Biomolecular Condensate Coronas from Nanoparticle Intracellular Processing Are Transferred Between Cells Allowing Cytosolic and Nuclear Access
Biomolecular Condensate Coronas from Nanoparticle Intracellular Processing Are Transferred Between Cells Allowing Cytosolic and Nuclear Access
HEK293 cells were seeded into T175 flask and cultured for 24 h. The culture medium was then removed, the cells were washed twice with pre-warmed PBS, and pre-warmed serum-free medium was added before incubating for a further 24 h. The conditioned serum-free medium was collected and centrifuged at 2,000 × g for 30 mins at 4 ℃ to remove cells, followed by 16,000 × g for 20 mins at 4 ℃ to remove cell debris. The resulting supernatant was ultracentrifuged at 160,000 × g for 1.5 h at 4 ℃. The pellet was resuspended in PBS and ultracentrifuged again at 160,000 × g for 2 h at 4 ℃. The final pellet was used for proteomics analysis.  The supernatant were ultracentrifuged at 160,000 g for 1.5h HEK293 cells are cultured in complete growth media Complete growth media were removed. HEK293 cells were treated with serum-free media for 24h Samples were initially lysed by adding 700 µL QIAzol Lysis Reagent (QIAGEN, Cat. No. 79306), followed by vortexing for 60 sec and incubation at RT (15–25 °C) for 5 min. Subsequently, 140 µL chloroform was added, and samples were shaken vigorously for 15 s. Phase separation was achieved by centrifugation at 14,000 × g for 5 min at RT using QIAGEN MaXtract High Density Tubes. The aqueous phase containing total RNA was collected, and the protocol described in Appendix A of the miRNeasy Micro Kit manual was followed to selectively recover large RNA species (>200 nucleotides) depleted from small RNAs including miRNAs. We used Invitrogen™ Collibri™ Stranded RNA Library Prep Kit for Illumina sequencing.