Extended Data Fig. 1: H4S47 O-GlcNAcylation regulates DNA replication.
From: H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins
a, Detection of H4S47 O-GlcNAcylation by mass spectrometry. Histone H4 extracted from HEK293T cells were analyzed by mass spectrometry. Tandem mass spectrum of peptides with O-GlcNAcylation on S47 of H4 is shown. The y and b fragmentations were used to map the O-GlcNAcylation site. b, Quantification of H4 O-GlcNAcylation. WB from Fig. 1a were quantified. n = 4 biologically independent experiments. P values were calculated by unpaired, two-tailed t-tests, means ± s.d., n.s., non-significant. a.u., arbitrary unit. c, Functional assessment of mutations at H4S47 in DNA replication. HEK293T cells were transfected with Flag-H4WT, Flag-H4S47A for 24 h and cultured with or without PUG (100 μM) for another 24 h prior to EdU staining and FACS analysis. d, e, Evaluation of replication efficiency in HT1080 and A549 cells. HT1080 (d) or A549 (e) cells were transfected and treated as in Fig. 1a before the analyse of replication efficiency by immunofluorescence. Representative images (left) and associated quantifications (right) are demonstrated. Cells positive for both PCNA and Flag were marked with dashed lines. From left, n = 1,981, 2,232, 1,553, 1,181 (d) and n = 821, 963, 827, 1,306 (e) cells. P values were calculated by unpaired, two-tailed t-tests. For all box plots, the bottom, middle line and top of the box and whiskers indicate the 25th, 50th, 75th and 10–90th percentiles, respectively and means were shown as red ‘+’ signs. n.s., non-significant. Scale bar, 10 μm. a.u., arbitrary unit.
