Ectopic expression of fluorescently tagged histones in mock-treated HFt cells.

HFt cells were stably transduced with lentiviral vectors encoding C-terminally tagged fluorescent (mEmerald; mEm) histones or eYFPnls (negative control) as indicated. (A) Cells were induced to express proteins of interest for 24 h with doxycycline (DOX) prior to whole cell lysate (WCL) collection and western blotting. Membranes were probed for GFP and endogenous (endog.) histones H2A or H3. (B) RPE cells were transfected with plasmids expressing eGFP, H3.1-mEm, or H3.3-mEm for 24 h prior to WCL collection and western blotting. Membranes were probed for GFP and histone H3. (A/B) Molecular mass markers shown. (C to E) HFt cells were DOX induced for 6 h prior to fixation and indirect immunofluorescence staining for PML (red). Nuclei were stained with DAPI (blue). (C) Confocal microscopy images of histone-mEm or eYFPnls localization at PML-NBs. Cut mask (yellow) highlights regions of colocalization between cellular proteins of interest and PML; weighted (w.) colocalization coefficient (coeff.) shown. Cells expressing high (H) and low (L) levels of H3.3-mEm and H4-mEm are indicated. (D) Quantitation of the percentage of cells that demonstrate histone-mEm or eYFPnls colocalization at PML-NBs. Means and SD shown. (E) Violin plots showing histone-mEm w. colocalization coeff. frequency at PML-NBs: median w. colocalization coeff., solid black line; 25 th to 75 th percentile range, dotted black lines; coincidence threshold (0.2), dotted grey line; high confidence threshold (0.7), solid grey line. Mann-Whitney U -test, P -value shown. (D/E) N ≥ 150 nuclei per sample condition. (A to E) Data derived from a minimum of three independent experiments. Raw values presented in S1 data . (TIF)