Supplementary Figure 9: HD and control neural cells show no difference in cell composition after Isx-9 treatment; representative pictures used for nuclear condensation assay; and cumulative risk of death for adult-onset-range differentiated iPSCs.

(a) Immunocytochemistry at day 56 of differentiation demonstrate that both HD and non-disease iPSC lines can generate neural progenitors (NESTIN, DOUBLECORTIN). Scale bar represents 100 um.(b) Differentiated iPSC cultures were treated with ISX-9 or BDNF for 48 h. Nuclei were stained with Hoechst 3343. The images were obtained on an Axiovert 200 inverted microscope and quantified using Volocity. Representative images are show for each condition. Inserts illustrate healthy nuclei (left panel) and condensed nuclei (right panel). (c) The cumulative risk of death of both the 46Qn1 (HR= 2.1, p= 4.16e-06) and 46Qn10 (HR= 2.9, p= 0.000243) are greater than the control 18Qn2 line. Addition of 20 μM ISX-9 decreases the cumulative risk of death of 46Qn1 (HR= 0.79, p= 0.004961) and in the 46Qn10 (HR=0.65, p= 5.42e-06). (d) Although we saw a trend in the survival curve of the 46Qn10, we did not see significant differences in the CRD between the 18Qn6 risk of death to the 46Qn1 (HR= 0.87, p= 0.4) and the 46Qn10 (HR= 1.2106, p= 0 0.367148) likely due to a low n as when we combine clones the difference becomes significant as in Figure 7. Control 18Qn2 +DMSO, n=69 cells, Control 18Qn +ISX-9, n= 78 cells, Control 18Qn6+DMSO, n=55 cells, Control 18Qn6+ISX-9, n= 66 cells, (n=2 experiments). 46Qn1 + DMSO, n= 414 cells, 46Qn1+ISX-9, n= 474 cells, (n=5 experiments); 46Qn10+DMSO, n= 289 cells, 46Qn10+ISX-9 n= 310 cells, (n=5 experiments). (e) We evaluated the cumulative risk of death of the 53Qn3 lines compared to the controls. We found evidence of non-proportionality for a subset of the lines so we used log rank tests to assess the differences of survival between the HD and controls lines and the effects of ISX-9 on the HD and control lines. All p values are reported from the log rank test; however, we report the hazard ratios as an estimate of the hazard from the Cox proportional hazards model. (f) The cumulative risk of death of the 18Qn2 is lower risk of death than the 53Qn3 (HR= 1.4, p= 0.06 approaching significance) and 53Qn5 (HR=1.3 p= 0.00572). Addition of 20 μM ISX-9 increases survival of 53Qn3 (HR= 0.74, p= 2.4e-06) and 53Qn5 (HR=0.83, p= 0.0198) i-neurons. ISX-9 does not significantly change cumulative risk of death for the control Q18n2 (p= 0.931). The cumulative risk of death of 18Qn6 is lower than the 53Qn3 (HR= 1.2, p= 0.0535 approaching significance) and 53Qn5 (HR=1.3, p= 0.00622). ISX-9 does not significantly change cumulative risk of death for the control Q18n6 (p= 0.197). Control 18Qn2 +DMSO, n=345 cells, 18Qn +ISX-9, n= 324 cells, (n=4 experiments) Control 18Qn6+DMSO, n=145cells, 18Qn6 + DMSO, n= 128 cells, (n=3 experiments); 53Qn3+DMSO, 695 cells, 53Qn3+ISX-9, n= 726 cells, (n=6 experiments); 53Qn5+DMSO, 352 cells, 53Qn5+ISX-9, 381cells (n=3 experiments).