Figure 4.

Topoisomerase Inhibitors Partially Rescue UBE3A Levels and Neuronal Function in AS hCOs

(A, D, F, and G) qRT-PCR measurements of mRNA levels of UBE3A (red) and UBE3A-ATS (gray) after vehicle (DMSO), 1 μM topotecan, or 1 μM indotecan treatment. Signals normalized to TATA-box binding protein (TBP) and ratioed to vehicle-treated AS hCOs.

(A) mRNA 3 days after a single drug treatment in 11 week hCOs.

(B) 11 week AS hCOs with CamKIIa-GFP neurons. Insets zoom in on arrowheads.

(C) Quantification of (B).

(D) mRNA 3 days after a single drug treatment in 4 week hCOs.

(E) mRNA after 1–9 days of continuous 1 μM indotecan treatment in 11 week AS hCOs, ratioed to untreated day 0 AS hCOs. No significant change in vehicle-treated samples (Figure S4C).

(F) mRNA 10 days after a single drug treatment in 11 week hCOs.

(G) mRNA 17 days after a single drug treatment in 11 week hCOs. Statistics: ^∗p < 0.05, n.s., not significant, full tick marks compared with half tick marks by one-way ANOVA with Tukey-Kramer post hoc. For (A, D, E, F, G) n = 3 independent experiments with three to five organoids in each; error bars, 95% confidence intervals. For (C) A.U., arbitrary fluorescence units; n = 3 independent experiments with two organoids in each.

(H–J) (H) Quantification of the calcium transient frequencies. (I) Quantification of the amplitudes of calcium transients. (J) Representative sets of calcium transient traces extracted from individual neurons of H9 DMSO, AS DMSO, and AS indotecan-treated hCOs.

Statistics for (H and I): ^∗p < 0.05, two-tailed unpaired Student's t test. n = 22, 41, and 34 neurons from H9 DMSO, AS DMSO, and AS indotecan-treated hCOs, respectively. Note: statistical comparison between H9 and AS hCOs shown only for completeness, as these are non-isogenic cell lines.

(See also Figure S4.)