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. 2017 Jun 19;26(18):3466–3481. doi: 10.1093/hmg/ddx227

Figure 4.

Figure 4

αS-3K::YFP inclusions colocalize with endosomes, lysosomes, and Golgi components in induced M17D cells. (A) Parental M17D cells or M17D-TR cells expressing YFP-tagged αS wt, 3K or KLK were transfected with cDNA for RFP and passaged for 10 days to achieve stable RFP expression in a subset of cells. Then αS expression was induced by dox for 90 h. Fluorescence microscopy for YFP (top), RFP (middle) plus merge images that include DAPI staining for nuclei (bottom). Scale bar, 10 µm. (B) Analogous to (A), but stable expression of RFP or indicated RFP-tagged markers in M17D-TR/αS-3K::YFP cells. Statistics were generated by first identifying inclusion-bearing double-positive (YFP and RFP) cells and then assigning ‘1’ to cells with apparent colocalization in at least one round inclusion and ‘0’ to cells without distinct colocalization in round inclusions. Samples were blinded during analysis. Three independent experiments, eight double-positive inclusion-bearing cells each (N = 3, n = 24); significance relative to RFP alone. Scale bars, 10 µm and 5 µm (zoom-in), respectively. (C) IncuCyte-based quantification of integrated YFP inclusion intensity and total RFP in induced (24 h) M17D-TR/αS-3K::YFP//RFP cells, plus representative images. 0.1% DMSO (vehicle), 300 µM brefeldin A and 80 µM dynasore were applied shortly after induction. Inclusion integrated intensities relative to DMSO (whose mean was set to 1 in each experiment) were 77.2 ± 15.0% (brefeldin A) and 73.2 ± 19.6% (dynasore). Three independent experiments in triplicates (N = 3, n = 9), significance relative to DMSO only. Scale bar, 50 µm. Integrated intensities of co-expressed RFP were not significantly changed. (D) Time-course of induced M17D-TR/αS-3K::YFP. IncuCyte analysis, YFP images were were taken in 2h intervals up to 96 h after induction, revealing mitotic events within this time frame for both 3K- and KLK-expressing cells. Scale bar, 10 µm. (E) Analogous to (D), but M17D-TR/αS-KLK::YFP. One-way ANOVA for statistical analyses. Bars are means +/− S.D. Criteria for significance: *P < 0.05, ****P < 0.01, ***P < 0.001, ****P < 0.0001.