DOI: 10.5281/zenodo.6052377
title: Raw confocal imaging and FRAP data for "Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription"

description: <strong>Raw confocal imaging and FRAP data of "Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription"</strong> Shasha Chong<sup>1</sup>, Thomas G.W. Graham<sup>2</sup>, Claire Dugast-Darzacq<sup>2,5</sup>, Gina M. Dailey<sup>2</sup>, Xavier Darzacq<sup>2,5</sup>, Robert Tjian<sup>2,3,4,5</sup>* <sup>1 </sup>Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA <sup>2 </sup>Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. <sup>3 </sup>Howard Hughes Medical Institute, University of California, Berkeley, CA, USA. <sup>4</sup><sup> </sup>Li Ka Shing Center for Biomedical & Health Sciences, University of California, Berkeley, CA, USA. <sup>5</sup><sup> </sup>CIRM Center of Excellence, University of California, Berkeley, CA. * Lead contact <strong>Overview</strong> This repository contains 1) raw three-color confocal fluorescence images of a transiently expressed protein (mNeonGreen-EWS, mNeonGreen, EGFP-TAF15, EGFP, mNeonGreen-EWS-NPM1, or mNeonGreen-NPM1), endogenously expressed EWS::FLI1-Halo labeled with JFX549 Halo ligand, and intron RNA fluorescence in situ hybridization (FISH) targeting <em>ABHD6</em>, <em>CAV1</em>, or<em> GAPDH </em>in genome-edited A673 cells, 2) raw fluorescence recovery after photobleaching (FRAP) movies of endogenously expressed EWS::FLI1-Halo labeled with TMR Halo ligand in genome-edited A673 cells in the presence and absence of transient expression of mNeonGreen-EWS-NPM1. The imaging data, after being processed, were used to generate Figure 1D-G (also S1A, S3, and S4), 2E-G (also S5A and S7), 3C-E (also S9), 4A, S2, S6, and S8 of the manuscript in the title. <strong>Method details</strong> 1. RNA fluorescence in situ hybridization (FISH) The genome-edited A673 cells (described in https://www.science.org/doi/10.1126/science.aar2555) were plated on 18 mm circular No. 1 cover glasses (VWR VistaVision, 16004-300) and transfected with a protein expression plasmid using Lipofectamine 3000. 24 hours after transfection, we stained the cells with 200 nM JFX549 HaloTag ligand following the protocol described above, fixed the cells, and then proceeded with RNA FISH. To measure nascent transcription levels of <em>ABHD6</em>, <em>CAV1</em>, and <em>GAPDH </em>genes, we performed intron RNA FISH following the published Stellaris RNA FISH protocol for adherent cells (https://biosearchassets.blob.core.windows.net/assets/bti_stellaris_protocol_adherent_cell.pdf) using Quasar 670-labeled FISH probes designed with the online software Stellaris Probe Designer (https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer) and purchased from LGC Biosearch Technologies. 2. Confocal fluorescence imaging of protein and nucleic acid distribution Two confocal microscopes were used to image intron RNA FISH samples. One is an inverted laser scanning confocal microscope (Zeiss, LSM 710 AxioObserver) equipped with 34-channel spectral detection, a motorized stage, a full incubation chamber maintaining 37°C and 5% CO<sub>2</sub>, a heated stage, an X-Cite 120 illumination source as well as several laser lines (405, 458, 488, 514, 561, 591, 633 nm). Images were acquired with a 40x Plan NeoFluar NA1.3 oil-immersion objective under control of the Zeiss Zen software. The other is an inverted laser scanning confocal microscope with Airyscan super-resolution capability (Zeiss, LSM 900 with Airyscan 2) and equipped with four laser lines (405, 488, 561, 640 nm). Images were acquired with a 40x oil objective (Zeiss Plan-Apochromat 40x/1.3 Oil DIC) in the confocal (CO) mode under control of the Zen software. We acquired z stacks of RNA FISH samples with a slice interval of 0.3 mm. 405 nm, 488 nm, 561 nm, and 633 or 640 nm lasers were used to excite the fluorescence of Hoechst-labeled nuclei, EGFP or mNeonGreen-labeled proteins, JFX549-labeled EWS::FLI1-Halo, and Quasar 670-labeled intron RNA FISH, respectively. Before acquiring any fluorescence image, we carefully set the laser intensity and microscope detectors to make sure that no pixel in the image was saturated. We used proper emission filters for sequential four-color imaging and ensured no bleed-through between the four channels by imaging cell samples that contain only one of the four fluorophores (Hoechst, EGFP or mNeonGreen, JFX549, and Quasar 670) under the four-color imaging settings. 3. Fluorescence recovery after photobleaching (FRAP) FRAP was performed on the inverted laser scanning confocal microscope (Zeiss, LSM 710 AxioObserver) described above. The 561 nm laser and the epi-illumination mode were used for FRAP measurements. Images were acquired with a 40x Plan NeoFluar NA1.3 oil-immersion objective. The knock-in A673 cells were grown on glass-bottom (No. 1.5, 14 mm diameter) 35 mm dishes (MatTek, P35G-1.5-14-C). To measure the FRAP dynamics of EWS::FLI1-Halo in the nucleolus, we transfected the knock-in cells with a plasmid encoding mNG-EWS-NPM1 and stained the cells with 500 nM HaloTag TMR ligand (Promega, G8251) following the protocol described above. We acquired 1000 frames at one frame per 0.3 seconds with the first 5 frames acquired before the bleach pulse for the measurement of baseline fluorescence of the bleach spot and the whole nucleus. We chose to photobleach a circular spot with a radius of 1 μm within a nucleolus using the 561 nm laser at maximum intensity. To measure the FRAP dynamics of EWS::FLI1-Halo in the nucleoplasm, we followed the same procedure as above, except that the knock-in cells were not transfected and a circular bleach spot with a radius of 1 μm was chosen within the nucleoplasm of a cell and at least 1 μm from nuclear and nucleolar boundaries.