Extended Data Figure 9.
The segmentation clock is sensitive to the rate of translation
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, 40nM, 80nM or 160nM cycloheximide (CHX) for 24 hours. 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), 40nM (n=4), 80nM (n=5) or 160nM (n=4) cycloheximide (CHX) for 24 hours. n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. 40nM p=7.6x10−5, control vs. 80nM p=4.5x10−6, control vs. 160nM p=4.2x10−5
d. Number of HES7-Achilles oscillations observed in 25 hours in human PSM cells treated with DMSO (n=6), 40nM (n=4), 80nM (n=5) or 160nM (n=5) cycloheximide (CHX) for 24 hours. n denotes independent experiments. Mean ±SD.
e. Mean amplitude expressed as a percent of control in human PSM cells treated with DMSO (n=6), 40nM (n=4), 80nM (n=5) or 160nM (n=4) cycloheximide (CHX) for 24 hours. 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 80nM 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 80nM 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 80nM cycloheximide (CHX, n=5). n denotes independent experiments. Mean ±SEM.
k. 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 100nM cycloheximide for 24 hours. 1μM Oligomycin, 1μM FCCP, and 0.5μM Rotenone + 0.5μM 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 100nM cycloheximide (CHX) for 24 hours. 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 100nM cycloheximide (CHX) for 24 hours. Mean ±SD. n=8 biological replicates. Unpaired two-sided t-test.