In subsequent experiments, after treatment with plain medium, empty vector, gC1qR vector, negative control siRNA vector or gC1qR siRNA vector for the indicated time periods, ROS generation was determined

using H2DCFDA fluorescence and quantified by flow cytometric analysis. The data showed that ROS generation reached a maximal level at 60 hr after the initial manipulation, and ROS levels in the gC1qR vector group were increased by approximately 3.18-fold compared with empty HM781-36B clinical trial vector-treated HTR-8/SVneo and HPT-8 cells (Fig. 3C). Cytosolic Ca2+ levels were determined using a fluorescent ELISA reader, and the results revealed a notable increase at 84 hr after the initial manipulation (Fig. 3D). At this time, the [Ca2+]i concentration

in the gC1qR vector group was 3.6-fold higher than that in the empty vector-treated HTR-8/SVneo and HPT-8 cells. The gC1qR siRNA vector group showed no changes compared with the negative siRNA vector-treated HTR-8/SVneo and HPT-8 cells. Time-dependent changes in relative Δψm values in gC1qR-overexpressing Selleckchem KU-60019 HTR-8/SVneo and HPT-8 cells were also explored. We used the JC-1 dye to monitor the estimated Δψm using the 590:527 nm emission ratio at specific time points from 0 hr to 84 hr following transfection. The value of Δψm in the gC1qR vector group decreased approximately 61.8% compared with the empty vector group at 84 hr. There was no difference in Δψm in HTR-8/SVneo and HPT-8 cells between the negative control siRNA and gC1qR siRNA groups after the initial manipulation (Fig. 3E). Additionally, we evaluated the production of ATP. As shown in Fig. 3F, the ATP concentration was notably decreased in cells transfected with the gC1qR vector compared with the empty vector group. In contrast, no significant change in the ATP concentration was observed in cells treated with the negative control siRNA and gC1qR siRNA

vectors (P > 0.05). To investigate whether the effects of the gC1qR gene on ROS generation and intracellular Ca2+ influx were interlinked, gC1qR vector-mediated gC1qR-overexpressing cells were treated either with the antioxidant Oxymatrine PDTC (25 μm) or with EGTA a Ca2+ ion chelator (30 μm). As shown in Fig. 4A, there was a twofold decrease in ROS generation in the presence of the Ca2+ ion chelator EGTA. Furthermore, the intracellular Ca2+ level was diminished by 68% after treatment with PDTC (Fig. 4B). The data indicated that inhibition of Ca2+ accumulation by EGTA diminished ROS generation. Similar results were also demonstrated in that blocking excess ROS generation with PDTC decreased the Ca2+ levels. Metformin can promote mitochondrial biosynthesis.