Halo-Tag Labeling of NUP96 in U-2 OS Cells

Human osteosarcoma cells of line U-2 OS, endogenously expressing NUP96 carboxy-terminally tagged with Halo-Tag^{23 (link)} (U-2 OS-CRISPR-NUP96-Halo clone no. 252, CLS GmbH) were grown in McCoy’s medium (16600082, Thermo Fisher Scientific) with 10% (v/v) FBS (S0615, Bio&SELL), 1% (v/v) sodium pyruvate (S8636, Sigma-Aldrich) and penicillin–streptomycin (P0781, Sigma-Aldrich) on coverslips. For fixation, the cells were treated with 8% (w/v) paraformaldehyde (PFA) in PBS at 37 °C for 5 minutes, quenched with 100 mM NH₄Cl in PBS for 10 minutes and permeabilized using 0.5% (v/v) Triton X-100 in PBS for 5 minutes. Samples were incubated with Halo-ONB-CP560 (Supplementary Fig. 5) at 1 µM in PBS for 1 hour, followed by several washes with PBS for 1 hour. For sample alignment on the microscope, the samples were incubated with primary antibody against NUP153 (ab24700, Abcam), bearing Alexa Fluor 488 (A11001, Invitrogen / Thermo Fisher Scientific, 1:2,000 dilution) for 30 minutes in 2% (w/v) BSA in PBS. Finally, the cells were washed with PBS.