Figure 4. ICM decreases neurite outgrowth and increases apoptosis on D6.

A, LUHMES cells were subcultured on D2 and exposed to ICM at 10²-10⁴ times [M] on D5. After 24 h, effects on cell viability and neurite outgrowth were evaluated. Caspase-3/7 activity was measured at 4 h and 16 h post-exposure. For neurite stability experiments, Brefeldin-A (Bf-A,10 μM) and U0126 (30 μM) were used as controls known to decrease neurite outgrowth (Supplementary Figure 10A). B, Representative photomicrographs of D6 LUHMES cells exposed to vehicle or ICM at 10³ X [M] co-stained using Hoechst (blue) and calcein-AM (green) and mask for automated analyses of neurite outgrowth in individual neurons. Bar=50 μm. Cell viability was measured as C, percentage of cells stained with calcein-AM and D, LDH release, normalized to vehicle, with lysis buffer (L, 9% Triton-X 100) as the positive control. E-F, Effects of ICM on neurite outgrowth measured in live cells as E, total neurite outgrowth and F, maximum process length per cell, both as percent of vehicle. G-H, Quantification of caspase-3/7 activity in D6 LUHMES cells exposed for 4 h or 16 h to ICM, EMA mix, individual cytokines, or 3 μM TPCA-1 ± ICM, normalized to vehicle. Data shown as mean ± SD (n=4 (C-G) or 5 (H) independent experiments with 12–24 (C-F) or 4–8 (G-H) replicate wells each). C-F, H, ^p<0.1, *p<0.05, ***p<0.0001 vs. vehicle as determined by one-way ANOVA; G, *p<0.05, **p<0.001 vs. vehicle as determined by 2-way ANOVA; H, ζp<0.01, ζζp<0.001 vs. ICM by one-way ANOVA; specific group differences identified by post hoc Dunnett’s multiple comparisons test. C-F, #p<0.05, ###p<0.0001 vs. vehicle as determined by unpaired t test; H, p=0.0596 for TPCA-1 vs. TPCA-1 + ICM by unpaired t test.