3D-Structured Illumination microscopy (3D-SIM) for super-enhancers

To visualize SEs, 3D-SIM experiments were performed. Cells were seeded on a chambered cover glass (Lab-Tek, Cat# 155411) and fixed with 4% PFA after 2 days culture. The samples were then permeabilized by 0.1% Triton and blocked with 3% BSA for 1 h. The samples were next blotted with MED1 antibody (Abcam, Cat#Ab60950) at 1:1000 at 4°C overnight and then blotted with Goat anti-Rabbit secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, Cat#A11008) after wash. Nuclei were counterstained with DAPI (1mg/ml) at 1:2000 dilution for 5 min at RT. ProLong Diamond Antifade Mountant (Invitrogen, Cat#{"type":"entrez-protein","attrs":{"text":"P36961","term_id":"547831","term_text":"P36961"}}P36961) reagent was added to the sample before super-resolution images were taken using 100× oil-immersion objective of A1R N-SIM N-STORM microscope (Nikon). To obtain optimal images, immersion oil with refractive indices of 1.516 was used at 25°C room temperature. Super-resolution image stacks were captured with a z-distance of 0.125 μm with five phases, three angles and 15 raw images per plane. The raw images were reconstructed into one image using NIS Elements software. All SIM images were cropped and processed by NIS Elements software. YAP antibody (Santa cruz, Cat#sc-101199) was used for YAP/MED1 co-immunostaining at 1:50 dilution and Goat anti-mouse Alexa Fluor 647 (Invitrogen, Cat#A21236) was used as a secondary antibody. Co-localization of the two channels was evaluated with Fiji Coloc 2 plugin. Imaris was used to render all the individual z-plane images and Med1 condensates were calibrated with spherical beads with mean short diameter > 0.7 μm.