Volocity

Strains that carried a gfp reporter were viewed using a Leica DMRXA microscope. In some cases, confocal microscopy was performed using a Leica DMFLS laser confocal microscope equipped with a 63 PC APO CS lens (1.40–0.60) or with a Nikon CSi laser confocal microscope. Confocal images were analyzed by processing confocal z-axis series using Volocity (Quorum Technologies) or ImageJ software (NCBI).

Cells were plated on poly-lysine treated coverslips and subjected to the standard 4-HT treatment to induce Taf1b deletion. At the indicated time points cells were rinsed with PBS, fixed in 4% PFA in PBS for 10 min and permeabilized with 0.5% Triton, PBS for 15 minutes. After a blocking step in PBS-N (PBS, 0.1% IGEPAL (Sigma)), 5% donkey serum, cover slips were incubated with primary antibodies in PBS-N, 5% donkey serum for ~16h at 4 deg. C. RPI was detected using a combination of mouse anti-A194 and A135 antibodies (#SC-293272, #SC-48385), fibrillarin with goat anti-FBL (#LS-C155047), and UBTF with rabbit anti-UBTF (in-house #8). Cells were incubated for ~ 2h at room temperature with the appropriate AlexaFluor or Dylight 488/568/647 conjugated secondary antibodies (ThermoFisher / Jackson ImmunoResearch) and counterstained with DAPI. After mounting in Prolong Diamond (ThermoFisher), epifluorescent 3D image stacks were acquired using a Leica SP5 II scanning confocal microscope and LAS-AF (Leica Microsystems) and Volocity (Quorum Technologies) software.

For fluorescence microscopy, cells were spotted on slides and fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, Burlington, MA) at 4ºC for 10 min, treated with 5% normal donkey serum (Jackson, USA) in PBS for 1 h, and washed with PBS. Cells were incubated with anti-EGFP (1:500, ThermoFisher Scientific), anti-CD11b (1:100, BD Pharmingen, San Diego, CA), and anti-p24 (1:50, H12 clone, NIAID U.S.A.). For secondary antibody binding, anti-chicken Alexa 488 (1:200; ThermoFisher Scientific) and anti-mouse Alexa 555 (1:100; ThermoFisher Scientific) were suspended in PBS and incubated for 1 h and washed with PBS three times then were conjugated, DAPI (ThermoFisher Scientific) staining was used as a nuclear marker and observed with fluorescence microscopy (Zeiss, Inwood, NY). Cells positive for specific markers were counted under fluorescence microscope for statistical analysis (Volocity, Quorum Technologies, Inc.). Briefly, 5 × 10³ DAPI positive cells were counted for EGFP expression and presented as percent positive. For p24 staining Catalyzed Signal Amplification System (Dako, Santa Clara, CA) was used with H12 Ab clone 183-H12-5c, then observed with inverted microscopy (Nikon, Garden City, NY). Experiments were repeated at least three times.