Extended Data Fig. 5: Effects of TSC2(S1365A) and TSC2(S1365E) on mTORC1 activation in response to phenylephrine; PKG1 activation of autophagy requires in part its phosphorylation of TSC2; and amplification of mTORC1 stimulation in cells expressing S1365A TSC2 mutation requires RHEB.
From: PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress
a, NRCMs expressing TSC2(WT), TSC2(S1365A) or TSC2(S1365E) (human TSC2(S1365) was modified) and exposed to vehicle or phenylephrine (PE, 100 mM) for 48 h. Left, example immunoblot for phospho-p70S6K and total protein. Right, summary data, normalized to TSC2(WT) without phenylephrine. Data are mean ± s.d., n = 6 biologically independent samples, Kruskal–Wallis Test with Dunn’s multiple comparisons test, *P = 0.003 versus corresponding vehicle; †P = 0.0013 versus TSC2(S1365E) and ET1. b, Top, example immunoblots for LC3-II and p62 in lysates obtained from Tsc2 knockout MEFs transfected with either human TSC2(WT) or TSC2(S1364A) plasmids and then treated with ET1 (10 nM) with or without cGMP (50 μM). n = 3 biologically independent samples. Bottom, summary data, normalized to TSC2(WT) without cGMP and without ET1; data are mean ± s.d., one-way ANOVA with Tukey multiple comparisons test, *P = 0.01 versus TSC2(WT) without ET1 without cGMP, P = 0.02 versus TSC2(WT) and ET1 with cGMP, †P < 1 × 10−6 versus TSC2(S1364A) without ET1 without cGMP and versus with ET1 with cGMP, #P = 2 × 10−6 versus TSC2(WT) with ET1, ‡P = 5 × 10−5 versus TSC2(WT) and ET1 with cGMP, ††P = 1 × 10−6 versus TSC2(WT) without ET1 without cGMP and versus ET1 with cGMP, **P = 1 × 10−6 versus TSC2(WT) without ET1 without cGMP, §P = 0.002 versus TSC2(WT) with ET1, §§P < 1 × 10−6 versus TSC2(WT) with ET1 with sildenafil. c, Summary results for Fig. 2d. Rat cardiomyocytes transfected with RHEB or scrambled (Scr) siRNA, transfected with TSC2(WT), TSC2(S1365E) or TSC2(S1365A) plasmids (human TSC2(S1365) modified) and stimulated with ET1 for 48 h. Effect of gene silencing of Rheb on RHEB protein expression (right) and on phosphorylated/total p70S6K protein expression (left). n = 4 biologically independent experiments, data are mean ± s.d., values normalized to TSC2(WT) treated with vehicle and scramble siRNA. Each plot was analysed by one-way ANOVA with pairwise Tukey multiple comparisons test, †P = 1 × 10−6 versus corresponding scramble siRNA without or with ET1, one-way ANOVA with Tukey multiple comparisons test, *P = 1 × 10−6 versus scramble siRNA and vehicle for TSC2(WT) and TSC2(S1365A), #P = 0.00002 versus scramble siRNA and ET1 for TSC2(WT) or TSC2(S1365A).
