mCherry-RPB1 protein stability measurement in HCT116 cells using bleach-chase

Description

mCherry-RPB1 half-life measurements were made using the ‘bleach-chase’ method. This involves partially bleaching cells expressing a fluorescent protein and monitoring the rate of fluorescence recovery over time to infer protein turnover dynamics. Imaging was performed on a Nikon Ti2 microscope with CSU-W1 spinning disk 72-90h after siRNA transfection, using a 20X/0.75NA objective and Hamamatsu ORCA-Fusion C14440-20UP camera (image pixel size 325nm x 325nm). The entire imaging experiment was automated using Nikon JOBS. Pre- and post-bleach imaging used 561nm laser excitation and a 617/73 nm bandpass emission filter, for ‘bleached’ and ‘unbleached control’ regions with the same well (9 imaging sites each). Bleach steps were performed using a widefield light source, from a mercury vapour lamp with 635/60 nm filter. In all cases, seven z-planes were acquired with a spacing of 2.0 µm. Two wells (ARMC5 siRNA and scrambled siRNA) were imaged sequentially for each timepoint. Allowing 10 min between frames resulted in a time between frames of approximately 14.4 min. Images were acquired for 6 frames before the bleach step and recovery was monitored for 9 h after the bleach step. The mean loss of mCherry-RPB1 intensity induced by the bleach pulse was 43 ± 1% – optimised to minimise bleach pulse duration and to retain the ability to visualise cells using mCherry signal after bleaching. An example movie is included. Experiments were repeated on four different days, with two replicates performed each day. Cell confluency was monitored post hoc by examining the change in nuclear area distributions over time (nuclear area decreases with cell number as cultures become close to confluent). Wells that showed a decrease in mean nuclear area with cell number during post-bleach acquisition were excluded, due to a confounding effect of cell morphology changes on mean fluorescence intensity measurements and the possibility of changes in cell growth rate at confluency. After excluding these data, four experimental replicates were used for protein half-life measurements, across three repeats of the experiment on different days.

Steps to reproduce

- Folders 20240208_085709_941, etc. contain single-cell quantifications over time (QUANTIFICATION subfolder) and imaging metadata (OME-TIFF-MIP subfolder) - 20X_mCherry-POLR2A contains the images and manual segmentations used to train the nuclear segmentation model, as well as the Cellpose 2.0 model file used for nuclear segmentation. - ARMC5_bleach_chase_example.avi is an example live-cell imaging time course. - bleach_chase_analysis.html gives the theoretical background and shows all data analysis steps from the single-cell data provided.

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