Figure 4.

CHX does not immediately deplete replicative polymerases but disrupts new production of histones. (A) U2OS cells were either left untreated or treated with CHX for the timepoints indicated followed by Western blot probed with the specified antibodies. (B) Schematic protocol and representative images for fluorescent SNAP-tag labeling of U2OS cells stably expressing histone H3.1 or H3.3. Cells were quenched by DMSO (panel 1) or 0.2 μM blocking agent for 20 minutes (panel 2 and 3), labeled with 1 μM TMR for 30 minutes after a 3 hour release into fresh media +/- 10 μg/ml CHX. The contrast of the images in the middle panel of this figure has been equally enhanced in order to visualize the production of new histones after release from the quenching agent (panel 2), whereas the upper panel is unaltered. Furthermore, figure S4B and C (panel 3) shows cells without enhanced contrast, where arrows instead indicate new histone production. (C) Effect of HU on new histone H3.1 and H3.3 production via quenching of pre-existing histones using 0.2 μM blocking agent for 20 minutes, labeling with 1 μM TMR for 30 minutes after a 3 hour release into fresh media +/- 2 mM HU. (D) U2OS cells were treated with 10 μg/ml CHX or 2 mM HU for 24 hours and stained for RPA32. (E) U2OS cells were treated with 10 μg/ml CHX for 24 hours and co-stained for RPA32 and PML. See also Figure S5.