Figure 5: Temporal resolution of Htt96Q oligomeric forms and insoluble aggregates shows correlation of UPR induction with the presence of oligomers (a–c).

Parallel samples of HEK 293 cells expressing myc-tagged Htt96Q for 16, 24 or 30 h were incubated with Dox (1 μg ml⁻¹) for another 24 h (40, 48 and 54 h, respectively). The soluble fraction of a larger amount of each sample (60% of total) was fractionated in glycerol gradients (a). Smaller amounts (20% of total) were subjected to protein (b) and mRNA (c) detection. Fractions 5–12 in a are defined as oligomers (Htt96Q monomer is around 50 kDa). P=pellet (insoluble aggregates) and SN=supernatant (monomers+oligomers) in b. Overnight treatment with tunicamycin (5 μg ml⁻¹, b,c) was used as a positive control for UPR induction. Values were corrected by GAPDH levels, used as a loading control as in Fig. 4. (d,e) The graphs summarize the results presented in a–c. SN+P in b corresponds to total Htt at each time point. Percent of total Htt was P × 100/(SN+P) for insoluble aggregates and for oligomers it was oligomers × 100/total (SN) at each time point in a multiplied by SN × 100/(SN+P) in b and divided by 100; the remainders are monomers (not plotted). Especially informative are time points of 48 h, when oligomers are preponderant, and 54 h, when there are insoluble aggregates but almost no oligomers. Increase in BiP, XBP1-s and GADD34 levels correlates with the accumulation of Htt96Q oligomers. Shown is a representative of three experiments.