# Vitamin D Regulation of a SOD1-to-SOD2 Antioxidative Switch to Prevent Bone Cancer
## 2 Results  
In order to establish the in vitro conditions to investigate the mechanism of action toward mitochondrial dynamics, MG-63 cellular response to a 10-fold serial dilution of 1,25D\({}_{3}\) was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay (Figure 1). MG-63 cells were treated with 1,25D\({}_{3}\) at concentrations ranging from 1 pM to 1 uM, and cell viability was assessed at 24 and 48 h post treatment. In addition, two different media conditions were compared to access the synergistic effects of liganded VDR signaling. For one, a charcoal-stripped fetal bovine serum (FBS)-based media was applied to assess the singular role of VDR signaling within MG-63 osteosarcoma cells. For the other, a standard complete media with normal FBS was applied to study the synergistic effects of VDR signaling with other hormone ligands in the system. Interestingly, when cultured with charcoal-stripped media, 1,25D\({}_{3}\) was unable to inhibit viability of MG-63 cells even at the highest concentration of 1 uM for both 24 and 48 h after treatment (Figure 1A). However, in complete media a mild suppression of cell viability was observed only after 24 h of 1,25D\({}_{3}\) treatment at the higher concentration range of 10-1000 nM (\(p<\) 0.05-0.001) (Figure 1A). This suppression in cell viability became even more pronounced after 48 h of 1,25D\({}_{3}\) treatment within the lower 0.1-1000 nM range (Figure 1A; \(p<\) 0.05-0.001). Based on these studies, it appears that 1,25D\({}_{3}\) treatment over time has a significant suppressive effect on MG-63 cell viability within human serum levels down to the pmol/L range [44]. Figure 1: (**A**) The impact of 1,25D\({}_{3}\) on MG-63 cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide) assay was performed in experiments with complete serum or charcoal-stripped serum supplementation for 24 and 48 h of incubation. The cells were treated with one addition of 1 pM to 1 uM 1,25D\({}_{3}\) before the determination of cell viability. \(p\leq\) 0.05 *, 0.01 **, 0.001 *** using the 2-way ANOVA with Bonferroni post hoc test (\(n=5\); error bars represent the SD). (**B**) Logarithmic dose vs. response graph (MTT assay 24–48 h). The calculated logIC\({}_{50}\) for 1,25D\({}_{3}\) in 48 h was 1.74 and the IC\({}_{50}\) was 55.43 nM. In this figure the Hill slope is 1.0, which describes the steepness of the curve. The value R\({}^{2}\) (R square) quantifies goodness of fit. It is a fraction between 0.0 and 1.0 and has no units. Higher values indicate that the model fits the data better. In this graph, R square is 0.8773 and degree of freedom = 34. (**C**) Degree of inhibition by 1,25D\({}_{3}\) on MG-63 osteosarcoma cells after 48 h of treatment. The maximal inhibition value was obtained with 1 uM 1,25D\({}_{3}\) and arbitrarily set to 100%. Based on our dose curve for MG-63 cells, the IC\({}_{50}\) value (i.e., the concentration that provides a response halfway between the maximal and the minimally inhibited response) for 1,25D\({}_{3}\) was determined (Figure 1A). The 1,25D\({}_{3}\) concentrations were transformed to log value, whereby the model assumes that the response curve has a standard slope equal to a Hill slope (or slope factor) of \(-\)1.0. As there were no maximal responses for the 24-hour complete media and 24/48-hour charcoal-stripped media treated samples, the IC\({}_{50}\) values could not be determined in these cases. For the 48-hour complete media condition, the best-fit IC\({}_{50}\) value based on non-linear regression was 55.43 nM (logIC\({}_{50}\) = 1.74), whereby the goodness of fit R\({}^{2}\) value was 0.8773, and correlated with the transformed percentinhibition data (Figure 1C). In order to study the involvement and regulatory mechanisms of 1,25D\({}_{3}\) on mitochondrial dynamics, 10 nM and 100 nM concentrations were further investigated which encapsulates the determined IC\({}_{50}\) value.