Downregulation of FTO mRNA expression in SH-SY5Y cells and its effects on ATP concentration.

<p>(<b>A</b>) SH-SY5Y cells were transfected with siRNA for 24 hours and then incubated for a total of 48 hrs (−81.8%) or 72 hrs (−69.8%) post-transfection. Additionally, SH-SY5Y cells were transfected with siRNA for 48 hrs and then differentiated for 1 week (−59.3%). GAPD mRNA was used as a positive internal control. (<b>B</b>) 3T3-L1 cells were transfected as described above and FTO expression was decreased by −80.3% at 48 hrs post-transfection, 65.3% at 72 hrs post-transfection, and 59.6% after 48 hrs of transfection and 1 week of differentiation. (<b>C, D</b>) FTO knockdown modulates ATP levels in SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA and then incubated for 48 hrs post-transfection or differentiated for 1 week. (<b>C</b>) ATP levels were increased in naïve SH-SY5Y cells treated with glucose free DMEM only (46%, p = 0.013), oligomycin (30%, p<0.0001), and differentiated SH-SY5Y cells treated with only glucose-free DMEM (16% p = 0.05), but decreased in differentiated SH-SY5Y cells treated with oligomycin (−16%, p = 0.19). (<b>D</b>) ATP levels were decreased in naïve 3T3-L1 cells treated with glucose free DMEM only (−93%, p = 0.0005), oligomycin (−76%, p = 0.00002), and differentiated 3T3-L1 cells treated with only glucose-free DMEM (−54% p = 0.0068), yet increased in differentiated 3T3-L1 cells treated with oligomycin (179%, p = 0.026). Values indicate percent of control. ATP results are average of 2 independent experiments performed in at least quadruplicate and analyzed by one-way ANOVA with post-hoc analysis with α at 0.05. (<b>E, F</b>) FTO knockdown does alter cell viability of SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA, incubated for 48 hrs and viable cells were collected and counted. (<b>E</b>) No difference was found between FTO siRNA treated SH-SY5Y cells and Control siRNA treated cells (p = 0.35). In addition, no difference in the cell viability was detected in FTO siRNA treated SH-SY5Y cells after exposure to oligomycin (p = 0.77). (<b>F</b>) There is no difference in FTO siRNA or Control siRNA treated 3T3-L1 cells (p = 0.35) or in Control siRNA treated 3T3-L1 cells treated with oligomycin compared to FTO siRNA treated cells (p = 0.131). Cell viability results are the result of experiments performed in quadruplicate and analyzed by repeated measures ANOVA with α at 0.05.