Fig 1: Age- dependent correlation of HOXA5 and PTEN protein expression. (A) HOXA5 and PTEN protein quantities are significantly down-regulated in the old cohort as determined by ELISA in total cell lysates of monocytes isolated from young and old blood donors expressed as ng per mg total protein. Error bars denote SEM (n = 30, average age: 23.0 yrs and 81.1 yrs, respectively). (B) Fifteen micrograms of total cell lysates from monocytes isolated from young and old blood donors were subjected to immuno-protein gel blotting with primary rabbit monoclonal anti-human PTEN antibody (1:10000), recognizing the 47 kDA PTEN protein. ß-actin served as loading control. (C) Delineates a moderate linear correlation between HOXA5 and PTEN protein expression as reflected in the respective correlation coefficient (r = 0.5887).
Fig 2: Age- dependent HOXA5 protein decline. HOXA5 protein levels are significantly down-regulated in monocytes from old blood donors. (A) HOXA5 protein quantities were determined by ELISA in total cell lysates of monocytes isolated from young and old blood donors expressed as ng per mg total protein. Error bars denote SEM (n = 30, average age: 23.0 yrs and 81.1 yrs, respectively). (B) Delineates a linear correlation between HOXA5 protein expression and the participants age showing decreasing protein levels with increasing age as reflected in the respective correlation coefficient (r = -0.5357). (C) Twenty-five micrograms of total cell lysates from monocytes isolated from young and old blood donors were subjected to immuno-protein gel blotting with rabbit monoclonal anti-human HOXA5 antibody ab140636 (1:500), recognizing the HOXA5 protein at 42 kDa (predicted molecular weight 29 kDa). ß-actin served as loading control. (D) delineates a linear correlation between HOXA5 expression (ng per mg total protein) and HOXA5 methylation in the target CpG site of the promoter region showing decreasing protein levels with increasing methylation as reflected in the respective correlation coefficient (r = -0.5070).
Fig 3: Age- dependent correlation of HOXA5, PTEN and p53 mRNA expression. (A) mRNA expression levels of HOXA5, PTEN and p53 are downregulated in the old subjects compared to the young cohort. (B) Moderate to strong linear correlations were observed between HOXA5 and PTEN mRNA expression (r = 0.7115) (C) between HOXA5 and p53 mRNA expression (r = 0.6294), and (D) between p53 with PTEN mRNA expression (r = 0.6685). Detection of mRNA expression was performed by qRT-PCR on total cellular RNA prepared from monocytes. Error bars denote SEM (n = 30, average age: 23.0 yrs and 81.1 yrs, respectively).
Fig 4: DNAm of the target CpG sites of DUSP3, HOXA5 and RYR1. Mean DNA methylation values of the differentially methylated CpG sites are demonstrated in young and old subjects. The associated genes were assigned to the target CpG site, respectively. Detection of mean DNA methylation was performed by microarray on bisulfite-converted DNA prepared from monocytes. Error bars denote SEM (n = 4, average age: 23.8 yrs and 81.4 yrs, respectively). P values are FDR adjusted.
Fig 5: Age- dependent HOXA5 mRNA decline. mRNA expression levels of DUSP3, HOXA5 and RYR1 are demonstrated in young and old subjects. While there was no significant difference in mRNA expression demonstrated for DUSP3 and RYR1, HOXA5 mRNA levels are significantly down-regulated in monocytes of old blood donors. Detection of mRNA expression was performed by qRT-PCR on total cellular RNA prepared from monocytes. Error bars denote SEM (n = 30, average age: 23.0 yrs and 81.1 yrs, respectively).
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