FIGURE 1.

Proteasome inhibition in NIH 3T3 cells results in R-CRT accumulation in SGs. A, NIH 3T3 cells were incubated with MG132 (10 μm) for 1 and 2 h (control: 0 h treatment). B, quantification of fluorescence intensity in immunofluorescence (A). Data are shown as the mean ± S.E. *, p < 0.05; ***, p < 0.001 for comparison with control. C, cells incubated in the absence or presence of MG132 (10 μm) and treated with Tg (2 μm) for 20 min (20′ TG) were analyzed 40 and 60 min after Tg removal (40′ Post-TG and 60′ Post-TG). D, quantification of fluorescence intensity in immunofluorescence data (C). The corrected total cell fluorescence was expressed as fold change value relative to control (0 h). Data are shown as the mean ± S.E. ***, p < 0.001 for intragroup comparison; i.e. black bar versus white bar within each of the four experimental groups. ###, p < 0.001 for intergroup comparison with control (0 h) group; i.e. white or black bar of 20′ TG, 40′ Post-TG, or 60′ Post-TG group versus the corresponding bar of the control group. Results shown are representative of 200 cells from at least three separate experiments for each condition. Scale bars, 20 μm. N.S., not significant. Cells were analyzed by double immunofluorescence using anti-R-CRT and SG marker anti-TIA-1 pAbs. Co-localization of proteins was monitored by confocal microscopy. SG formation was observed upon MG132 treatment, and R-CRT was found to be co-localized with SGs as shown by yellow pseudo-color in the merge images.