Cell viability curves of the increasing concentration of DMKG in control (CTNS^WT) and CRISPR‐generated cystinotic (CTNS^−/−) cells after 24 h of incubation in fed and starved condition, respectively (n = 3).

Cell viability test in CTNS^WT, CTNS^−/− cells treated with the increasing concentrations of cysteamine, bicalutamide, luteolin, genistein, 8‐bromo‐cAMP, disulfiram, and a combination of cysteamine and bicalutamide (100 and 35 µM, respectively), respectively (n = 3).

Heat map analysis of the measured metabolites in CTNS^−/− cells upon treatment with cysteamine or cysteamine–bicalutamide combination treatment (n = 3). Metabolites significantly decreased were displayed in green, while metabolites significantly increased were displayed in red.

Heat map and REACTOME analysis of the altered proteins in CTNS^−/− cells upon treatment with cysteamine, bicalutamide and cysteamine–bicalutamide combination treatment (n = 3). Proteins significantly decreased were displayed in green, while metabolites significantly increased were displayed in red.

Proteomic analysis of CTNS^−/− cells treated with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide, respectively (n = 3). AKGDH: Alpha‐ketoglutarate dehydrogenase, GLUD1: GLUD2; Mitochondrial glutamate dehydrogenase 1/2, IGF2R; Cation‐independent mannose‐6‐phosphate receptor, GSTK1: Glutathione S‐transferase kappa 1, COX6B1: Cytochrome c oxidase subunit 6B1, ACACA: Acetyl‐CoA carboxylase 1‐Biotin carboxylase, GLS: Glutaminase kidney isoform, mitochondrial, CASP3: Caspase‐3, NPC1: Niemann‐Pick C1 protein.

Western blotting and densitometric analyses for LC3‐II/LC3‐I ratio in CRISPR‐generated CTNS^−/− cells treated with cysteamine (100 µM), bicalutamide (35 µM), and a combination of cysteamine and bicalutamide (100 and 35 µM, respectively). β‐Actin was used as a loading control (n = 3).

Quantification of TFEB‐GFP nuclear translocation in CTNS^WT, and CTNS^−/− cells upon treatment with bicalutamide (35 µM) (n = 3).

TFEB mRNA expression of the CTNS^WT cells upon starvation and treatment with bicalutamide (35 µM) (n = 3).

Heat map analysis of top 25 metabolites distinctively expressed in control and CTNS^−/− cells. Metabolites significantly decreased (P < 0.05) were displayed in green, while metabolites significantly increased (P < 0.05) were displayed in red (n = 3).

Global test pathway enrichment analysis of the intracellular metabolic interactions distinctively affected in CTNS^−/− cells compared to healthy control cells (n = 3). Larger circles further from the y‐axis and orange‐red colour show higher impact of pathway.

List of metabolites that were shared and significantly altered in both the CTNS^−/− and CTNS^Patient cells compared to control cells (n = 6).

Plasma levels of αKG (µM) in healthy individuals (n = 4) and cystinotic patients (n = 6). All cystinotic patients were on cysteamine treatment at the time of blood sampling. For this experiment, non‐parametric Mann–Whitney t‐test was used to demonstrate the significance.

Principal component analysis (PCA) of the measured proteins in CTNS^WT, CTNS^−/− and CTNS^Patient (n = 3).

Volcano plot illustrates differentially abundant proteins (n = 3). The ‐log₁₀ (Benjamini–Hochberg corrected P‐value) is plotted against the log₂ (fold change: CTNS^WT/CTNS^−/−).

Gene ontology (GO) analysis illustrates classes of proteins differing between CTNS^WT and CTNS^−/− cells (n = 3).

List of proteins that were significantly upregulated and downregulated in CTNS^−/− cells compared to control cells (n = 3). AKGDH; Alpha‐ketoglutarate dehydrogenase, LIPA; Lysosomal acid lipase, ACP2; Lysosomal acid phosphatase, CTSS; Cathepsin S, CTSC; Dipeptidyl peptidase, IGF2R; Cation‐independent mannose‐6‐phosphate receptor, SORT1; Sortilin, CAT; Catalase, CTSA; Lysosomal protective protein and SOD; Superoxide dismutase.

Quantification of cystine levels (nmol/mg protein) by HPLC‐MS/MS in control and cystinotic tubuloids (n = 3).

Quantification of cystine levels (nmol/mg protein) by HPLC‐MS/MS in two different patient‐derived cystinotic tubuloids in the absence of the drugs (NT) or upon treatment with cysteamine (100 μM), bicalutamide (35 μM) or cysteamine (100 μM)‐bicalutamide (35 μM) combination treatment (n = 3).

αKG levels measured in patient‐derived cystinotic tubuloids (CNTS^Patient‐1 and CTNS^Patient‐2) in the absence of the drugs (NT) or upon treatment with cysteamine, bicalutamide or cysteamine–bicalutamide combination treatment using metabolomics (n = 3).

Representative images of control and cystinotic zebrafish.

Quantification of cystine levels (nmol/mg protein) by HPLC‐MS/MS in control and cystinotic zebrafish (n = 3).

Quantification of cystine levels (nmol/mg protein) by HPLC‐MS/MS in cystinotic zebrafish after treatment with cysteamine (1,000 µM), bicalutamide (10 µM), and a combination of cysteamine and bicalutamide (1,000 and 10 µM, respectively) (n = 40 embryos per group).

Survival rates in ctns^−/− zebrafish upon treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 40 embryos per group).

Deformity rates in ctns^−/− zebrafish after treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 40 embryos per group).

Hatching rates in surviving ctns^−/− zebrafish evaluated at 72‐ and 96‐h post‐fertilization (hpf) with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 40 embryos per group).

Sanger sequencing chromatogram shows resulting sequence in CRISPR‐generated cystinotic cells (CTNS^−/−).

Quantification of cystine levels (nmol/mg protein) by HPLC‐MS/MS in control (CTNS^WT), CRISPR‐generated cystinotic cells (CTNS^−/−; line 3, 7 and 35, and patient‐derived cystinotic cells (CTNS^Patient) (n = 3–6).

Quantification of cystine levels (nmol/mg protein) by HPLC‐MS/MS in CTNS^−/− lines (3, 7 and 35), and CTNS^Patient cells upon treatment with cysteamine (100 µM) (n = 3–6).

Western blotting and densitometric analyses for LC3‐II/LC3‐I ratio in DMKG (2 mM)‐treated CTNS^WT and CTNS^−/− cells upon pre‐treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 3).

Relative reactive oxygen species (ROS/mg protein) production in DMKG‐treated CTNS^WT and CTNS^−/− cells upon pre‐treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 3).

Representative confocal micrographs and quantification of DQ‐BSA in CTNS^WT, and CTNS^−/− cells upon treatment with bicalutamide. Scale bars are 20 µm (n = 8–14 quantified images).

Quantification of cystine levels (nmol/mg protein) by HPLC‐MS/MS in CTNS^−/− in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 6), bicalutamide (n = 6), and a combination of cysteamine and bicalutamide (n = 6).

Quantification of cystine levels (nmol/mg protein) by HPLC‐MS/MS in CTNS^Patient in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 4), bicalutamide (n = 4), and a combination of cysteamine and bicalutamide (n = 6).

Brightfield images of cystinotic tubuloids and healthy control tubuloids at the start of treatment and after 5 days of cysteamine (100 μM)‐bicalutamide (35 μM) combination treatment or treatment with medium only (negative control). Scale bars are 2,000 µm.

Bicalutamide safety screening in cystinotic tubuloids. Tubuloid viability upon treatment with cysteamine (100 μM) in combination with increasing concentrations of bicalutamide was compared to treatment with cysteamine alone (= 100% viability) (per donor n = 4 replicates for each dose).

Survival rates in wild‐type zebrafish upon treatment with bicalutamide (10 µM), and a combination of cysteamine and bicalutamide (1,000 and 10 µM, respectively) (n = 40 embryos per group).

Deformity rates in wild‐type zebrafish upon treatment with bicalutamide (10 µM), and a combination of cysteamine and bicalutamide (1,000 and 10 µM, respectively) (n = 40 embryos per group).

Hatching rates in surviving wild‐type zebrafish evaluated at 72‐ and 96‐h post‐fertilization (hpf) with bicalutamide (10 µM), and a combination of cysteamine and bicalutamide (1,000 and 10 µM, respectively) (n = 40 embryos per group). Drugs were administered at 48‐h post‐fertilization in all experiments dissolved in the swimming water with the specified concentrations.

ROS production (ROS/mg protein) in CTNS^WT, CTNS^−/− and CTNS^Patient cells upon starvation for 4 h in the presence and absence of DMKG (4 h), respectively (n = 3).

Viability test in starved (−AA) CTNS^WT, CTNS^−/− and CTNS^Patient cells in the presence or absence of DMKG (2 mM) for 24 h. Results are shown relative to the fed condition (n = 3).

Representative confocal micrographs (scale bars are 20 µm) and immunofluorescence analysis of caspase 3/7 activation in DMKG (2 mM)‐treated CTNS^WT, CTNS^−/− and CTNS^Patient cells for 24 h (n = 3).

Western blotting and densitometric analyses for LC3‐II/LC3‐I ratio in CTNS^WT, CTNS^−/− and CTNS^Patient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 h, respectively (n = 3).

Quantification of transcription factor EB (TFEB)‐GFP nuclear translocation in CTNS^WT, CTNS^−/−, and CTNS^Patient, respectively. Data are demonstrated as the ratio between number of the cells with nucleus‐TFEB positive over the total number of TFEB‐transfected cells. The ratios were then presented as a fold change compared to control cells (n = 3).

TFEB mRNA expression in CTNS^−/− and CTNS^Patient cells compared to control cells (n = 3).

Representative confocal micrographs and quantification of LC3‐II accumulation in CTNS^WT and CTNS^−/− cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 h, respectively (n = 3). Scale bars are 20 µm.

Western blotting and densitometric analyses for LC3‐II/LC3‐I ratio and SQSTM1 protein levels in CTNS^WT, CTNS^−/− and CTNS^Patient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3).

Representative confocal micrographs and quantification of DQ‐BSA and BSA in CTNS^WT, CTNS^−/− and CTNS^Patient cells, respectively (n = 7–14 quantified images). Scale bars are 20 µm.