Deltavision fluorescence microscope

Cells were fixed by adding formaldehyde to a final concentration of 3.7% in PBS, then permeabilized and blocked in PBS/0.1% TritonX-100/4% (w/v) BSA. Incubation was performed at 4°C overnight with primary antibodies at the following concentrations: goat anti-brachyury 1 µg ml⁻¹ (AF2085, R&D), rabbit anti-Sox2 5 µg m⁻¹ (ab5603, Millipore), rabbit anti-β-III-tubulin 1 µg ml⁻¹ (T2200, Sigma-Aldrich), mouse anti-HB9 1.75 µg ml⁻¹ (81.5C10, Developmental Studies Hybridoma Bank) and rabbit anti-islet 1 2.5 µg ml⁻¹ (ab20670, Abcam). Fluorochrome-conjugated secondary antibodies used were the following: anti-goat Alexa647-conjugated 4 µg ml⁻¹ (A21447, Invitrogen), anti-rabbit Alexa488-conjugated 4 µg ml⁻¹ (A21206, Molecular Probes) and anti-mouse Alexa594-conjugated 4 µg ml⁻¹ (A11032, Molecular Probes). Observations were carried out with a DeltaVision fluorescence microscope (GE Healthcare) and images were acquired using softWoRx software, except images in Fig. S8, which were captured on a Zeiss LSM 710 confocal microscope.

To monitor recruitment of SCAI to IR-induced foci, 400,000 U-2 OS Flp-In/T-REx cells expressing either GFP alone or GFP-SCAI were seeded on glass coverslips in a 6-well plate in doxycycline-containing media to induce protein expression. Approximately 24 h after seeding, cells were mock- or IR-treated. Cells were fixed with 4% PFA/2% sucrose for 15 min at room temperature, washed with PBS, permeabilized with CSK buffer (100 mM NaCl, 300 mM sucrose, 10 mM PIPES (pH 6.8), 3 mM MgCl₂, 0.5% Triton-X-100), stained with DAPI, and mounted on microscopy slides for imaging. Microscopy was performed using a DeltaVision fluorescence microscope equipped with SoftWorx (GE Healthcare). Images were analyzed using ImageJ.

FISH probes that specifically recognize a locus on human chromosome 3 were made as described previously [50 (link)]. HeLa cells expressing shCtrl (HeLa C1) or shDDX11 (HeLa 5–5) were transfected with plasmid vectors that express wild type or mutant DDX11. After a thymidine-block for 16–18 hr, cultures were released to fresh medium and incubated for 4 hr. Then, cells were harvested by treatment with Trypsin, treated with 75 mM KCl hypotonic solution for 25 min at 37°C and fixed with ice-cold methanol and acetic acid (3:1, v:v). Fixed cells were dropped onto pre-warmed slides, in situ hybridized at 80°C with DNA probes and incubated at 37°C overnight. Slides were sequentially washed with 0.1% (w:v) SDS in 0.5x SSC at 70°C for 5 min, PBS at room temperature for 10 min and 0.1% (v:v) Tween 20 in PBS at room temperature for 10 min. Slides were then mounted with ProLong Gold (Life Technologies) and viewed with a 100 x objective on a DeltaVision fluorescence microscope (GE Healthcare). Image processing and quantification were performed with the ImageJ software.