Extended Data Fig. 9: The segmentation clock is sensitive to the rate of translation.
From: Metabolic regulation of species-specific developmental rates
a. Mass-specific OPP-Puromycin incorporation as a measure of global translation rate in MSGN1-Venus+ PSC-derived mouse and human PSM cells. OPP-Puromycilated peptides were detected by click chemistry with AlexaFluor647-Picoyl Azide. Mean ±SD. n = 3 biological replicates. Unpaired two-sided t-test. b. Translation rate as measured by puromycin incorporation expressed as percent of control for human PSM cells treated with DMSO, 40 nM, 80 nM or 160 nM cycloheximide (CHX) for 24 h. Mean ±SD. n = 3 biological replicates. One-way ANOVA with Dunnett correction. c. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with DMSO (n = 6), 40 nM (n = 4), 80 nM (n = 5) or 160 nM (n = 4) cycloheximide (CHX) for 24 h. n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. 40 nM p = 7.6 × 10−5, control vs. 80 nM p = 4.5 × 10−6, control vs. 160 nM p = 4.2 × 10−5. d. Number of HES7-Achilles oscillations observed in 25 h in human PSM cells treated with DMSO (n = 6), 40 nM (n = 4), 80 nM (n = 5) or 160 nM (n = 5) cycloheximide (CHX) for 24 h. n denotes independent experiments. Mean ±SD. e. Mean amplitude expressed as a percent of control in human PSM cells treated with DMSO (n = 6), 40 nM (n = 4), 80 nM (n = 5) or 160 nM (n = 4) cycloheximide (CHX) for 24 h. n denotes independent experiments. Mean ±SD. One-way ANOVA with Dunnett correction. f. HES7-Achilles oscillatory profile for human PSM cells treated with DMSO-control (n = 3) or 80 nM cycloheximide (CHX; n = 5). n denotes independent experiments. Mean ±SEM. g. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation for human PSM cells treated with DMSO-control (n = 3) or 80 nM cycloheximide (CHX, n = 5). n denotes independent experiments. Mean ±SEM. h. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation for human PSM cells treated with DMSO-control (n = 3) or 80 nM cycloheximide (CHX, n = 5). n denotes independent experiments. Mean ±SEM. i. Translation rate as measured by incorporation of the methionine analog AHA in human PSM cells treated with either DMSO control or 1mM sodium azide for one hour. Mean ±SD. n = 3 biological replicates. Unpaired two-sided t-test. j. Oxygen consumption rate measured over the course of the mitochondrial stress test for human PSM cells treated with DMSO control or 100 nM cycloheximide for 24 h. 1 μM Oligomycin, 1 μM FCCP, and 0.5 μM Rotenone + 0.5 μM Antimycin A were added at the timepoints marked by dotted lines. Mean ±SD. n = 9 biological replicates. k. Spare respiratory capacity in human PSM cells treated with vehicle control (DMSO) or 100 nM cycloheximide (CHX) for 24 h. Mean ±SD. n = 8 biological replicates. Unpaired two-sided t-test. l. Coupling efficiency shown as the percent of basal oxygen consumption that is linked to ATP production in human PSM cells treated with vehicle control (DMSO) or 100 nM cycloheximide (CHX) for 24 h. Mean ±SD. n = 8 biological replicates. Unpaired two-sided t-test.
