Slide 8 well glass bottom plates

The viability was measured with a Live/Dead assay, as a complementary experiment to the AlamarBlue assay, in order to study the cells’ viability. 200 µL of hydrogels was prepared inside µ-Slide 8-well glass bottom plates (Ibidi GmbH, Grafelfing, Germany) with embedded hFBs (160,000 cells/mL) and measured at 3 replicates per condition: 48 h and 7 days as time points.
Live/Dead^® staining was performed by adding calcein AM and ethidium homodimer from Live/Dead Viability/Cytotoxicity kit (Thermofisher, Frederick, MA, USA) at final concentrations of 0.5 µL/mL and 2 µL/mL in PBS, respectively. 100 µL of this staining was added to the hydrogels and incubated during 40–50 min at 37 °C in the incubator. After this time, the reagent was removed and substituted by PBS. Finally, the hydrogels were observed in a confocal microscope (Leica-SPE (Leica, Wetzlar, Germany)) in the biomedical investigation area in the Gregorio Marañón Hospital. The visualization of the hydrogels in the confocal microscope shows the viability of the cells.

To study the XPO1-mediated nuclear export, HAP1/SpCas9⁺ cells were transfected with a plasmid encoding for the NLS-AcGFP-NES reporter construct and a plasmid pool encoding 4 sgRNAs targeting XPO1 for knockout. Cells were transfected with the Neon Electroporation system as described above and plated in µ-slide 8-well glass bottom plates (IBIDI). Two days after transfection, cells were treated with DMSO or 1 µM selinexor for 3 h. After incubation, the AcGFP reporter construct was visualized in live cells using a Leica SP5 confocal microscope employing a CX PL APO 63x (NA 1.2) objective. AcGFP was excited at 488 nm (argon laser) and emission was detected at 492–560 nm.