Figure 6.

Link between the cellular localization of the Hsp72 protein, the disassembly of SGs and translation reactivation during prolonged UPS inhibition. (A) The expression level of Hsp72 protein is enhanced during UPS inhibition. HeLa cells were treated with or without 10 μM MG132 for the indicated periods of time. The expression of Hsp72 was then analyzed by Western blot. (B) The overexpression of Hsp72 prevents SG formation. HeLa cells transfected with a construct encoding Flag-Hsp72 were treated with or without 10 μM MG132 for 3 h as indicated. The localization of Flag-Hsp72 was verified by immunofluorescence using the anti-Flag antibody. The formation of SGs was verified by immunofluorescence using antibodies against G3BP. Shown are representative images of three independent experiments. Cells overexpressing Flag-Hsp72 and did not form SGs are indicated with arrowheads. (C and D) RNAi-mediated Hsp72 depletion prevents SG disassembly after 7 h of treatment with MG132. HeLa cells incubated with an siRNA targeting Hsp72 or a control siRNA (Ctr) for 48 h were subsequently treated with 10 μM MG132 for 7 h and then lysed, and protein extracts were prepared and analyzed by Western blot for Hsp72 expression (C, HuR levels are included as a loading control). (D) The same experiment as in C was performed, and cells were fixed and used for immunofluorescence experiment with anti-FMRP and anti-Hsp72 antibodies. Cells that did not uptake the Hsp72-siRNA and lacking SGs are indicated with arrowheads. Representative images of two independent experiments are shown. (E and F) The knockdown of Hsp72 correlates with the absence of translation recovery upon treatment with MG132 for 7 h. (E) HeLa cells incubated with an siRNA targeting Hsp72, or a control siRNA, were treated with or without 10 μM MG132 for 3 or 7 h (h) and then subjected to ³⁵S-methionine labeling for 30 min to monitor de novo protein translation. Proteins were resolved by SDS-polyacrylamide gel, stained with Coomassie blue (bottom panel), and detected by autoradiography (top panel). (F) The results were plotted as a bar graph. The intensity of the signals was defined as described in Figure 2B.