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. 2014 May 15;9(5):e97670. doi: 10.1371/journal.pone.0097670

Figure 5. Identification of lipoprotein particles as a colony-spreading stimulator.

Figure 5

(A) Protein from each purification step was electrophoresed by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (B) The final fraction (Fraction IV) was serially diluted 2-fold, and the colony-spreading stimulatory activity was measured. (C) Elution profile of DEAE-cellulose column chromatography using Fraction III. Open circles indicate diameters of colony-spreading. Filled circles indicate absorbance at 280 nm. 500-µl aliquots of each sample were applied to the soft agar medium and their colony-spreading stimulatory activity was measured. (D) SDS-PAGE analysis of DEAE-cellulose column chromatography fractions. The 25-kDa protein coincided with colony-spreading stimulatory activity in fractions 46-58. The 25-kDa protein was identified as apolipoprotein A1 by peptide-mass fingerprinting. (E) Colony-spreading stimulation by HDL particles. Purified HDL, delipidated HDL, or recombinant human apolipoprotein A1 (Wako Chemicals, cat. no. 019-20731) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. Human apolipoprotein A1 shares 78% amino acids identity with calf apolipoprotein A1. (F) Agarose gel electrophoresis analysis of calf serum, HDL, LDL, and lipoprotein-depleted serum. Agarose gel electrophoresis was performed according to a previous report [23]. Each fraction (4 µg phospholipid) was electrophoresed in agarose 0.5%, and then lipoprotein bands were detected by lipid-specific staining using Oil Red O. The electrophoresed protein amount of calf serum, HDL, LDL, and lipoprotein-depleted serum was 500 µg, 5 µg, 5 µg, and 1800 µg, respectively. (G) Colony-spreading stimulation by phosphatidylcholine. Purified HDL, lipid extract from HDL, phosphatidylcholine from egg yolk (99% purity, Sigma, cat. no. P3556), or cholesterol (99% purity, Sigma, cat. no. C8667) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. (H) Colony-spreading stimulation by HDL, LDL, and VLDL. Fractionated HDL, LDL, or VLDL was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured.