mCherry-RPB1 FRAP - ARMC5, and/or INTS8 knockdown, triptolide ± CB5083 treatment

Description

FRAP data generated in HCT116 cells expressing mCherryPOLR2A (mCherry RPB1) on a Zeiss LSM900 confocal microscope. FRAPData1 - Scrambled vs ARMC5 siRNA FRAPData2 - Scrambled + vehicle vs ARMC5 siRNA + vehicle vs ARMC5 siRNA + triptolide FRAPData3 - Vehicle vs triptolide alone vs triptolide + CB5083 FRAPData4 - Scrambled vs ARMC5 siRNA vs INTS8 siRNA vs ARMC5 + INTS8 siRNA FRAPData5 - Fixed cells, scrambled or ARMC5 siRNA 3 days after plating cells in 8-well chamber-slides, regular media was exchanged for 360 μL imaging media, McCoy’s 5A phenol red-free (Cytiva SH30270.01) + 10% FBS (Moregate Biotech) + 1% penicillin and streptomycin (Sigma-Aldrich P0781) via a 2x wash on HCT116 mCherry-RPB1 cells. Where included, triptolide, triptolide plus CB-5083, and vehicle-only were made up in imaging media at 10x concentrations and added into wells 60 minutes before commencing imaging of that well, 40 μL onto 360 μL. Final concentrations were 1 μM triptolide, ± 10 μM CB-5083, in a consistent 0.044% DMSO vehicle in all wells. All FRAP traces were collected on a Zeiss LSM900 point-scanning confocal with Plan-Apochromat 63x oil immersion objective, NA 1.40, at 37 ̇C and 5% CO2, in a window of 60 – 90 minutes after compound treatment where relevant. For each cell, an initial image of the whole nucleus was collected, before two circular regions were imaged with a diameter of 1.8 μM (18 pixels) and an area of 2.45 μM (255 pixels). Both regions were imaged within a 1 s frame for 120 s. After a 10 frame baseline, one region was bleached with 100% laser power for approximately 5 s. Control traces were collected under identical conditions from cells fixed in 4% PFA for 15 minutes. Each trace was individually normalised as a percentage of the pre-bleach baseline values. FRAP traces collected from fixed cells did not show substantial recovery, reflecting almost completely immobilised mCherry-RPB1. Further rescaling of FRAP traces was performed, with 0% being defined by the post-bleaching intensity in fixed cells, and 100% being defined by the intensity of the respective unbleached control region at each timepoint. Normalised, rescaled FRAP traces were fit in Prism 9 (Graphpad Software) with a two-phase association model, fitting either each cell individually, or the mean of each experimental day (Supplemental Figure 3F). The initial value was constrained to the 0% value defined by fixed cells, and the plateau was constrained to the 100% value defined by the unbleached control region. To calculate fluorescence intensity-adjusted Pol II fractions, estimates of the bound percentage of Pol II were multiplied by the normalised baseline fluorescence intensity of each condition within each experiment relative to the corresponding control.

Steps to reproduce

Each dataset is saved as long csv files with fluorescence intensity over time for each cell, best readable in pandas python package. Raw and normalised data are in separate files for each dataset. Jupyter notebook FRAPData_ExamplePlotting.ipynb provides plots of all data, raw and normalised, and shows how to interpret the data file structure. Prism files FRAPDataXX_Fitting_Plotting.pzfx show fitting of the data to two-phase association curves, and plotting of the results. Layouts in each show final plots used in Cacioppo et al. FRAPData_FI_AdjustedFractions_PerDay.xlsx shows calculation of total Pol II bound/free amounts by using bound/free fraction and mean fluorescence intensity of the cells in the dataset.

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