Fig. 3: Hyperactive mTORC1 drives metabolic switch in CTNS-defective/cystine accumulating PT cells.

a Schematic of the experimental workflow for multi-omics integration, and pathway/network enrichment analysis. b, c Volcano plot of (b) proteome and (c) metabolome changes induced by CTNS loss in the PT cells. Black and blue dots show the differentially produced proteins and metabolites, respectively. [Log₂ (fold change) <−0.5 and Log₂ (fold change) >1; and P value < 0.05]. (d, left) Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis; n = 3 biologically independent experiments. Black dashed line shows the threshold of significant enrichment. Black circles show the size of enriched proteins and metabolites, respectively. P value was calculated from the enrichment analysis using Omicsnet2.0. d (right) Upstream regulators of mTORC1 signaling activity and downstream-regulated pathways. e, f Immunoblotting and quantification of the indicated proteins in Ctns mPTCs; n = 3 biologically independent animals. g Immunoblotting and quantification of the indicated proteins in input samples and corresponding Lyso-IP fractions from Ctns^WT and Ctns^KO mPTCs; n = 4 biologically independent experiments. h Confocal microscopy and quantification of RagC/Lamp1⁺ structures (expressed as percentage of total lysosomes; n = 15 randomly selected fields of views from 3 biologically independent animals). Dotted white squares contain images at high magnification. i Confocal microscopy and quantification of ^pS235/236S6 MFI in Aqp1⁺ (green) PT segments of Ctns rat kidneys; n = 227 Ctns^WT and n = 228 Ctns^KO PT segments pooled from 3 biologically independent animals. j, k Immunoblotting and quantification of the indicated proteins in (j) 24-week-old Ctns rats or (k) 5 days post fertilization (dpf)-ctns zebrafish. For rat kidneys: n = 10 animals in each experimental group and for zebrafish: n = 7 biologically independent samples (with each sample representing a pool of 10 zebrafish larvae). Plots represent mean ± SEM. Statistics calculated by unpaired two-tailed Student’s t test. Nuclei counterstained with DAPI (grey). Scale bars are 10 μm in h and 50 μm in i. Source data is provided as a Source Data file. Images in a and d were drawn using pictures from Servier Medical Art, which is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).