gclc  (Cell Signaling Technology Inc)

Journal: Bioactive Materials

Article Title: A multimodal ROS logic-gated therapeutic platform disrupts the vicious cycle of senescence to promote aged bone defect repair

doi: 10.1016/j.bioactmat.2026.02.002

Figure Lengend Snippet: Transcriptomic and molecular analysis of the potential pathways involved in MMBOx-mediated BMSCs rejuvenation. (A) Circular heatmap showing differentially expressed genes (DEGs) associated with cell senescence, inflammation, and osteogenesis in senescent BMSCs treated with MMBOx@GPP compared to GPP. (B) Gene Ontology (GO) enrichment analysis of upregulated DEGs. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs. (D) Gene Set Enrichment Analysis (GSEA) plots of the glutathione metabolic process (ES: enrichment score; NES: normalized enrichment score; FDR: false discovery rate). (E) Heatmap of DEGs enriched in aging-related GO terms. (F) Western blot analysis of Keap1, Nrf2, Nqo1, Gclc, and GAPDH protein expression in BMSCs. (G) Quantitative analysis of protein band intensities ( n = 3). (H) Representative flow cytometry plots of ThiolTracker™ fluorescence staining indicating intracellular glutathione levels. (I) Quantification of intracellular GSH/GSSG ratio in BMSCs ( n = 3). (J) Schematic diagram illustrating the proposed mechanism by which MMBOx attenuates BMSCs senescence via Nrf2 pathway activation and glutathione metabolism enhancement. Data are expressed as mean ± SD; ∗ P < 0.05, ∗∗∗ P < 0.001.

Article Snippet: Western blotting was employed to evaluate protein expression of key targets, including Keap1 (Affinity, AF5266; 1:1000), Nrf2 (Proteintech, 16396-1-AP; 1:1000), Nqo1 (Abcam, ab80588; 1:10,000), Gclc (Proteintech, 12601-1-AP; 1:25000) and GAPDH (Proteintech, 60004-1-Ig; 1:50,000).

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Staining, Activation Assay

Journal: Nature

Article Title: Catabolism of extracellular glutathione supplies cysteine to support tumours

doi: 10.1038/s41586-026-10268-2

Figure Lengend Snippet: a , Schematic of the tumour-specific Gclc knockout mouse model. Autochthonous tumours from Gclc f/f Rosa26 creERT2 MMTV-PyMT mice were excised and orthotopically transplanted into mammary fat pads of WT C57BL/6 mice. C57BL/6 mice were treated with vehicle (corn oil; WT) or 50 mg kg –1 tamoxifen for 5 days (KO). b , Relative Gclc mRNA levels of WT and KO tumours ( n = 5 representative animals from 3 independent experiments). c , GSH levels of WT and KO tumours ( n = 6 representative animals from 3 independent experiments). d , e , Volume over time ( d ) and mass at end point ( e ) of WT tumours ( n = 11) and KO tumours ( n = 12) from a representative of 3 independent experiments. f , g , Immunoblot of GCLC protein levels ( f ) and GSH levels ( g ) in HCC-1806 human breast cancer cells transduced with lentiCRISPRv2 containing non-targeting guides (sgNTC) and guides against GCLC (sgGCLC 1 and sgGCLC 2) ( n = 3 representative samples from 2 independent experiments). For gel source data, see Supplementary Fig. . h , i , Volume over time ( h ) and mass at end point ( i ) of orthotopically implanted HCC-1806 breast cancer cell treated with sgNTC, sgGCLC 1 or sgGCLC 2 ( n = 7 representative animals from 2 independent experiments). j , Concentration of tGSH (GSH + 2 × GSSG) in serum ( n = 11) and the TIF ( n = 4) from Gclc WT MMTV-PyMT autochthonous tumours from a . k , Concentration of tGSH in serum ( n = 8) and the TIF ( n = 6) from patients with breast cancer. Significance was assessed by two-tailed unpaired t -test ( b , c , e ), Mann–Whitney test ( j , k ), ordinary two-way analysis of variance (ANOVA) ( d , h ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( g , i ). Data are the mean ± s.e.m.

Article Snippet: Gclc f/f mice were crossed with the MMTV-PYMT (The Jackson Laboratory, 022974) and Rosa26 creERT2 (The Jackson Laboratory, 008463) mouse strains.

Techniques: Knock-Out, Western Blot, Transduction, Concentration Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Catabolism of extracellular glutathione supplies cysteine to support tumours

doi: 10.1038/s41586-026-10268-2

Figure Lengend Snippet: a , Single-nucleus RNAseq (snRNAseq) of Gclc transcripts from the indicated cells present in human mammary tissue, from previously published data . b-e , Autochthonous tumors from MMTV-PyMT Gclc f/f Rosa26-CreERT2 mice were excised and orthotopically transplanted into mammary fat pads of wild-type C57BL/6 mice, which were treated with vehicle (corn oil; WT) or 50 mg/kg tamoxifen for 5 days (KO). Representative immunohistochemistry (b) and immunofluorescence (c) staining of indicated proteins in Gclc WT and Gclc KO mammary tumors (Scale bar = 50 µm). Relative mRNA expression of Gclm (p = 0.0026), Hmox1 , Nqo1 , and Slc7a11 (d) and representative immunohistochemistry of indicated proteins (e) in Gclc WT and Gclc KO tumors (n = 6 representative from 3 independent experiments). f-j , Volcano plot of metabolic differences ( f) and levels of GSSG (g) , ophthalmic acid (h) , glutamate (i) , cysteine (NEM-cysteine) (j) from Gclc WT and Gclc KO tumors. Statistical significance was assessed by unpaired two-tailed t-test in d, f, g-j . Data represented as mean ± s.e.m., *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001; ns, not significant.

Article Snippet: Gclc f/f mice were crossed with the MMTV-PYMT (The Jackson Laboratory, 022974) and Rosa26 creERT2 (The Jackson Laboratory, 008463) mouse strains.

Techniques: RNA sequencing, Immunohistochemistry, Immunofluorescence, Staining, Expressing, Two Tailed Test

Journal: Nature

Article Title: Catabolism of extracellular glutathione supplies cysteine to support tumours

doi: 10.1038/s41586-026-10268-2

Figure Lengend Snippet: a-c , Autochthonous tumors from MMTV-PyMT Gclc f/f Rosa26-CreERT2 mice were excised and subcutaneously transplanted into flanks of wild-type C57BL/6 mice, which were treated with vehicle (corn oil; WT) or 50 mg/kg tamoxifen for 5 days (KO). Tumor volume over time (WT, n = 7; KO, n = 8 representative from 2 independent experiments, p = 0.9527) (a) , total tumor mass (WT, n = 6; KO, n = 7 representative from 2 independent experiments, p = 0.7441) (b) , tGSH levels (p = 0.001, WT, n = 4; KO, n = 3 representative from 2 independent experiments, p = 0.0001) (c) . Statistical significance was assessed by by ordinary two-way ANOVA in a , and by unpaired two-tailed t-test in b , c . Data represented as mean ± s.e.m., ***p-value < 0.001; ns, not significant.

Article Snippet: Gclc f/f mice were crossed with the MMTV-PYMT (The Jackson Laboratory, 022974) and Rosa26 creERT2 (The Jackson Laboratory, 008463) mouse strains.

Techniques: Two Tailed Test

Journal: Nature

Article Title: Catabolism of extracellular glutathione supplies cysteine to support tumours

doi: 10.1038/s41586-026-10268-2

Figure Lengend Snippet: a-c , Concentration of tGSH and cystine in plasma (n = 17) and tumor interstitial fluid (n = 7) from the LSL-KrasG12D/+ Trp53f/f Pdx-1-Cre (KP -/- C) mice bearing pancreatic ductal adenocarcinoma (PDAC) (GSH: ****p < 0.0001, Cys: *p = 0.0114) (a) , in the plasma (n = 27) and tumor interstitial fluid (n = 46) from renal cell carcinoma (RCC) human patients (GSH: ****p < 0.0001, Cys: p = 0.1078) (b) , in cell culture media formulations (c) . d-i , Autochthonous tumors from MMTV-PyMT Gclc f/f Rosa26-CreERT2 mice were excised and orthotopically transplanted into the mammary gland or subcutaneously transplanted into the flank of wild-type C57BL/6 mice, which were treated with vehicle (corn oil; WT) or 50 mg/kg tamoxifen for 5 days (KO). tGSH and cystine levels in TIF from orthotopic (WT, n = 4; KO, n = 6; GSH: p = 0.0621, Cys: p = 0.0735) (d) or flank (WT, n = 4; KO, n = 3; GSH: p = 0.0001, Cys: p = 0.7050) (e) tumors. tGSH in TIF from orthotopic and flank Gclc WT MMTV-PyMT tumors (n = 4; p = 0.7607) (f) . tGSH and cystine levels in serum of mice with orthotopic (WT, n = 11; KO, n = 12; GSH: p = 0.3512, Cys: p = 0.6212) (g) or flank (WT, n = 4; KO, n = 5; GSH: p = 0.7398, Cys: p = 0.4937) (h) tumors. Serum tGSH and cystine in serum of mice without Gclc WT MMTV-PyMT tumors (n = 8; p = 0.2805) (i) . j-k , Serum tGSH (no tumor vs. orthotopic: *p = 0.0412; no tumor vs. flank: p = 0.1310) (j) and cystine (no tumor vs. orthotopic: p = 0.3725; no tumor vs. flank: p = 0.6482) (k) levels in mice bearing orthotopic (n = 11), flank (n = 4), or no Gclc WT tumors (n = 8). l-n , Glutamate levels in plasma and TIF from Fig. (*p = 0.0153) (l) , from a (p < 0.0001) (m) , and from b (p < 0.0001) (n) . Total GSH (tGSH) was calculated as the sum of reduced glutathione (GSH) and twice the amount of oxidized glutathione (GSSG). Statistical significance was assessed by an unpaired two-tailed t-test in a - i , l - n , and by Ordinary One-Way ANOVA followed by Dunnett’s multiple comparisons test in j , k . Data represented as mean ± s.e.m., *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001; ns, not significant.

Article Snippet: Gclc f/f mice were crossed with the MMTV-PYMT (The Jackson Laboratory, 022974) and Rosa26 creERT2 (The Jackson Laboratory, 008463) mouse strains.

Techniques: Concentration Assay, Clinical Proteomics, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: Catabolism of extracellular glutathione supplies cysteine to support tumours

doi: 10.1038/s41586-026-10268-2

Figure Lengend Snippet: a , Schematic of the different mechanisms of cysteine acquisition and use. b , Concentration of cystine in serum ( n = 11) and the TIF ( n = 4) from Gclc WT MMTV-PyMT autochthonous tumours from Fig. . c , Concentration of cystine in serum ( n = 8) and the TIF ( n = 6) from patients with breast cancer. d – f , HCC-1806 breast cancer cells were grown in the following medium: control (208 µM cystine; Cys 2 ); cystine-free (Cys 2 -free); cystine-free with GSH supplemented (750 µM, Cys 2 -free + GSH); or cystine-free with cysteinylglycine (CysGly) supplemented (750 µM). Cell numbers ( n = 3 technical replicates representative of 4 independent experiments) ( d ), percentages of proliferative cells (positive for bromodeoxyuridine (BrdU + ) at t = 48 h; n = 6 technical replicates from 3 independent experiments) ( e ) and apoptotic cells (Annexin V + at t = 72 h; n = 9 technical replicates from 3 independent experiments) ( f ) were determined at the indicated time points. g , Levels of extracellular CysGly in the medium at 72 h after HCC-1806 breast cancer cells were grown in the indicated medium ( n = 3 representative technical replicates from 2 independent experiments). h , Schematic of the 13 C-cystine stable-isotope labelling approach and metabolomics. i , Per cent labelling of 13 C-cystine stable-isotope in the indicated species at 72 h ( n = 5 technical replicates). j , Schematic of the 15 N 13 C-cystine stable-isotope labelling approach and proteomics. k , Per cent labelling of 15 N 13 C-cystine stable-isotope in the indicated species at 96 h (average of n = 3 technical replicates). TXN1, thioredoxin 1; TXN2, thioredoxin 2. Significance was assessed by unpaired two-tailed t -test ( b , c ), ordinary two-way ANOVA followed by Šídák’s multiple comparisons test ( e , f ), Dunnett’s multiple comparisons test ( d ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( g ). Data are the mean ± s.e.m.

Article Snippet: Gclc f/f mice were crossed with the MMTV-PYMT (The Jackson Laboratory, 022974) and Rosa26 creERT2 (The Jackson Laboratory, 008463) mouse strains.

Techniques: Concentration Assay, Control, Two Tailed Test

Journal: Nature

Article Title: Catabolism of extracellular glutathione supplies cysteine to support tumours

doi: 10.1038/s41586-026-10268-2

Figure Lengend Snippet: a , GGT gene family table showing all 13 GGT-related genes in the human genome. GGT1, 5, 6, and 7 are predicted to have enzymatic activity, but only two (GGT1,5) have been functionally characterized. b , mRNA expression (Expression Public 24Q2) of cancer cell lines (n = 1,517) from DepMap’s Cancer Cell Line Encyclopedia (CCLE). c , Protein levels from cancer cell lines from DepMap’s CCLE (Proteomics GGT1: P19440 (n = 339), GGT5: P36269 (n = 108), GGT6: Q6P531 (n = 45), GGT7: Q9UJ14 (n = 375)). d , mRNA and protein levels of GGT isoforms in the cell line HCC-1806, using data from (b) and (c) . e , Histogram (splined curves) of gene dependency of cancer cell lines from Dependency Map (DepMap) (Public 24Q2+Score Chronos). A lower score suggests a gene is more likely to be essential in a cell line. A score of 0 indicates a non-essential gene, while -1 reflects the median for essential genes. MTOR and GAPDH are shown as examples of common essential genes. f , mRNA expression of GGT enzymes was analyzed in human tumors from 32 different TCGA projects. g , GGT1 mRNA expression levels in human tumoral and non-tumoral tissues from the Cancer Genome Atlas Program (TCGA) and the Genotype-Tissue Expression (GTEx) databases. Black lines represent median values. Statistical significance was analyzed by two-tailed unpaired t-test. ns, not significant; ****p-value < 0.0001; **p = 0.0029 (papillary renal cell carcinoma); **p = 0.0038 (clear cell renal cell carcinoma); ***p = 0.0005 (hepatocellular carcinoma). h , The mutational landscape and somatic mutation frequence (SMF) of each GGT gene was analyzed in 105,260 cancer patient samples across 226 non-redundant studies using the cBioPortal platform revealing a low mutation frequency (mean = 0.15%). i , Autochthonous tumors from MMTV-PyMT Gclcf/f Rosa26-CreERT2 mice were excised and orthotopically transplanted into the mammary gland or subcutaneously transplanted into the flank of wild-type C57BL/6 mice, which were treated with vehicle (corn oil; WT) or 50 mg/kg tamoxifen for 5 days (KO). Immunohistochemistry of GGT1 protein expression in Gclc WT and KO orthotopic and flank tumors (n = 4 animals representative of 2 independent experiments). Numbers displayed on the top left of each panel represent the animal identification number. Scale bar = 50 µm.

Article Snippet: Gclc f/f mice were crossed with the MMTV-PYMT (The Jackson Laboratory, 022974) and Rosa26 creERT2 (The Jackson Laboratory, 008463) mouse strains.

Techniques: Activity Assay, Expressing, Two Tailed Test, Mutagenesis, Immunohistochemistry

Journal: bioRxiv

Article Title: Systematic Evaluation Defines the Limits of Ferroptosis in Cancer Therapy

doi: 10.64898/2026.03.11.711115

Figure Lengend Snippet: A , Left, correlation between the reported number of damaging mutations in each gene (DepMap) and nAUC measurement for BSO sensitivity (+Fer-1 condition, ) and associated P value, TXNRD1 highlighted. Right, correlation between number of number of damaging mutations in TXNRD1 in each cell line and corresponding nAUC for BSO sensitivity (+Fer-1), SKMES1 cell line highlighted. B , Immunoblot for indicated proteins from lysates of A549 cells and two clones in which TXNDR1 is deleted. MW markers indicated. C , Viability (ATP) of a TXNRD1 KO clones from B upon treatment with BSO, with or without βMe (50 μM), or Fer-1 (10 μM), 3 days. Data report mean ± SEM of n=3 biological replicates. D , Viability (ATP) of A549 cells, A549 TXNRD1 knockout clones, or SKMES1 cells expressing the indicated TXNRD1 cDNAs and selenocysteine (U498C) or SEICS (Δ3’UTR) mutants, treated with BSO as indicated, 3 days. Data report mean ± SEM of n=3 biological replicates. E , Schematic of glutathione synthesis and BSO sensitivity for GCLC versus GshF. F , Viability (ATP) of HT-1080, A549, A549 TXNRD1 knockout clones, or SKMES1 cells expressing S. thermophilus GshF, treated with BSO as indicated, 3 days. Data report mean ± SEM of n=3 biological replicates. G , Viability (ATP) of SKMES1 cells expressing exogenous GCLC cDNA and GCLC mutants resistant to BSO identified in , treated with BSO as indicated, 3 days. Data report mean ± SEM of n=3 biological replicates. H , Schematic of BSO (L-buthionine sulfoximine) and sulfoxide or methionine derivatives. Differences in buthionine vs methionine (blue circle) or sulfoximine vs sulfoxide (red circle) indicated. I , GSH content of HT-1080 cells upon treatment with BSO (red line) and indicated related species, 3 days. Data are mean of two technical replicates. J-K , Viability (ATP) upon treatment with BSO (red line) and indicated related species for A549 or A549 TXNRD1 knockout clones ( J ), or HT-1080 and SKMES1 cells, including additional Fer-1 treatment (10 μM, K ), 3 days. Data are mean of two technical replicates. L , Viability (ATP) of HT-1080, A549, A549 TXNRD1 knockout clones, or SKMES1 cells expressing the indicated GPX4 cDNAs and selenocysteine (U46C) or SEICS (Δ3’UTR) mutants, treated with BSO as indicated, 3 days. Data report mean ± SEM of n=3 biological replicates.

Article Snippet: TXNRD1 (SC-28321), GCLC (SC-166345) from Santa Cruz Goat Anti-mouse IgG (H+L), HRP (Invitrogen; 62-6520) Goat Anti-rabbit IgG (H+L), HRP (Invitrogen; 31460).

Techniques: Western Blot, Clone Assay, Knock-Out, Expressing