Fig. 2: Proteasome integrity is required for foci formation upon nutrient deprivation.

a Immunostaining of proteasome components in IMR90 human fibroblasts showing that component of the CP and RP co-localize in nuclear foci following 6 hrs incubation in HBSS. The green (endogenous PSMD11 or PSMB7), red (endogenous PSMB1, PSMB2, PSMB4, or PSMD4), and blue (DAPI) signals were merged to indicate foci localization within the nucleus. The inset at the bottom right of each picture corresponds to a magnification of a small portion of the nucleus as indicated. Graphs in the bottom represent relative signal intensity of the indicated line for each component of the proteasome (representative from 3 independent experiments). b siRNA-mediated depletion of components of the RP or CP abolishes PSMD7 or PSMB4 foci formation. IMR90 cells were transfected twice with the indicated siRNAs. After three days, cells were treated with HBSS for 6 hrs and harvested for immunostaining of endogenous PSMB4 and PSMD7 proteins. The images at the bottom represent magnification of the merge as indicated by white arrows (representative from 3 independent experiments). c Cell counts of PSMD4, PSMB4, or PSMD7 foci following depletion of individual components of the proteasome (n = 3 independent experiments). d Immunostaining of PSME3 in IMR90 cells showing that this subunit of the 11S RP localizes with PSMD7 in nuclear foci following 6 hrs incubation in HBSS (representative from 5 independent experiments). e siRNA-mediated depletion of components of the RP or CP abolishes PSME3 foci formation. IMR90 cells were transfected with siRNAs of components of the RP or CP. After 3 days, cells were incubated in HBSS for 6 hrs and harvested for immunostaining of PSME3 (n = 3 independent experiments). f Depletion of PSME3 by siRNA does not affect PSMD4 or PSMB4 foci formation. IMR90 cells were transfected with siRNAs of components of the RP or CP. After 3 days, cells were incubated in HBSS for 6 hrs and harvested for immunostaining of PSME3 (n = 3 independent experiments). Data in c, e, f represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant; 2-sided unpaired Student’s t-test with Welch correction (c, e, f). Source data are provided as a Source data file.