Dmi8 microscopy

Approximately 1 × 10⁴ cells in suspension were washed with PBS, centrifuged in a Cytospin 4 Centrifuge (Thermo Scientific, Waltham, MA, USA) at 350 rpm for 10 min, and fixed over microscopy slide with 4% w/v PFA for 15 min. After fixation, cells were washed with PBS and incubated with the InsidePerm Buffer (Inside Stain Kit, Miltenyi Biotec, Madrid, Spain) for permeabilization and as antibody diluent. Immunostaining was done incubating with the antibodies against E-Cadherin (1:200, ab40772; Abcam, Cambridge, UK) and Vimentin (D21H3) XP^®® Rabbit mAb (1:100, #5741; Cell Signalling, Danvers, MA, USA) for 1 h at RT. After incubation with the primary antibodies, cells were washed and incubated with the secondary antibody Anti-Rabbit AlexaFluor 647 (1:1000, ab150079; Abcam, Cambridge, UK). Nuclei were stained with DAPI. Fluorescence images were acquired with a DMi8 microscopy (Leica).

For in vitro phase separation, purified recombinant p62 or DAXX was centrifugated at 16,000 × g for 5 min to remove any potential protein aggregates. p62 droplet phase separation was carried out in a glass-bottomed well in a 384-well plate or a microcentrifuge tube, in the buffer containing 40 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT, at room temperature. Imaging was performed on a 384-well plate or a glass slide. p62 was stocked in the buffer containing 40 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM DTT, 10% glycerol or the buffer containing 40 mM Tris-HCl pH 7.4, 1 mM DTT, 10% glycerol. Purified recombinant DAXX was stocked in the buffer containing 40 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT. For in vitro phase separation, the stock of p62 protein was adjusted to the phase separation buffer conditions. Images were acquired with Leica DMi8 microscopy or confocal microscopy SP8.

For in vitro phase separation, purified recombinant p62 or p62-N was centrifuged at 16,000 × g for 5 min to remove any potential protein aggregates. p62-droplet phase separation was carried out in a glass-bottomed well of a 384-well plate in the buffer containing 40 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT and 10% glycerol at room temperature. p62 was stocked in the buffer containing 40 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM DTT and 10% glycerol. Purified recombinant p62-N was stocked in the buffer containing 40 mM Tris-HCl pH 7.4, 1 mM DTT and 10% glycerol. For in vitro phase separation, protein mixtures were adjusted to the phase separation buffer conditions, and the concentration of p62 or p62-N was kept consistent across the relevant samples. Images were acquired on a 384-well plate with Leica DMi8 microscopy.

5x10⁴ cells were seeded on 22x22 mm coverslips pretreated with 2% type A from porcine skin gelatin (Sigma Aldrich, St. Louis, USA); after 24 h, culture media were replaced by wo FBS DMEM (Euroclone, Milan, Italy) or HFF CM containing the indicated concentration of drug. After 24 h from treatments, cells were fixed in 4% formaldehyde in 1X PBS for 10 min, permeabilized in 1X PBS containing 0.5% Triton X-100 for 5 min at room temperature. Coverslips were incubated with primary and secondary antibodies as previously described (11 (link)). The used antibodies are reported in Supplementary Materials. Images were taken using Leica DMi8 microscopy (Leica, Solms, Germany) and Zeiss LSM 880 Confocal Microscope (Carl Zeiss, Oberkochen, Germany) equipped with ×63 lenses and operated by Zen 09 black software (Carl Zeiss).