Fig 1: Western blot analysis of mitochondrial proteins. (a) 20 µg of total cellular proteins from various cybrid cell lines was electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with respiratory complex subunits in mutant and control cells with VDAC as a loading control. (b) Quantification of 6 respiratory complex subunits. The levels of ND6, ND4, ATP6, CO2, and ND1 in two mutant cell lines and two control cybrid cell lines were determined. The error bars indicate two standard errors of the means. *P < 0.05 and **P < 0.01 indicate the significance, according to the t-test, of the differences between mutant and control cybrid cell lines.
Fig 2: In vitro treatment with Na2SeO3 attenuated intracellular OS and restored the mitochondrial functions of OP-BMMSCs. (A) Na2SeO3 increased the enzyme activity of total GPx in a dose-dependent manner, n = 4. (B) Gpx1 gene expression was quantified with real-time RT-PCR, n = 4. (C) The protein level of GPx1 was determined using Western blot, n = 3. (D) Treatment with Na2SeO3 significantly reduced the intracellular ROS, n = 3. (E) Treatment with Na2SeO3 attenuated mitochondrial superoxide level, n = 3. (F) ATP content was measured in Na2SeO3-treated OP-BMMSCs, n = 3. (G, H) Treatment with Na2SeO3 improved MMP in OP-BMMSCs, n = 3. (I) The gene expressions of Nd4, sdha, Cox4 and Atp5a were quantified with real-time RT-PCR, n = 4. (J) The protein levels of ND4, SDHA, COX4 and ATP5A were determined using Western blot, n = 3. Values represent mean ± SEM. Statistically significant differences are indicated by * where P < 0.05 or ** where P < 0.01 between the indicated groups.
Fig 3: BMMSCs derived from OVX rats (OP-BMMSCs) exhibited decreased osteogenic potential, increased OS and impaired mitochondrial functions. (A) BMMSCs derived from normal (N-BMMSCs) and OVX rats (OP-BMMSCs) were induced toward osteogenic differentiation for 21 days. Representative images of mineralized extracellular matrix stained by ARS. Scale bar = 200 µm. (B) Comparison of matrix mineralization between N-BMMSCs and OP-BMMSCs, n = 4. (C) The gene expressions of osteoblast-specific markers were quantified with real-time RT-PCR, n = 4. (D) The enzyme activity of total GPx was compared between N-BMMSCs and OP-BMMSCs, n = 4. (E) The transcript level of Gpx1 was quantified with real-time RT-PCR, n = 4. (F) The protein level of GPx1 protein was determined using Western blot, n = 3. (G) The intracellular ROS was measured by flow cytometry, n = 3. (H) The mitochondrial superoxide level was compared between N-BMMSCs and OP-BMMSCs, n = 3. (I) ATP content was evaluated, n = 3. (J) MMP level was determined by flow cytometry, n = 3. (K) The gene expressions of Nd4, sdha, Cox4 and Atp5a were quantified with real-time RT-PCR, n = 4. (L) The protein levels of ND4, SDHA, COX4 and ATP5A were determined using Western blot, n = 3. Values represent mean ± SEM. Statistically significant differences are indicated by * where P < 0.05 or ** where P < 0.01 between the indicated groups.
Fig 4: Inhibition of Gpx1 expression by siRNA abrogated the protective effect of Na2SeO3 on the osteogenic potential and mitochondrial antioxidant functions of OP-BMMSCs. OP-BMMSCs were pre-treated with siRNA to inhibit Gpx1 expression and then exposed to 100 nM Na2SeO3. (A) RT-PCR revealed that transfection with Gpx1-siRNA inhibited the gene expression of Gpx1, n = 4. (B) Western blot confirmed that the protein level of GPx1 in siRNA-treated OP-BMMSCs was significantly down-regulated, n = 3. (C) The enzyme activity of total GPx was significantly decreased after siRNA transfection, n = 4. (D) The intracellular ROS has increased in siRNA-transfected cells even exposure to Na2SeO3, n = 3. (E) Inhibition of Gpx1 increased mitochondrial superoxide production, n = 3. (F) Transfection with Gpx1-siRNA reduced the ATP content in Na2SeO3-treated cells, n = 3. (G) Transfection with Gpx1-siRNA decreased the MMP in Na2SeO3-treated cells, n = 3. (H) Inhibition of Gpx1 significantly down-regulated the gene expressions of Nd4, sdha, Cox4 and Atp5a, n = 4. (I) Western blot confirmed that treatment with Gpx1-siRNA decreased the protein levels of ND4, SDHA, COX4 and ATP5A, n = 3. (J) Inhibition of Gpx1 suppressed the matrix mineralization of Na2SeO3-treated cells. Scale bar = 200 μm, n = 4. (K) Quantification of the stained mineral layers, n = 4. (L) Inhibition of Gpx1 down-regulated the gene expressions of osteoblast-specific markers in Na2SeO3-treated cells, n = 4. Values represent mean ± SEM. Statistically significant differences are indicated by * where P < 0.05 or ** where P < 0.01 between the indicated groups.
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