Figure 6.

Intranuclear Shuttling of RPL27-GFP

(A) Small nucleoplasmic regions (shown as white circles) in live or paraformaldehyde-fixed HeLa^RPL27-GFP cells were photobleached every 4 s for 50 times. The cells were imaged immediately after each bleaching event, and GFP fluorescence in the nucleoli (red circles) was measured. The scale bars represents 5 μm.

(B) Quantitation of nucleolar RPL27-GFP fluorescence relative to prebleach levels in live HeLa^RPL27-GFP cells (red), unbleached neighboring HeLa^RPL27-GFP cells in the same experiment (green), and paraformaldehyde-fixed HeLa^RPL27-GFP cells (blue). Average ± SD from five cells in each category.

(C and D) Effect of prior treatment with actinomycin D on the response of HeLa^RPL27-GFP cells to proteasome inhibitor MG132. HeLa^RPL27-GFP cells were treated with either 1/5000 (v/v) DMSO (C) or 0.5 μg/ml actinomycin D (stock solution: 2.5mg/ml in DMSO) (D) for 1 hr before the addition of 25 μM MG132. In both cases, the same cell was imaged before (left panel) and 150 min after (right panel) MG132 treatment. The scale bar represents 5 μm.

(E) Changes in RPL27-GFP fluorescence intensities in nucleoli, the nucleoplasm, and the cytoplasm (relative to the intensities before treatment. Average ± SD of five cells). Green represents cells pretreated with DMSO before MG132 treatment. Red represents cells pretreated with actinomycin D before MG132 treatment.