRNA or Orai1 siRNA, the ARS-853 stock solution was diluted to a concentration of 25 nM in the cell culture medium. DMSO alone was dissolved as negative (vehicle) control. After that, western blotting and cell proliferation and cell cycle assays were performed after incubation with or without the Ras signaling pathway inhibitor ARS-853 (25 nM) for an appropriate period. b After incubation with or without ARS-853, the amounts of p-p65-NF-κB and cyclin D1 were evaluated in the MC3T3-E1 cells transfected with either the control siRNA or Orai1 siRNA. c After incubation with or without ARS-853, the proliferation rate of the MC3T3-E1 cells (transfected with either the control siRNA or Orai1 siRNA) was evaluated. d After incubation with or without ARS-853, the cell cycle distribution was investigated for the MC3T3-E1 cells transfected with either the control siRNA or Orai1 siRNA. Data are presented as means ± SD from three independent experiments. *p < 0.05 as compared with the control