Mouse embryonic fibroblasts (MEFs) and primary cortical neurons (PNCs) were isolated from E15.5 embryos from Cln1R151X, Cln2R207X, Cln3Δex7/8, Cln6nclf, and Cln8mnd mouse lines. Cln11−/− fibroblasts were isolated from adult Cln11−/− mice. All cell lines were dosed with drug-containing media on day in vitro (DIV) 3 and DIV5 and analyzed on DIV7 using the CellInsight CX7 High-Content Screening Platform (CX7) (A) Representative ASM accumulation in Cln3Δex7/8 MEFs and PNCs. Upon treatment with AF38469 (40 nM, 400 nM, 4μM), ASM accumulation was (B-F) reduced in Cln1R151X Cln2R207X, Cln3Δex7/8, Cln6nclf, and Cln8mnd MEFs; (B-D, F) reduced in Cln1R151X Cln2R207X, Cln3Δex7/8, and Cln8mnd PNCs; and (E, G) increased in Cln6nclf PNCs and Cln11−/− MEFs. Mean ± S.E.M.. One-way ANOVA with a Šidák post-hoc test, */#p<0.05, **/##p<0.01, ***/###p<0.001, ****/####p<0.0001. Hashsigns indicate comparison to WT, asterisks indicate comparison to mutant vehicle. MEFs and PNCs n=1000-4000 and 4000-20000 cells/treatment group, respectively. Scale bar, 100 μm, Inset scale bar, 50 μm.