The total number of subjects in each diagnostic category.

<p>(AD: Alzheimer’s disease; OD: Other dementia; AD/PD: Alzheimer’s disease with Parkinson’s disease; MCI: Mild cognitive impairment; PD: Parkinson’s disease; PD D: Parkinson’s disease with dementia; OPTIMA: Oxford Project to Investigate Memory and Ageing; PD GEN: Parkinson’s disease DNA bank).</p><p>The total number of subjects in each diagnostic category.</p

DNA distribution histograms from HEK293 cells transfected with p21^cip1.

<p>Representative DNA distribution histograms of single cells from the Acumen cytometer (black bars). The debris and multiple cells (white bars) were excluded from the analysis. <b>Panel A</b>: Typical DNA content histogram of single cells following the transfection with the empty vector (EV NC). <b>Panel B</b>: Typical DNA content histogram of single cells following transfection with the common variant of p21^cip1. <b>Panel C:</b> Typical DNA content histogram of single cells following transfection with the rare variant of p21^cip1.</p

Kaplan-Meier probability distribution of disease free survival prior to the age of 75.

<p>The disease in question was dementia in Parkinson’s disease. The graph shows the disease free survival probability of subjects prior to the age of 75, in subgroups defined by the p21^cip1 genotype. Prior to the age of 75, the variant p21^cip1 was significantly associated with a reduction in the disease free survival compared to the common p21^cip1 (hazard ratio: 3.239, p-value < 0.001). The x-axis represents the age in years. The y-axis represents the survival probability expressed as a percentage. The solid black line represents subjects that were homozygous for the common p21^cip1. The broken grey line represents subjects that were heterozygous or homozygous for the variant p21^cip1.</p

Odds ratios for disease in relation to the rare p21^cip1 variant.

<p>The OPTIMA and PD Gen cohorts were separated into groups defined by diagnosis based on the diagnostic criteria outlined in the methods. Odds ratios in relation to the p21^cip1 variant were calculated for the disease groups compared to age-matched controls.</p><p>Odds ratios for disease in relation to the rare p21^cip1 variant.</p

The effect of the p21^cip1 genotype on beta-actin expression.

<p>The graph shows the amount of beta-actin detected per cell expressed as a percentage change relative to the empty vector negative control. Beta-actin expression was determined by Acumen Cytometry with beta-actin immunostaining: with the total amount of beta-actin detected divided by the total number of cells in the sample. The cells were not double stained for p21^cip1 protein, so we were unable to differentiate the p21^cip1 positive population (transfected cells) from the p21^cip1 negative population (non-transfected cells). X-axis: EV NC: Empty vector negative control; com p21: cells transfected with common p21^cip1; var p21: cells transfected with variant p21^cip1. The y-axis represents the percentage change in beta-actin expression per cell relative to the EV NC. The top of the bars represent the mean. The error bars represent the SEM.</p

The effect of the p21^cip1 genotype on the age of onset of AD.

<p>Of the subjects with advanced AD at post-mortem, the subjects with the variant p21^cip1 had a significantly lower age of onset than subjects with the common p21^cip1 (p-value: 0.016). The x-axis represents the p21^cip1 genotype, with com and var representing subjects with the common and variant p21^cip1 respectively. The y-axis represents the age at onset of AD in years. The top of the bars represent the mean. The error bars represent the standard error of the mean (SEM). Statistical test: one-way ANOVA.</p

The allele frequency of the p21cip1 variant in groups defined by the severity of AD.

<p>The diagnosis was defined according to the Braak staging system: with entorhinal, limbic and neocortical stage subjects in a pre-clinical, mild and advanced stage of AD respectively.</p><p>The allele frequency of the p21cip1 variant in groups defined by the severity of AD.</p

The ratio of p21^cip1 protein to mRNA in the cells transfected with the different variants of p21^cip1.

<p>The p21^cip1 protein and mRNA results were normalised to the equivalent value for beta-actin.</p><p>The ratio of p21^cip1 protein to mRNA in the cells transfected with the different variants of p21^cip1.</p

The effect of the p21^cip1 genotype on the expression of tau pathology in the brain.

<p><b>Panel A and B</b>: The graphs shows the mean p-tau levels in the brain of subjects in subgroups defined by the severity of AD and the p21^cip1 genotype (Panel A: frontal lobe, Panel B: occipital lobe). The x-axis represents the severity of AD as defined by Braak: E = entorhinal stage, L = limbic stage, N = neocortical stage. The y-axis represents the amount of p-tau detected in the relevant lobe by ELISA with a marker for AT8 (arbitrary units). Light grey bars: subjects with common p21^cip1; dark grey bars: subjects with variant p21^cip1. Statistical test: Kruskal Wallis. <b>Panel C, D, E and F</b>: Z-scores were calculated for the amount of p-tau and NFT detected in the temporal, frontal and occipital lobe of each subject, taking into account the severity of AD as defined by Braak. This eliminated the need for subgroups defined by the disease severity. The graphs show the mean z-scores in subgroups defined by the p21^cip1 genotype. Panel C: p-tau in the frontal lobe. Panel D: p-tau in the occipital lobe. Panel E: NFT in the frontal lobe. Panel F: NFT in the occipital lobe. The x-axes represent the p21^cip1 genotype, with com and var representing subjects with common and variant p21^cip1 respectively. The y-axes represents the p-tau or NFT content of the relevant brain region as determined by ELISA with markers for AT8 and DC11 respectively (arbitrary units). Statistical test: one-way ANOVA. The top/bottom of the bars represent the mean. The error bars represent the SEM.</p

HEK293 cells transfected with p21cip1. Image of cells acquired from the Acumen cytometer.

<p>The p21^cip1 was labelled by immunohistochemistry (green fluorescence). The DNA is labelled red by the PI. <b>Panel A:</b> The cell with the blue edge is an example of what we would accept as cytoplasmic protein expression. <b>Panel B</b> is the green fluorescence intensity histogram (as measured by the cytometer) over the surface of the same cell (with the blue edge in panel A) indicating a relatively uniform protein distribution in the cytoplasm around the nucleus. <b>Panel C</b> is the distribution histogram of the red fluorescence (DNA) from the same cell (with the blue edge in panel A). The distribution of the DNA is characteristic for a single cell. <b>Panel D</b>: The cell with the blue edge is an example of what we would accept as nuclear protein expression. <b>Panel E</b> is the green fluorescence intensity histogram (protein content as measured by the cytometer) over the surface of the same cell (with the blue edge in panel D). <b>Panel F</b> is the red fluorescence (DNA) from the same cell (with the blue edge in panel D). The overlap and identical distribution patterns of the protein (green label) and DNA (red label) is characteristic for nuclear proteins.</p