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. Author manuscript; available in PMC: 2019 Jul 30.
Published in final edited form as: Nature. 2019 Jan 30;566(7743):264–269. doi: 10.1038/s41586-019-0895-y

Extended Data Figure 4: TSC2 huS1364 or huS1365 mutated to glutamatic acid (SE) suppresses endothelin-1 stimulated myocyte hypertrophy and mTORC1 activation, whereas mutation to alanine (SA) amplifies both.

Extended Data Figure 4:

a) Nppb mRNA expression (pathological hypertrophy gene marker) in myocytes transfected with WT, SA, or SE TSC2 (huS1365 mutated), and then exposed to 48-hrs ET1 (to induce hypertrophy) or to vehicle (Veh). Activation of PKG by sildenafil (Sil) reduces ET1-stimulated Nppb in WT expressing cells, but not in cells expressing SA or SE TSC2 mutants. SE expression depresses Nppb rise with ET1 stimulation, whereas SA expression enhances it. These results are nearly identical to those shown in Figure 2A in which the huS1364 (first serine of the duplet) was mutated. This shows that genetic modulation of either serine results in the same biological modulation of ET1 stimultion on growth and mTORC1 activity. Mean±SEM, n=6 biologically independent experiments, 1WANOVA with Tukey multiple comparisons test. *p<1E-5 vs other WT groups; † p=0.001 vs SE, p<1E-6 vs WT+ET1, ‡ p<1E-6 vs SA, WT-ET1, SE+ET1. b) Summary analysis of immunoblots displayed in Figure 1b. Values are normalized to WT/Veh; Mean SEM, n=4 (LC3-II) or 6 (others) biologically independent experiments; 1WANOVA, Tukey multiple comparisons test: *p≤ 7E-6 vs Vehicle control; † p<1E-6 vs SE-ET1; ‡p<5E-6, #p=0.01 vs WT-ET1, c) Example immunoblots from the same experiment as in Panel a and b, showing changes in mTORC1 signaling proteins, p62, and LC3-II. ET1 stimulates phosphorylation of mTORC1 targets (p70S6K, 4EBP1, Ulk1) and increases LC3-II and p62–consistent with mTORC1 activation and enhanced autophagy. SE (huS1365E) reduces mTORC1 activation and p62, and increases LC3-II, whereas SA (huS1365A) does the opposite. This is identical to responses found using huS1364 mutants (Fig 2b, Extended Data panel 4b) confirming functional equivalency from either serine modification. Experiment replicated 2–4 times, providing n=4–8 biologically independent samples. d) Summary data for this experiment. Values normalized to WT/Veh; Mean±SEM, n=8 independent replicates for p70S6K and 4EBP1, n=6 for Ulk-1, and n=4 for p62 and LC3-II. 1-WANOVA with Tukey multiple comparisons test, *p≤1E-6 vs WT vehicle and SE-ET1, †p<1E-6 vs SA-ET1, §p=0.0001, ‡p=0.003, ¶p<1E-6 vs SA-ET1, #p=0.003 vs. SA-Veh.