Extended Data Fig. 6: Tsc2S1365A/S1365A knock-in mouse genotyping; expression of TSC2 with or without pressure overload; and effect on pressure-overload-stimulated hypertrophy, autophagy and mTORC1 activation.
From: PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress
a, Mouse Tsc2S1365A/S1365A knock-in genotyping by PCR detects a unique sequence based on the mutated residue as a 206-base pair (BP) fragment. This signature was used for genotyping. b, Immunoblot of TSC2 protein from Tsc2S1365A/S1365A and Tsc2WT/WT (littermate controls) for sham and pressure-overload-treated groups. There is no difference in expression levels among these groups or conditions. Experiments were repeated independently three times with identical results. c, Nppa mRNA expression normalized to Gapdh mRNA expression in Tsc2WT/WT versus Tsc2S1365A/S1365A myocardium before and after chronic pressure overload. n = 8 biologically independent experiments, data are mean ± s.d., one-way ANOVA with Tukey multiple comparisons test, *P = 2 × 10−5 versus sham, †P < 1 × 10−6 versus sham, P = 3 × 10−6 versus Tsc2WT/WT pressure overload (PO). d, Immunoblots of LC3-II from Tsc2WT/WT, Tsc2S1365A/WT and Tsc2S1365A/S1365A myocardium from mice subjected to sham or pressure overload and treated with vehicle or sildenafil. Pressure-overload-stimulated LC3-II expression is lacking in Tsc2S1365A/WT and Tsc2S1365A/S1365A, but is recovered in Tsc2S1365A/WT mice after sildenafil treatment. It increases further in Tsc2WT/WT mice after pressure overload with sildenafil treatment. Experiments were replicated twice providing 4 biologically independent samples, data are mean ± s.d.; data normalized to the mean of Tsc2WT/WT sham, one-way ANOVA with Tukey test for multiple comparisons, *P < 1 × 10−6 versus respective sham and versus pressure overload in other two groups, †P < 1 × 10−6 versus respective sham and versus Tsc2S1365A/S1365A pressure overload and sildenafil, P = 0.0013 versus Tsc2S1365A/WT pressure overload and sildenafil, ‡P < 1 × 10−6 versus sham and Tsc2S1365A/S1365A pressure overload and sildenafil. e, Summary data for immunoblots displayed in Fig. 3e, f. n = 4 biologically independent experiments, data are mean ± s.e.m., data normalized to mean of Tsc2WT/WT sham; one-way ANOVA with Tukey multiple comparisons test, *P < 1 × 10−6 versus respective sham and pressure overload with sildenafil treatment; †P = 0.0002 versus sham, ‡P < 1 × 10−6 versus respective sham and Tsc2WT/WT and pressure overload, #P < 1 × 10−6 versus Tsc2WT/WT and Tsc2S1365A/WT and pressure overload with sildenafil treatment.
