Article Figures & Data
Figures
- Fig. 1.
Dynamic chromatin association of DNA damage repair proteins during interphase progression. A-C, iTRAQ ratio indicating the relative abundance of proteins obtained by partial MNase digestion of chromatin samples collected during G1,G1/S and G2/M phases of the cell cycle. D, Western blot showing the quantity of proteins Ku70, Ku80 and histone H4 detected in the same chromatin digests. E, Western blot showing the protein expression levels of Ku70, Ku80, histone H4 and actin during G1,G1/S and G2/M phases.
- Fig. 2.
Dynamic chromatin association levels of the chromatin organization proteins and transcriptional regulators during interphase progression. Proteins were grouped by relative abundance into three distinct clusters (a–c) that each contained two separate subclusters (1 and 2). “Cluster a” proteins were maximally associated with chromatin in early G1 phase but also contained distinct subclusters that displayed differential association patterns in G1/S and G2/M. “Cluster b” proteins were maximally associated with chromatin in early G1/S phase and included subgroups of proteins with distinct association profiles in G1/M, G2/M and early G1. “Cluster c” proteins were enriched in early G2/M phase chromatin samples and incorporated subclusters with differential association characteristics in early G2/M, G1 and G1/S. The combined iTRAQ ratios of matching pellet and supernatant fractions were used for the clustered analysis and the color code reflect the relative binding levels of the proteins.
- Fig. 3.
Release profiles of chromatin-associated transcriptional regulators and chromatin organization proteins upon partial MNase digestion. iTRAQ ratio indicating the relative abundance of proteins in the different chromatin fractions. Supernatant fractions G1-S, G1/S-S, and G2/M-S, and pellet fractions G1-P, G1/S-P, and G2/M-P were obtained by partial MNase digestion. Differential chromatin association patterns of “cluster a” proteins (A–B), “cluster b” proteins (C–D), and “cluster c” proteins (E–F).
- Fig. 4.
Western blot of HP1BP3 protein in different chromatin fractions. A, Distinct chromatin association levels of HP1BP3 protein during G1,G1/S and G2/M phases. B, Relative association level of HP1BP3 with G1,G1/S and G2/M phase chromatin.
- Fig. 5.
HP1BP3 depletion alters higher-order chromatin structure. A, MNase finger-printing of mock- and HP1BP3-depleted chromatin preparations. Chromatin samples were digested with different concentrations of MNase (0, 5, and 10 U) and separated using 1% agarose gels. B, Model of HP1BP3-mediated heterochromatin packing.
- Fig. 6.
HP1BP3 depletion modulates host cell protein expression. A, Distribution of molecular weight (MW) and isoelectric point (pI) of the identified proteins. B, iTRAQ ratio indicating differential protein expression in HP1BP3-depleted cells. Protein differential expression was classified according to subcellular localization (C) and cellular function (D). E, Differential expression levels of proteasome subunits in HP1BP3-depleated cells as determined by iTRAQ quantitation of relative protein abundance.
- Fig. 7.
HP1BP3 regulates cell proliferation. A, MTT colorimetric assay showing viable cell numbers in mock- and HP1BP3-depleted samples. B, Clonogenic assay showing mock- and HP1BP3-depleted cell colony staining with crystal violet. C, Graph representing the number of colonies formed by mock- and HP1BP3-depleted cells together with the corresponding absorbance value of the crystal violet colony staining. D, Progression of cell cycle during 4–12h window after release from double thymidine block. E, Proportion of S phase cells detected in cultures harvested between 4–12 h (n = 3 experimental replicates).
- Fig. 8.
Nuclear size is increased by HP1BP3 depletion. A–B, Mock- and HP1BP3-depleted A431 cells were plated onto glass cover slips and stained with Alexa 594-conjugated WGA (red cell membrane staining) and with DAPI to identify nuclei (blue) prior to immunofluorescence imaging (scale bar = 10 μm). C, Comparison of mock- and HP1BP3-depleted A431 cell sizes by flow-cytometry. D, Distribution of nuclear diameter measurements in mock- and HP1BP3-depleted A431 cells. E, Differential expression levels of nuclear transport proteins in HP1BP3-depleted cells as determined by iTRAQ quantitation of relative abundance.
Tables
Sample Number of proteins with significant change in iTRAQ ratio (p value <0.05) Total number of unique proteina G1/S-S G2/M-S G1-P G1/S-P G2/M-P iTRAQ ratio 114:113 115:113 117:113 118:113 119:113 Total 148 117 245 233 153 334 Ratio>1.3 63 44 108 107 65 219 Ratio<0.75 59 45 131 121 72 209 ↵a The number of unique proteins with at least one ratio with p < 0.05.
Supplemental Data
Files in this Data Supplement:
- Supplementary Figures - Supplementary Figures.pdf
- Protein Summary - Cell Cycle Progression.xlsx - Protein Summary - Cell Cycle Progression.xlsx
- Peptide Summary - Cell Cycle Progression.xlsx - Peptide Summary - Cell Cycle Progression.xlsx
- Protein Summary - Mock vs HP1BP3 depleted.xlsx - Protein Summary - Mock vs HP1BP3 depleted.xlsx
- Peptide Summary - Mock vs HP1BP3 depleted.xlsx - Peptide Summary - Mock vs HP1BP3 depleted.xlsx
- SRM validation of iTRAQ results.xlsx - SRM validation of iTRAQ results.xlsx
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