Fig. 1: Nutrient starvation induces the formation of proteasome foci in the nucleus of mammalian cells.

a Schematic representation of the mammalian proteasome composed by the 20S catalytic particle (CP) and 19S or 11S regulatory particles (RP). b Protein levels of proteasome components and other factors following nutrient deprivation in IMR90 cells. Cells were incubated in HBSS solution and harvested at different time points for immunoblotting. 4EBP1 phosphorylation is included as a control for starvation (representative from 2 independent experiments). c IMR90 and HCT116 cells form nuclear foci following nutrient deprivation. IMR90 and HCT116 cells were incubated in HBSS for 6 hrs or 8 hrs, respectively, and harvested for immunostaining (representative from 3 independent experiments). d Fractionation of IMR90 and immunoblotting for components of the proteasome. IMR90 cells were fractionated by hypotonic lysis and cytoplasmic and nuclear fractions were used for immunoblotting. LDH and PARP1 proteins were detected as markers of cytoplasmic and nuclear fractions, respectively (representative from 2 independent experiments). e Fractionation of HCT116 cells and immunoblotting for components of the proteasome. HCT116 was fractionated by quick lysis with 0.1 % NP-40 detergent and nuclear and cytoplasmic fractions were obtained. The wash fraction corresponds to resuspension of the nuclear pellet in detergent-free buffer followed by an additional centrifugation. LDH and PARP1 were detected as markers of cytoplasmic and nuclear fractions, respectively (representative from 2 independent experiments). f Subcellular localization of PSMB4-GFP following incubation of IMR90 cells in HBSS. IMR90 cells were transduced with lentivirus particles expressing PSMB4-GFP. Following four days post-infection, cells were incubated in HBSS for 6 hrs and harvested for fluorescence microscopy (representative from 3 independent experiments).