Fig 1: Rapamycin treatment reverses the metabolic profiling of Tsc1 KO PTCs through a direct effect on the arginine metabolism pathway(A) Principal-component analysis plot showing WT mice, Tsc1 KO mice, and Tsc1 KO mice treated with rapamycin. Each dot represents an independent biological samples.(B) Heatmap and dendrogram generated via hierarchical clustering of samples and all (58) detected metabolites.(C) Pathways enriched with significantly altered metabolites. The metabolite concentration tables of the selected groups were analyzed by using MetaboAnalyst 4.0 (https://www.metaboanalyst.ca), applying no filtering, quantile normalization, log transformation, and no scaling.See also Figure S2.
Fig 2: Rapamycin treatment in vivo alleviates TSC-associated cyst development and inhibits ASS1 expression in a mTORC1-dependent manner(A and B) Kidney sections of WT and Tsc1 KO mice treated with either vehicle or rapamycin, as indicated, were H&E stained (A) or immunostained for ASS1 (B) or pS6 as a marker for mTORC1 activation (D). WT pups treated with vehicle (n = 3 biological replicates), Tsc1 KO pups treated with vehicle (n = 3 biological replicates), or rapamycin (n = 3 biological replicates). Scale bar = 500 μm.(C) Relative quantification of sum fluorescence intensity as in (B). ∗p < 0.05.
Fig 3: Arginine depletion in vitro induces cell-cycle arrest and attenuates TSC-associated signaling(A) Control and Tsc1 KO HK2 cells were incubated with either control or arginine-free medium for 10 days. Cells were harvested and fixed, and the cell cycle was monitored by propidium iodide flow cytometry-based analysis.(B) Quantification of data in A (n = 6 biological replicates), ∗p < 0.05.(C) Western blot analysis for ASS1, pS6, c-Myc, P65, and actin protein expression in control and Tsc1 KO HK2, representative of 2 independent experiments with similar results.See also Figures S4 and S5.
Fig 4: Arginine depletion ameliorates cyst development in Tsc1 KO mice(A) Representative H&E staining of kidney sections from WT (Tsc1fl/fl) and Tsc1 KO pups from Tsc1fl/fl mothers fed either control or arginine-deficient diets at P0, showing reduced cyst formation in kidneys of Tsc1 KO pups fed an arginine-deficient diet. Scale bar: 500 μm.(B–E) Quantification of the cyst area per section in the different groups as in (A). WT pups were fed a control diet (n = 5 biological replicates) and an arginine-deficient diet (n = 4 biological replicates). Tsc1 KO pups fed a control diet (n = 5 biological replicates) and arginine-deficient diet (n = 5 biological replicates), ∗p < 0.05. Kidney sections, as in (A), were immunostained for pS6 (C) and for the proliferation marker Ki67 (E). Scale bar: 50 μm.(D) Quantification of sum fluorescence intensity as in (C). ∗p < 0.05.(F) Quantification of Ki67-positive cells compared with total cells (DAPI positive cells) as in (E). ∗p < 0.05.See also Figures S6, S7, and S9.
Fig 5: Rapamycin treatment reverses the metabolic profiling of Tsc1 KO kidneys(A) Principal-component analysis plot of WT mice and Tsc1 KO mice with or without rapamycin treatment during pregnancy. Each dot represents an independent biological sample.(B) Scatterplot (ggplot2) showing log2 fold changes between the change in metabolites levels in Tsc1 KO and WT (x axis) and with and without rapamycin in Tsc1 KO mice (y axis), implying a reversal of mutation effects by rapamycin.(C) Venn diagram (ggVennDiagram) showing the overlap in dysregulated (significantly upregulated or downregulated) metabolites across the two pairwise comparisons.(D) Heatmap and dendrogram generated via hierarchical clustering of samples and metabolites that were significantly altered in at least 1 of the comparisons depicted in (C).(E) Venn diagram showing the overlap of metabolic pathways enriched with significantly altered metabolites in either of the depicted comparisons (KO, Tsc1 KO; KO+r, Tsc1 KO treated by rapamycin).(F) Scatterplot showing a correlation of the negative log10 FDR (false detection rate) of pathways affected by the experimental interventions (as in E).(G) Representative pathways enriched with significantly altered metabolites.See also Figure S1.
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