Figure 1.
IGFBP3 expression is down-regulated in an APP-mutant cell line and transgenic mice IGFBP3 mRNA expression in normal and APP-Swedish mutant H4 cells (A), and the brain of wild type and PSEN1-APP transgenic mice (B6C3-Tg(APP695)85Dbo Tg(PSEN1)85Dbo) (C) was measured by transcriptome sequencing analyses and qPCR.
Expression of IGFBP3 protein in normal and APP-Swedish mutant H4 cells was detected using western blot analyses (B). IGFBP3 protein in APP-Swedish mutant H4 cells is expressed relative to IGFBP3 protein levels in normal H4 cells. Data are represented as the mean ± standard deviation (SD) of triplicate experiments. Statistical analyses were performed using t-tests (* indicates p<0.05). H4-sw, APP-Swedish mutant H4 cells; Hippo, hippocampus; FC, frontal cortex; CB, cerebellum.
Figure 2.
IGFBP3 protects cells from Aβ1–42 induced apoptosis H4 and H4-sw cells were treated for 24 h in serum-free media (SF) with 5 µM soluble Aβ1–42 in the presence or absence of exogenous recombinant human IGFBP3 (BP3) protein.
IGFBP3 knockdown H4 cells generated by siRNA transfection or non-targeted siRNA-transfected H4 cells were treated with or without 5 µM soluble Aβ1–42 in SF media for 24 h. Apoptosis was evaluated after propidium iodide staining using FACS analyses. Compiled FACS results from three independent experiments are graphically illustrated in (A) and (B). Caspase 3 activation was examined in H4 cells by detecting cleaved forms of caspase 3 using western blot analyses. Representative results are illustrated and graphical values from densitometric analyses after normalization to β-actin are reported as relative values to that of untreated control (C). Rat hippocampal neuronal primary cells in supplements-free media were treated for 24 h with 500 nM oligomeric Aβ1–42 in the presence or absence of exogenous recombinant human IGFBP3 (BP3) protein. Caspase 3 activation was determined by detecting cleaved forms of caspase 3 using western blot analyses (D). Data are the mean ± SD of three independent experiments. Statistical analyses were performed using two-way analysis of variance (ANOVA) and Bonferroni post-tests. * indicates p<0.05. H4-sw, APP-Swedish mutant H4 cells; BP3, IGFBP3; siNC, non-targeting siRNA; siBP3, IGFBP3 siRNA.
Figure 3.
Hypermethylation of CpG islands within the IGFBP3 promoter in APP-Swedish mutant cells Schematic diagram of the genomic region (+60 to −290) of IGFBP3 that was analyzed for methylation status.
The CpG island is represented as a box (−240 to +10). Thin horizontal lines indicate each CpG site. The bent arrow indicates the transcription start site (+1) and the thick vertical solid line indicates the target CpG sites for pyrosequencing analyses (A). Methylation status analyses were conducted using bisulfite sequencing analyses, the 454 GS-FLX system, and bisulfite pyrosequencing. Individual bars represent the percentage of methylation at the corresponding CpG site within the IGFBP3 promoter (A). Representative pyrograms are shown for each sample with the percentage methylation at each of the five CpG sites tested (B). Average percent methylation of triplicate pyrosequencing analyses at each of the five CpG sites are presented graphically (B). Data are shown as the mean ± SD of triplicate experiments. Statistical analyses were performed using t-tests (* indicates p<0.05). H4-sw, APP-Swedish mutant H4 cells.
Figure 4.
IGFBP3 expression changes following demethylation in APP-Swedish mutant cells H4-sw cells were treated for 3 days with 10 µM 5-aza-2′-deoxycytidine.
DNA methylation status at specific CpG sites were analyzed using bisulfite pyrosequence analyses. The average percent methylation of triplicate pyrosequencing analyses from each of the five CpG sites are presented graphically (A). After treatment with 5-aza-dC, IGFBP3 mRNA expression was measured using qPCR (B). IGFBP3 protein expression was detected using western blot analyses. Representative results are illustrated and values from densitometric analyses after normalization to β-actin are reported relative to that of untreated controls (C). Data are shown as the mean ± SD (n = 3). Statistical analyses were performed using t-tests (* indicates p<0.05). 5aza, 5-aza-2′-deoxycytidine.
Figure 5.
CpG island methylation is altered in the IGFBP3 promoter region in H4 cells treated with Aβ1–42 H4 cells were treated for 5 days with different concentrations of Aβ1–42.
DNA methylation at the -164 and -173 CpG sites was analyzed using bisulfite sequencing analyses (A). Each circle represents CpG dinucleotides. The methylation status of each CpG site is illustrated by black (methylated) and white (unmethylated) circles. The total percentage of methylation at specific CpG sites is indicated as a pie graph. The black segment of the pie graph indicates methylated CpG percentage whereas the white segment represents the unmethylated CpG percentage (A). IGFBP3 expression after treatment with Aβ1–42 was determined using qPCR (B) and western blot analyses (C). Graphs depict compiled data from three independent experiments and values are relative to those of untreated controls. Data are shown as the mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA and Bonferroni post-tests (* indicates p<0.05).