Extended Data Fig. 1: Extended Data Figure. 1 related to Fig. 1. H3K9me3 accumulates in cancer cells and upon persistent replication stress condition.
From: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability
(a) Bar chart representing global chromatin profiling for enrichment of deacetylated H3K9me1/K14ac0 (red), H3K9me2/K14ac0 (green) and H3K9me3/K14ac0 (purple) epigenetic marks in >40 different ovarian cancer cell lines (the name of each cell line is indicated on the x axis) were analyzed from CCLE database 23. For each chromatin mark, the fold change relative to median value of the respective ovarian cancer cell lines is shown. (b) Analysis of S phase cells undergoing active replication. Cells were pulse-labeled with BrdU for 45 min at the indicated time. BrdU incorporating fractions of S-phase cells were determined by flow cytometry. The percentages of BrdU positive cells are indicated as the mean +/- SD of three independent experiments. (c) Immunoblot analysis of DNA damage and checkpoint signaling. Phosphorylated histone H2AX (γH2A.X), Ser317-phosphorylated Chk1 (Chk1p) and Ser15-phosphorylated p53 (p53-S15p). This experiment was reproduced independently three times with similar results. (d) Persistent replication stress induces senescence, as demonstrated by high senescence-associated-β-galactosidase (SA-β-Gal) activity. TIG3 cells expressing the oncogene B-RAF (OIS: Oncogene induced senescence) were used as positive control for the presence of SA-β-Gal positive cells. This experiment was reproduced independently three times with similar results. (e) Analysis of mitotic cells. Histone H3 serine 10 phosphorylation (H3S10p) was analyzed by flow cytometry of TIG3 cells treated as indicated. The percentages of H3S10p positive cells are indicated as the mean +/- SD of three independent experiments. (f) Analysis of histone PTM levels in proliferating (grey), quiescent (blue) and HU-treated (pink) TIG3 fibroblasts. The graph show the average of three biological replicates with error bars indicating SD. Unpaired two-sided t-test: (****) P < 0.0001; (***) P < 0.001; (**) P < 0.01; (*) P < 0.05. Source numerical data and unprocessed blots are available in source data.
