Extended Data Fig. 10: Protein stability differences between mouse and human PSM cells.
From: Metabolic regulation of species-specific developmental rates
a. Pulse-chase experiment tracking the degradation of puromycilated peptides over the course of 12 h in MSGN1-Venus+ PSC-derived mouse and human PSM cells following a 1-hour pulse with puromycin. Solid line represents best one-phase decay fit with the 95% confidence intervals shown as shaded regions. n = 3 independent experiments. b. Mean amplitude expressed as a percent of control in human PSM treated with DMSO control (n = 9), 2.5 nM (n = 6), 5 nM (n = 13) or 10 nM (n = 12) bortezomib, or 1 μM lactacystin (n = 8). n denotes independent experiments. Mean ±SD. One-way ANOVA with Dunnett correction. c. Number of HES7-Achilles oscillations observed in 25 h in human PSM treated with DMSO control (n = 17), 2.5 nM (n = 6), 5 nM (n = 13) or 10 nM (n = 12) bortezomib, or 1 μM lactacystin (n = 9). n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. 5 nM bortezomib p = 2.4 × 10−19, control vs. 10 nM bortezomib p = 1.8 × 10−22. d. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with DMSO control (n = 10) or 2.5 nM bortezomib (BTZ, n = 6). n denotes independent experiments. Mean ±SD. Unpaired two-sided t-test. e. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with DMSO control (n = 9) or 1 μM lactacystin (n = 8). n denotes independent experiments. Mean ±SD. Unpaired two-sided t-test. f. HES7-Achilles oscillatory profile for human PSM cells treated with DMSO-control (n = 9), 5 nM bortezomib (n = 13), or 1 μM lactacystin (n = 8). Mean ±SEM. g. stantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation for human PSM cells treated with DMSO-control (n = 9), 2.5 nM bortezomib (n = 6), or 1 μM lactacystin (n = 8). Mean ±SEM. h. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation for human PSM cells treated with DMSO-control (n = 9), 5 nM bortezomib (n = 13), or 1 μM lactacystin (n = 8). Mean ±SEM. i. Proteasome activity cells as measured by cleavage of a luminogenic proteasome substrate in human PSM treated with DMSO control, 2.5 nM, 5 nM or 10 nM bortezomib, or 1 μM lactacystin for 24 h. Mean ±SD. n = 6 biological replicates. One-way ANOVA with Šidák correction: control vs. 2.5 nM bortezomib p = 5.8 × 10−8, control vs. 5 nM bortezomib p = 1.2 × 10−9, control vs. 10 nM bortezomib p = 4.3 × 10−13, control vs. 1 μM lactacystin p = 2.2 × 10−10. j. Proteasome activity cells as measured by cleavage of a luminogenic proteasome substrate in human PSM treated with DMSO control, 100 nM antimycin A, or 1 mM sodium azide for 24 h. Mean ±SD. n = 3 biological replicates. One-way ANOVA with Šidák correction: control vs. antimycin p = 1.3 × 10−5, control vs. azide p = 3.1 × 10−7. k. Pulse-chase experiment tracking the degradation of puromycilated peptides over the course of 12 h in human PSM cells treated with DMSO control or 1 mM sodium azide following a 1-hour pulse with puromycin. Solid line represents best one-phase decay fit with the 95% confidence intervals shown as shaded regions. n = 3 independent experiments. l. Pulse-chase experiment tracking the degradation of AHA-labeled proteins over the course of 30 h in human PSM cells treated with DMSO control of 1 mM sodium azide following a 1-hour pulse with AHA. Solid line represents best one-phase decay fit with the 95% confidence intervals shown as shaded regions. n = 3 independent experiments.
