Immunofluorescence analysis and microscopy.

Immunofluorescence analysis was performed on SMARCA2- and SMARCA2ΔC-transfected SW-13 cells. Cells were plated on coverslips in 24-well plates, and 48 h after transfection cells were fixed in 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X-100–PBS for 5 min, followed by three wash steps with PBS. Cells were incubated with a specific SMARCA2 antibody detecting the N terminus (ab15597; Abcam) in 5% NGS–PBS at a 1:100 dilution for 1 h at room temperature. After four consecutive washing steps with PBS, the secondary anti-rabbit Cy3-conjugated antibody (Jackson Research) in 5% NGS–PBS was added at a 1:500 dilution and incubated in the dark for 40 min at RT. Subsequently the slides were washed three times with PBS, while the second washing step included a nuclear staining with DAPI (4,6-diamidino-2-phenylindole; 1:10 000 in PBS). The slides were mounted with ProLong Diamond antifade mountant (Life Technologies, Carlsbad, CA, USA) onto a glass slide.

For analysis of SMARCA2-protein distribution, pictures were taken at ×25 magnification as Z stacks off at least 32 vertical slices (x-z plane) with an LSM-I-NLO ZEISS LSM 880 using ZEN Black 2.3 SP1 (Zeiss) software. At least 125 cells per condition and approach were counted and grouped into cells showing nuclear SMARCA2 signals or cells showing both nuclear and cytoplasmic SMARCA2 signal. Additionally, pictures as Z stacks of at least 20 vertical slices (x-z plane) were taken at ×63 magnification, and a maximum intensity projection was calculated for every picture with ZEN Black 2.3 SP1 (Zeiss) software. Protein distribution of SMARCA2 and overlap with that of DAPI as a nuclear marker was analyzed with intensity profiles along the indicated arrows.

Phase contrast images at ×10 magnification of cells present in culture plates were taken with an Axio Observer.Z1 (Zeiss) microscope using AxioVision software.