Extended Data Fig. 8: Generation of Tsc2S1365E/S1365E knock-in mice and impact on pressure-overload-induced mTORC1 activation, autophagy and autophagic flux in vivo.
From: PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress
a, Strategy and guide RNA for CRISPR–Cas9 protocol to generate Tsc2S1365E/S1365E knock-in mice. b, Summary data for Fig. 4f. Phosphorylated/total p70S6K (n = 6 biologically independent experiments), p62/α-tubulin and LC3-II/total protein (n = 4 biologically independent experiments, data are mean ± s.d., values are normalized to Tsc2WT/WT sham), two-way ANOVA with Sidak’s multiple comparisons test, *P < 1 × 10−6 versus Tsc2WT/WT sham, P = 0.0012 versus Tsc2S1365E/WT with pressure overload, and P = 0.0001 versus Tsc2S1365E/S1365E with pressure overload, †P = 0.0008 versus Tsc2S1365E/WT sham, ‡P = 0.001 versus Tsc2S1365E/S1365E sham; for p62, #P ≤ 1 × 10−6 versus all other groups, for LC3-II, §P = 0.018 and P = 0.0003 versus pressure overload Tsc2S1365E/WT and pressure overload Tsc2S1365E/S1365E, respectively, **P = 0.02 versus pressure overload Tsc2S1365E/S1365E and P = 0.0002 versus sham, ##P < 1 × 10−6 versus sham. c, Example immunoblot and summary results for Tsc2WT/WT, Tsc2S1365E/S1365E and Tsc2S1365A/S1365A mice treated with BFA or vehicle. Myocardium was assayed for LC3-II, with higher expression in the presence of BFA, indicating greater autophagic flux. Summary data, values normalized to Tsc2WT/WT vehicle, data are mean ± s.d., n = 4 biologically independent experiments; two-way ANOVA: P < 1 × 10−6 for BFA effect, P = 0.003 for Tsc2 genotype effect, and P = 0.002 for interaction; Sidak’s pairwise multiple comparisons test: *P = 0.0008 versus Tsc2WT/WT vehicle, †P < 1 × 10−6 versus Tsc2S1365E/S1365E vehicle and P = 0.008 versus Tsc2S1365A/S1365A + BFA, ‡P = 0.05 versus Tsc2WT/WT + BFA.
