Time- and radiation-dose dependent changes in the plasma proteome after total body irradiation of non-human primates: Implications for biomarker selection - Fig 3

<p><b>Hierarchical cluster of significantly differentiating proteins in a dose dependent manner from days 4 (A), 7 (B), and 12 (C) post-irradiation.</b> Proteins were clustered using the average linkage method and Euclidean distance metric. Proteins were considered significant by Kruskal-Wallis p-value corrected using Benjamini-Hochberg. Red indicates elevated levels, while blue indicates lower levels of the given protein.</p

Dose-dependent analysis.

<p>Dose-dependent analysis.</p

Significantly differentiating proteins from days 4, 7, and 12 at both gamma-irradiation doses of 6.7 Gy and 7.4 Gy.

<p>Significantly differentiating proteins from days 4, 7, and 12 at both gamma-irradiation doses of 6.7 Gy and 7.4 Gy.</p

Venn diagrams show overlapping proteins identified in both plasma and urine samples from gamma-irradiated non-human primates.

<p>A) Total proteins identified in both sample types. B) From the 414 commonly identified proteins between plasma and urine, the Venn diagram shows how many were found to be significant in each sample type by ANOVA with a p-value < 0.05. Ten proteins are identified as significant from both the plasma and urine proteomic analyses.</p

The plasma proteome from non-human primates following gamma-irradiation was analyzed by high-resolution mass spectrometry.

<p>A) Representative gel image of replicate samples from the day 4 time point under varying gamma-irradiation doses of 0 Gy, 6.7 Gy, and 7.4 Gy. Plasma was first depleted of the top 20 most abundant plasma proteins, resolved by SDS-PAGE and visualized by Coomassie-staining. B) The Venn diagrams show the number of shared and unique proteins at days 4, 7, and 12 post-irradiation at both 6.7 Gy and 7.4 Gy radiation exposures. C) The number of shared and unique proteins identified in a dose-dependent analysis (non-irradiated, 6.7 Gy, and 7.4 Gy exposures) at each time point (days 4, 7, 12).</p

Time-dependent analysis.

<p>Time-dependent analysis.</p

Significantly differentiating proteins by gamma-irradiation dose levels of 6.7 Gy and 7.4 Gy at days 4, 7, and 12.

<p>Significantly differentiating proteins by gamma-irradiation dose levels of 6.7 Gy and 7.4 Gy at days 4, 7, and 12.</p

Loss of Sc65 results in dermal tears, abnormal collagen fibrils and skin fragility.

<p>a) H&E stained sections of WT and <i>Sc65KO</i> skin. Note the decreased density of collagen, the frayed dermis indicated by arrows and the reduced thickness of the muscle layer in the <i>Sc65-null</i> samples. b) Serial skin sections were stained with Sirius red. <i>Sc65-null</i> skin exhibits fewer large collagen fibers (red staining) and greater number of smaller collagen fibers stained in green compared to WT counterparts. c) Electron micrographs of 7 month-old mouse skin biopsy from WT and <i>Sc65KO</i> mice. Collagen fibrils, shown in cross-section, from <i>Sc65-null</i> skin tended to be smaller and have a decreased range of fibril diameter compared to WT fibrils. Loss of Sc65 also resulted in the presence of collagen fibrils with irregular profile and several large “cauliflower-like” fibrils (red arrow) which indicate abnormal fibrillogenesis (scale bar represents 500nm). d) Distribution of collagen fibril diameter in WT and <i>Sc65KO</i> mouse skin as measured from electron microscopy images. Measurements were collected from three different mice/genotype and >200 fibril/mouse. e) Skin EMs from <i>Sc65KO</i> mice also exhibited significantly more empty space among collagen fibrils compared to WT mice indicating a less densely packed collagen (*p = 0.01). Five electron micrograph images of non-overlapping areas were quantified from each mouse. f-h) Skin samples from WT and <i>Sc65KO</i> mice were subjected to a biomechanical skin loading test to measure tensile strength. Skin that lacks SC65 expression ruptured at a significantly lower peak load compared to WT skin indicating significant skin fragility (*p<0.01).</p

SC65 directly interacts with prolyl 3-hydroxylase 3 (P3H3).

<p>a) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control were used for IP experiments utilizing a Flag antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 10% SDS-PAGE gel, blotted and probed with antibodies against FLAG and P3H3. The reciprocal interaction of SC65-Flag with P3H3 is confirmed in both experiments. b) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and <i>Sc65KO</i> 3 day-old mice (N = 2) showing significantly decreased levels of P3H3 protein in <i>Sc65KO</i> samples. Densitometric quantification of P3H3 protein normalized to β-actin from the western blot shown above (#p<0.01; *p<0.05; error bars represent SD). All experiments were performed at least 3 times.</p

Increased electrophoretic mobility and altered cross-linking of type I collagen from <i>Sc65-null</i> skin.

<p>a) SDS-6%PAGE of type I collagen extracted from skin and decalcified bone of <i>Sc65KO</i> and WT mice shows increased mobility of α-chains and reduced ratio of cross-linked β to γ components in the <i>Sc65KO</i> skin extracts. An acetic acid extract from skin of the original Sc65-null mouse¹⁹ (1 mo.) created by gene-trap insertion is compared with that from the new <i>Sc65KO</i> (6 mo.) and their respective WT controls. Total heat denatured extracts of skin and bone collagens from new <i>Sc65KO</i> mice are shown on the right for comparison. Bone collagen from <i>Sc65KO</i> mice does not show the differences from WT in β/γ intensities evident for skin collagen. The strong (lower) γ band in SC65 skin extracts was identified as γ _112. Both original and new <i>Sc65KO</i> mice showed the same altered pattern of chain intensities from WT most pronounced in the acetic acid extracts of skin with an apparent increase in γ₁₁₂ at the expense of β_12. b) Densitometric analysis of collagen bands on SDS-PAGE. Densitometry was performed on bands 1–8 (counted from top to bottom) of acetic acid extracts from 1mo and 6mo skin samples of both original and new <i>Sc65KO</i> mice using NIH imageJ software. Values are means ± SD, n = 6; *p<0.01.</p