Extended Data Fig. 5: Effect of electron transport chain inhibitors on the segmentation clock.
From: Metabolic regulation of species-specific developmental rates
a. Basal oxygen consumption rate in human PSM cells treated with vehicle control (DMSO), 20nM rotenone, 50 nM atpenin A5, 100 nM antimycin A, 1 mM sodium azide, 1 μM oligomycin or 1 μM FCCP. Mean ±SD. n = 10 biological replicates. One-way ANOVA with Šidák correction: control vs. rotenone p = 5.4 × 10−12, control vs. atpenin p = 4.3 × 10−11, control vs. antimycin p = 3.8 × 10−18, control vs. azide p = 1.04 × 10−20, control vs. oligomycin p = 5.9 × 10−8, control vs. FCCP p = 0.999. b. Number of HES7-Achilles oscillations observed in 25 h for human PSM cells treated with vehicle control (DMSO, n = 29), 20 nM rotenone (n = 10), 50 nM atpenin A5 (n = 17), 100 nM antimycin A (n = 11), 1 mM sodium azide (n = 15), 1 μM oligomycin (n = 11), or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. rotenone p = 1.1 × 10−10, control vs. atpenin p = 2.3 × 10−12, control vs. antimycin p = 6.1 × 10−15, control vs. azide p = 2.0 × 10−15, control vs. oligomycin p = 4.7 × 10−10. c. Mean amplitude expressed as a percent of control for HES7-Achilles oscillations in human PSM cells treated with vehicle control (DMSO, n = 53), 20 nM rotenone (n = 10), 50 nM atpenin A5 (n = 18), 100 nM antimycin A (n = 11), 1 mM sodium azide (n = 14), 1 μM oligomycin (n = 11), or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SD. One-way ANOVA with Dunnett correction: control vs. oligomycin p = 3.4 × 10−8. d. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with vehicle control (DMSO, n = 53), 20 nM rotenone (n = 10), 50 nM atpenin A5 (n = 18), 100 nM antimycin A (n = 11), 1 mM sodium azide (n = 14), 1 μM oligomycin (n = 11), or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. rotenone p = 8.9 × 10−21, control vs. atpenin p = 1.8 × 10−18, control vs. antimycin p = 2 × 10−15, control vs. azide p = 1.4 × 10−33. e. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 10) or 20 nM rotenone (n = 11). n denotes independent experiments. Mean ±SEM. f. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 12) or 50 nM atpenin A5 (n = 18). n denotes independent experiments. Mean ±SEM. g. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 11) or 100 nM antimycin A (n = 13). n denotes independent experiments. Mean ±SEM. h. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 9) or 1 mM sodium azide (n = 15). n denotes independent experiments. Mean ±SEM. i. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 12) or 1 μM oligomycin (n = 17). n denotes independent experiments. Mean ±SEM. j. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 3) or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SEM. k. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 10) or 20 nM rotenone (n = 11). n denotes independent experiments. Mean ±SEM. l. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 12) or 50 nM atpenin A5 (n = 18). n denotes independent experiments. Mean ±SEM. m. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 11) or 100 nM antimycin A (n = 13). n denotes independent experiments. Mean ±SEM. n. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 9) or 1 mM sodium azide (n = 15). n denotes independent experiments. Mean ±SEM. o. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 12) or 1 μM oligomycin (n = 17). n denotes independent experiments. Mean ±SEM. p. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 3) or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SEM. q. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 10) or 20 nM rotenone (n = 11). n denotes independent experiments. Mean ±SEM. r. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 12) or 50 nM atpenin A5 (n = 18). n denotes independent experiments. Mean ±SEM. s. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 11) or 100 nM antimycin A (n = 13). n denotes independent experiments. Mean ±SEM. t. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 9) or 1 mM sodium azide (n = 15). n denotes independent experiments. Mean ±SEM. u. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 12) or 1 μM oligomycin (n = 17). n denotes independent experiments. Mean ±SEM. v. Instantaneous HES7-Achilles oscillatory amplitude over the time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 3) or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SEM. w. Inner mitochondrial membrane potential (ΔΨm) in human PSM cells under control conditions or treated acutely with 1 μM FCCP. TMRM fluorescence was normalized by mitochondrial content (MitoTracker Green) following flow cytometry. Mean ±SD. n = 3 biological replicates. Unpaired two-sided t-test: p = 6.4 × 10−7.
