Figure 4
Upregulation of RNF182 in AD brains and in NT2 neurons treated the cell death-inducing stresses. A. Changes in mRNA levels of RNF182 transcript in control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from pooled mRNA of 4 AD and 5 age-matched control subjects. The value of the control sample was set at 100%. The percentage of the AD sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between the AD sample and the control sample. The experiments were performed in triplicate. Asterisk indicates a significant difference (ρ < 0.005; t-test). B. Changes in mRNA levels of RNF182 transcript in individual control and AD brains were determined by qRT-PCR. The cDNA samples were prepared from mRNA of 5 AD and 5 age-matched control subjects. The qRT-PCR results were calculated against the average result (control mean) of the control samples, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between each sample and the control mean. The experiments were performed in triplicate. C. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD and OGD with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). D. Changes in RNF182 protein levels in NT2 neurons treated with OGD and OGD plus 16 h recovery were determined by Western blotting with anti-RNF182 antibody using 120 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. E. Changes in mRNA levels of RNF182 transcript in NT2 neurons treated with OGD plus 20 μM β-amyloid peptide and OGD plus 20 μM β-amyloid peptide with 16 h recovery were determined by qRT-PCR. The samples were measured against the cDNA of untreated NT2 neurons as a control, set at 100%. Percentage of each treated sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between treated sample and the control sample. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). Insert: Nuclear morphology of OGD plus Aβ treated cells was examined under an Olympus B x 50 fluorescence microscope after fixing and staining the cells with DAPI.