Figure 7.

Nuclear-enriched fraction proteomics identified novel proteins associated with VIC calcification in OM. (A) Confirmation of nuclear enrichment using two nuclear markers, prelamin-A/C and histone H1.5; n = 4 donors/fraction, ^****P < 0.0001 analyzed by t-test, error bars indicate SD. (B) XINA clustering analysis of protein abundances in NM, OM, and PM. Clusters with increased protein abundance at passage one (P1) and decreased abundance at passage four (P4) in OM indicated (clusters #1–3). Clusters with decreased protein abundance at P1 and increased abundance at P4 in OM indicated by purple box (clusters #25–30); n = 4 donors. (C) Venn diagram showing total number of proteins detected for the four donors analyzed (donors 1, 2, 6, 7) found in the three identified clusters in which abundance is increased at P1 and decreased at P4 in OM. Calcification-prone donors (calcified at P1 in OM; donors 6 and 7) indicated by red color, and calcification-resistant donors (did not calcify at P1 in OM; donors 1 and 2) indicated by blue color. Proteins detected in the three clusters of the two calcification-prone but not in the calcification-resistant donors indicated: heat shock protein family B (HSPB6), NIMA related kinase 9 (NEK9). (D) Venn diagram showing total number of proteins detected for the four donors analyzed (donors 1, 2, 6, 7) and found in the six identified clusters in which abundance was decreased at P1 and increased at P4 in OM.