Live cell imaging was performed on a 3i Marianas SDC spinning disk confocal imaging system (Intelligent Imaging Innovations Inc.) using a similar microscopy setup previously described (34). The system is based on an Axio Observer Z1 inverted microscope (Carl Zeiss MicroImaging Inc., Germany) equipped with a Yokogawa CSU-X1 spinning disk confocal head (Yokogawa Electric, Tokyo, Japan) and 100-mW solid-state lasers (Coherent Inc., Santa Clara, CA) coupled to an acousto-optic tunable filter. The axial position of the sample was controlled with a piezo-driven stage (Applied Scientific Instrumentation, Eugene, OR). Each MatTek dish was placed in an incubation chamber (PeCon P-Set 2000, PeCon GmbH, Erbach, Germany) mounted on the microscope stage and connected to CO2 (CO2 module S, PeCon) and humidity (Heating Device Humidity 2000, PeCon) controllers. The whole microscope body excluding lasers, camera, and spinning disk head was maintained inside a large plexiglass environmental chamber (PeCon, Erbach, Germany). The temperature in both the microscope and top stage incubation chambers was controlled by a common unit and set to 37°C. The environment inside the top stage incubation chamber was further set to 5% CO2 and 100% humidity. Samples were illuminated with λ = 488 nm for GFP, λ = 561 nm for TagRFPt, and λ = 640 nm for iRFP. Images were acquired using a 100× (Plan Apo, 1.4 numerical aperture) oil immersion objectives (Carl Zeiss MicroImaging Inc.) under the control of SlideBook 6.0 software (Intelligent Imaging Innovations, Denver, CO). Three-dimensional (3D) time-lapse image stacks of 12 to 16 optical slices separated by 0.4 μm were collected every 30 s for 50 to 60 min, with exposure acquisition times between 40 and 50 ms. Digital images (16-bit) were acquired using a back thinned air-cooled electron-multiplying charge-coupled device camera (Evolve 512, Photometrics, Tucson, AZ). 3D time-lapse sequences were analyzed with the spot tracking and quantification software tool, STaQTool, as described previously (34, 35). Briefly, this tool was used to track single transcription sites in the cell nucleus over time and calculate the total fluorescence intensity (TFI) for each diffraction-limited spot in a time-lapse sequence by performing a 2D Gaussian fit on the volume of interest at the Z plane corresponding to the highest intensity value (35). ImageJ was used to quantify labeling intensities of a selected ROI defined around the transcription site to determine MDC1 accumulation. Briefly, time-lapse frames in both green (GFP) and red (TagRFPt) were first corrected for fluorescence loss due to photobleaching, as described previously (36). A circular ROI was used to define the transcription site in the RFP channel. The same ROI was then duplicated in the GFP channel and used to determine the average intensity over time of MDC1 in the transcription site for the whole time-lapse sequence.

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