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. 2014 Apr 1;111(15):E1481–E1490. doi: 10.1073/pnas.1321811111

Fig. 1.

Fig. 1.

CME is reduced in cells with protein aggregates. Internalization of Alexa555-conjugated transferrin was assayed by fluorescence confocal microscopy in cells transiently (A–H) or stably (I–K) expressing various aggregation-prone proteins fused to GFP. (A and B) Internalized Alexa555-transferrin (Tf, Right) in human PC-3 cells transiently expressing soluble or aggregation-prone Q19-EGFP or Q82-EGFP, respectively (Left). Images shown are maximal projections of a z-series of fluorescence confocal slices through the entire cell volume. Arrows indicate cells containing aggregates. Note the aggregate-containing cells in B have reduced fluorescence levels of transferrin compared with untransfected or soluble Q82-expressing cells. (Scale bar, 10 μm.) (C) Quantification of the effect of relative Q82 expression levels, as measured by sum fluorescence intensities of EGFP divided by the cell area, on transferrin internalization. Quantification for each cell was measured by taking the sum fluorescence intensity of the z-series through the total cell volume, divided by the area of the cell. Shown are the mean values, with error bars representing SDs. P values were determined using a Student t test. (D) Scatterplot depicts quantification of internalized Alexa555-transferrin in untransfected and Q19-EGFP–, or Q82-EGFP–expressing cells. (E and F) As in A and B, but in PC-3 cells expressing WT (wt) or aggregation-prone mutant (A4V) SOD1-AcGFP, respectively. Arrows indicate cells containing aggregates. The cell labeled with an asterisk contains minute aggregates and is further magnified in Fig. S1E. (G) Quantification, as in C, of internalized transferrin in SOD1-expressing cells. (H) Effect of relative expression levels on transferrin uptake, as in D, in SOD1(A4V)-expressing cells. (I and J) As in A and B, but in Neuro2a cells induced to express stably a single integrated copy of EGFP-fused Q2- or Q82-expanded Atx-1. (J) Two separate image samples are shown for GFP (1, 2) and transferrin (1′, 2′) fluorescence. Arrows indicate cells containing aggregates. (K) Quantification, as in C, of internalized transferrin in Neuro2a cells induced to express Atx-1 Q2 or Q82. Dox, doxycycline.