![Figure 1. UPS impairment occurs above a critical, polyglutamine length-dependent concentration of mutant htt. (A) Flow cytometry analysis of UPS function in HEK293 cells. Two-color scatter plots of HEK293 cells (top row) or HEK293 cells stably expressing UbG76V-GFP (bottom row). Cells were analyzed 72 h after transient transfection of N-htt(Q25)–chFP or N-htt(Q91)–chFP as indicated. Control experiments included cells incubated overnight with 10 µM MG132 or left untreated (control [Ctrl]). Graphs are presented as pseudocolor density plots with “hot” colors (i.e., red) indicating high frequency areas and “cold” colors (i.e., blue) indicating rare events. (B) UbG76V-GFP accumulates above a critical concentration threshold of mutant htt. Relationship between UbG76V-GFP fluorescence (vertical axis) and the fluorescence of N-htt(Q25)–chFP, N-htt(Q91)–chFP, or the difference between the Q91 and Q25 curves derived from flow cytometry as in A. a.u., arbitrary unit. Details of data transformation are presented in Fig. S2. (C) Effect of polyglutamine length on dose response of chFP-CL1 fluorescence to N-htt(Qn)–GFP expression, in which n = 25 (•), 48 (■), 72 (◆), and 91 (▴). (D) Effect of equivalent levels of expression of mutant N-htt on UbG76V-GFP accumulation becomes more severe with increasing expression time. HEK293 cells stably expressing UbG76V-GFP were transfected with N-htt(Q91)–chFP or N-htt(Q25)–chFP and analyzed by flow cytometry. The Q91-specific mean reporter fluorescence was calculated as in B at 48 (•), 72 (■), 96 (◆), or 120 (▴) h after transfection. The data shown are from single representative experiments out of at least two independent repeats.](https://cdn.rupress.org/rup/content_public/journal/jcb/196/5/10.1083_jcb.201110093/6/jcb_201110093_fig1.jpeg?Expires=1771625638&Signature=OkBy-CPmqobsbMSdkVlmJnO3P~P6-ESgojrX7DREvJ5AmNJgJwxI~Whqn5sYQHRnSrkGVA4elC7ibLYxGjM-SuZodXcW9iqLhcFqD7y0Wb28EWAKRygYZnJQw0JtsmYXK7~qy8o8pi~1qcrfNv7Vaq17u3tFupFF3s9g98pxiDpWL9Lz4ypGUt0WkQ8iowo3-VRI~vLhxyTLtCRdeK1VCHqC2oKXfc4XSEPLW88SL6E-tZhzylxP6P5BN5LbOpSQSbi3xUbb57Edex~HDWnX7DQuRibfpL5MxpZ3xX-vfdx7IShY6W~poI~Qxzb9HkanEf~tlhwopq8X7S4bDSDbaA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
UPS impairment occurs above a critical, polyglutamine length-dependent concentration of mutant htt. (A) Flow cytometry analysis of UPS function in HEK293 cells. Two-color scatter plots of HEK293 cells (top row) or HEK293 cells stably expressing UbG76V-GFP (bottom row). Cells were analyzed 72 h after transient transfection of N-htt(Q25)–chFP or N-htt(Q91)–chFP as indicated. Control experiments included cells incubated overnight with 10 µM MG132 or left untreated (control [Ctrl]). Graphs are presented as pseudocolor density plots with “hot” colors (i.e., red) indicating high frequency areas and “cold” colors (i.e., blue) indicating rare events. (B) UbG76V-GFP accumulates above a critical concentration threshold of mutant htt. Relationship between UbG76V-GFP fluorescence (vertical axis) and the fluorescence of N-htt(Q25)–chFP, N-htt(Q91)–chFP, or the difference between the Q91 and Q25 curves derived from flow cytometry as in A. a.u., arbitrary unit. Details of data transformation are presented in Fig. S2. (C) Effect of polyglutamine length on dose response of chFP-CL1 fluorescence to N-htt(Qn)–GFP expression, in which n = 25 (•), 48 (■), 72 (◆), and 91 (▴). (D) Effect of equivalent levels of expression of mutant N-htt on UbG76V-GFP accumulation becomes more severe with increasing expression time. HEK293 cells stably expressing UbG76V-GFP were transfected with N-htt(Q91)–chFP or N-htt(Q25)–chFP and analyzed by flow cytometry. The Q91-specific mean reporter fluorescence was calculated as in B at 48 (•), 72 (■), 96 (◆), or 120 (▴) h after transfection. The data shown are from single representative experiments out of at least two independent repeats.
