Extended Data Fig. 5: OGT promotes DNA replication through H4S47 O-GlcNAcylation.
From: H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins
a, Quantification of H4 O-GlcNAcylation levels. WB from Fig. 2d were quantified. n = 4 biologically independent experiments. P values measured by unpaired, two-tailed t-tests, means ± s.d., n.s., non-significant. a.u., arbitrary unit. b, Evaluation of replication efficiency by EdU incorporation. HEK293T cells were transfected with p3 × Flag-CMV-10 (empty vector) or Flag-OGT for 48 h prior to the detection of replication efficiency with immunofluorescence analysis. HU (3 mM) treatment for 3 h was used as a positive control. Representative images (left) and quantifications of EdU intensities of PCNA-positive cells (right) were demonstrated. From left, n = 1,551, 1,063, 1,234 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. Scale bar, 10 μm. a.u., arbitrary unit. c, Analysis of protein O-GlcNAcylation. HEK293T cells were incubated with or without OSMI-1 (50 μM) for 24 h and analyzed by WB with CTD110.6 antibody. d, Examination of EdU intensity by FACS. HEK293T cells were treated as described in (c) and pulsed with EdU for 30 min. HU treatment (3 mM) for 3 h was included as a positive control. e, Representative images from Fig. 2e. Cells positive for Flag (H4) were selected for the quantification of EdU intensities. Scale bar, 10 μm. f, g, DNA fiber assay. Experimental setup (f, top), representative images (f, bottom) as well as frequency distribution of CIdU track lengths (g, left) and inter-origin distances (g, right) for Fig. 2f were shown. Scale bar, 10 μm.
