Fig. 3
Super-resolution imaging of RNA Pol II clusters after nuclear O-GlcNAc perturbation. A Schematic representation of the Tet-ON inducible transgene of bacterial OGA BtGH84, fused to a localization peptide (NLS) and the knock-in of the epitope tags HA and SPOT to endogenous Polr2a gene encoding RNA Pol II. B Western blot (WB) detection of the kinetic depletion of O-GlcNAc (detected by WGA, top), following ectopic expression of the bacterial OGA homolog BtGH84 fused to a localization peptide, namely BtGH84-NLS. The bottom panels show the WB detection of OGT and Lamin A/C (loading control) at the indicated time points following doxycycline induction of BtGH84-NLS expression. C Quantification by normalized optical density of the WB detection of SOX2-O-GlcNAc at the indicated time points after expression of BtGH84-NLS. The blots are shown in Additional file 1: Fig. S2D. D Immunoprecipitation of endogenous RNA Pol II using the ESC line described in A whereby Polr2a was targeted with a knock-in SPOT epitope tag. The immunoprecipitation was performed with magnetic beads coated with anti-SPOT antibodies. The efficiency of RNA Pol II IP and RNA Pol II O-GlcNAc levels were probed by WB analysis at different time points after Dox-induction of BtGH84-NLS expression. E Quantification of the western blot shown in D by the normalized optical density of O-GlcNAc on immunoprecipitated RNA Pol II. The ratio IP RNA Pol II / O-GlcNAc is plotted at different time points after BtGH84-NLS expression. F Representative micrographs of RNA Pol II and DNA (DAPI) acquired by stimulated emission depletion (STED) microscopy in ESCs and NPCs before and after prolonged nuclear O-GlcNAc depletion by expression of BtGH84-NLS (48 h and 5 days, respectively). Scale bars indicate 2 μm. G Distribution of fluorescence intensity quantification of RNA Pol II clusters from STED images before and after BtGH84 induction in ESCs and NPCs (t-test. Not-significant (ns): p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001)