Journal: Cell Death & Disease

Article Title: MTHFR functions as a metabolic checkpoint in NSCLC through SLC25A26-mediated SHMT2 inhibition

doi: 10.1038/s41419-026-08874-z

Figure Lengend Snippet: A Schematic diagram of the folate-driven one-carbon metabolism pathway, highlighting the folate cycle and methionine regeneration. B qPCR analysis of key one-carbon metabolic genes in control and MTHFR-overexpressing (MTHFR-OE) cells. MTHFR overexpression significantly downregulates SHMT2 and PHGDH mRNA levels. C Immunoblot analysis of SHMT2 and PHGDH protein expression in control and MTHFR-OE cells. MTHFR overexpression suppresses SHMT2 but not PHGDH protein levels. β-actin serves as a loading control. D Schematic diagram of the U- 13 C-serine isotope tracing workflow for MTHFR-mediated one-carbon metabolic flux analysis. Quantification of ¹³C incorporation into key downstream one-carbon metabolites (glycine, 5,10-CH₂-THF, SAM, dNTPs) in MTHFR-OE A549 ( E ) and H520 ( F ) cells, detected by U- 13 C-serine isotope tracing assay. G Analysis of SHMT2 expression in NSCLC and normal tissues from the TIMER database. H Negative correlation between MTHFR and SHMT2 mRNA expression in LUAD samples from the TCGA dataset (Pearson correlation). I Cell proliferation was assessed by a CCK-8 assay in cells expressing vector control, MTHFR alone, or MTHFR + SHMT2. J Cell cycle distribution analyzed by flow cytometry in the indicated groups. K Apoptosis rates measured by flow cytometry in the indicated groups. L Immunoblot analysis of pro-survival (p-AKT Ser473, p-ERK1/2, and Bcl-2) and pro-apoptotic (cleaved caspase-3/8) signaling molecules in the rescue experiment. β-actin serves as a loading control. Means ± SEM, * P < 0.05; ** P < 0.01; *** P < 0.001; t -test.

Article Snippet: The cells were incubated overnight at 4 °C with a primary antibody against SHMT2 (1:100, Cat# A21803, ABclonal, Wuhan, China), followed by three washes with 0.05% PBST (PBS containing 0.05% Tween 20).

Techniques: Control, Over Expression, Western Blot, Expressing, CCK-8 Assay, Plasmid Preparation, Flow Cytometry

Journal: Cell Death & Disease

Article Title: MTHFR functions as a metabolic checkpoint in NSCLC through SLC25A26-mediated SHMT2 inhibition

doi: 10.1038/s41419-026-08874-z

Figure Lengend Snippet: A , B Immunofluorescence analysis showing a significant reduction in SHMT2 fluorescence intensity (*** P < 0.001) in H520 cells overexpressing SLC25A26 (SLC25A26-OE) compared with that in the empty vector (EV) controls. Scale bars: 50 μm. C Restoration of SHMT2 protein levels in SLC25A26-OE cells treated with the proteasome inhibitor MG132 (10 μM, 6 h). D , E Cycloheximide (CHX, 100 μg/mL) chase assays demonstrating accelerated SHMT2 protein degradation in SLC25A26-OE cells. Half-life quantification is shown (*** P < 0.001 vs. EV). F , G Ubiquitination assays: SHMT2 was immunoprecipitated from cell lysates using anti-ubiquitin (Ub) antibodies, followed by immunoblot analysis with anti-SHMT2. SLC25A26-OE cells exhibited enhanced SHMT2 polyubiquitination. RT‒qPCR analysis of MYB mRNA levels in MTHFR-OE ( H ) and SLC25A26-OE ( I ) cells ( n = 3 biological replicates). Luciferase reporter assays demonstrating MYB binding to the SHMT2 promoter ( J ) and SLC25A26-mediated suppression of MYB transcriptional activity ( K ). Means ± SEM, * P < 0.05; ** P < 0.01; *** P < 0.001; t -test.

Article Snippet: The cells were incubated overnight at 4 °C with a primary antibody against SHMT2 (1:100, Cat# A21803, ABclonal, Wuhan, China), followed by three washes with 0.05% PBST (PBS containing 0.05% Tween 20).

Techniques: Immunofluorescence, Fluorescence, Plasmid Preparation, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Luciferase, Binding Assay, Activity Assay

Journal: Cell Death & Disease

Article Title: MTHFR functions as a metabolic checkpoint in NSCLC through SLC25A26-mediated SHMT2 inhibition

doi: 10.1038/s41419-026-08874-z

Figure Lengend Snippet: SHMT2 co-overexpression rescues MTHFR-mediated tumor suppression: Representative images ( A ) and quantification ( B ) of subcutaneous xenograft weights in nude mice injected with control (EV), MTHFR-overexpressing (MTHFR-OE), or MTHFR-OE + SHMT2-OE H520 cells ( n = 5 mice/group). C , D Haematoxylin‒eosin (HE) and immunohistochemical (IHC) staining of tumor tissues showing increased stromal infiltration (black arrows) and increased Ki67-positive proliferating cells (brown nuclei) in MTHFR-OE + SHMT2-OE tumors compared with those in tumors in which MTHFR-OE alone was used. Scale bars: HE = 50 μm; IHC = 100 μm. E , F Partial restoration of tumor growth in MTHFR-OE xenografts with SLC25A26 knockdown (MTHFR-OE + SLC25A26-KD): Representative images ( E ) and endpoint tumor weights ( F ) ( n = 5). G , H HE(G) and IHC(H) staining demonstrating enhanced stromal infiltration and Ki67 positivity in MTHFR-OE + SLC25A26-KD tumors. Means ± SEM, * P < 0.05; ** P < 0.01; *** P < 0.001; t -test.

Article Snippet: The cells were incubated overnight at 4 °C with a primary antibody against SHMT2 (1:100, Cat# A21803, ABclonal, Wuhan, China), followed by three washes with 0.05% PBST (PBS containing 0.05% Tween 20).

Techniques: Over Expression, Injection, Control, Immunohistochemical staining, Immunohistochemistry, Knockdown, Staining

Journal: Nature Metabolism

Article Title: ALDH1L2 regulates reactive oxygen species and acinar-to-ductal metaplasia in the pancreas

doi: 10.1038/s42255-026-01456-5

Figure Lengend Snippet: A ) SHMT2, MTHFD2 and MTHFD1L RNA normalized expression (nTPM) from human tissue samples extracted from the consensus dataset of the human protein atlas . B ). Shmt2 and Mthfd1l mRNA expression measured by RT-qPCR in subpopulation of healthy mouse pancreas isolated by flow cytometry. Acinar cells were obtained by CD45 − Epcam + CD133 + SSC hi gating, ductal cells by CD45 − EpCAM + CD133 + SSC low gating, stromal cells as CD45 − EpCAM − Pdpn + . Remaining non-immune cells are characterized as CD45 − EpCAM − Pdpn − endothelial cells. Data represented as the mean ± SEM of individual mice pooled from 2 independent experiments. CD45 − EpCAM − Pdpn − (n = 10), acinar (n = 6), ductal (n = 5), stromal (n = 8). C ) mRNA expression of ALDH1L2 in 3D ex-vivo cultures of human acinar cells at day 0 (n = 13) or at day 6 (n = 15) of ADM, extracted from available RNAseq data set ( GSE179248 ). Data shown as mean ± SD. Unpaired t test with Welch correction, ***p-value = 0.001. D ) mRNA expression of Cytokeratin 19 ( Krt19 ), amylase ( amy2b ) and Aldh1l2 expression in 2D ex-vivo cultures at day 0 (n = 3) and day 4 (n = 3) of mouse acinar cells extracted from available RNAseq data set ( GSE240106 ). Data shown as mean± SD. Unpaired t test, ****p-value < 0.0001, **p-value = 0.0077, *p-value = 0.0138. E ) mRNA expression of cytosolic enzymes ( G6pdx , glucose-6-phosphate dehydrogenase; Me1 , malic enzyme 1; Nadk , NAD + Kinase; Gclc , glutamate cysteine ligase) in 2D ex-vivo cultures at day 0 (n = 3) and day 4 (n = 3) of mouse acinar cells extracted from available RNAseq data set ( GSE240106 ). Data shown as mean ± SD. Unpaired t test, ****p-value < 0.0001, ***p-value = 0.006, *p-value = 0.0176. F ) mRNA expression of mitochondrial enzymes ( Me2 , malic enzyme 2; Mthfd2 , methylenetetrahydrofolate dehydrogenase 2; Idh2 , isocitrate dehydrogenase 2; Nadk2 , mitochondrial NAD + kinase) in 2D ex-vivo cultures at day 0 (n = 3) and day 4 (n = 3) of mouse acinar cells extracted from available RNAseq data set ( GSE240106 ). Data shown as mean ± SD. Unpaired t test, ****p-value < 0.0001, ***p-value = 0.003, *p-value = 0.0112.

Article Snippet: A ) SHMT2, MTHFD2 and MTHFD1L RNA normalized expression (nTPM) from human tissue samples extracted from the consensus dataset of the human protein atlas .

Techniques: Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Ex Vivo, RNA sequencing

Journal: Nature Metabolism

Article Title: ALDH1L2 regulates reactive oxygen species and acinar-to-ductal metaplasia in the pancreas

doi: 10.1038/s42255-026-01456-5

Figure Lengend Snippet: a , A scheme of cytosolic and mitochondrial one-carbon metabolism. Briefly, Serine hydroxymethyltransferase 1 (SHMT1) in the cytoplasm or SHMT2 in the mitochondria convert serine into glycine by donating a one-carbon unit to THF, producing 5,10-methylene-THF. Methylenetetrahydrofolate dehydrogenase enzymes (MTHFD2 and MTHFD2L in the mitochondria) oxidize 5,10-methylene-THF to 10-formyl-THF, regenerating NADPH. Mitochondrial 10-formyl-THF can then either be used to recycle THF by reducing NADP + into NADPH by ALDH1L2 or can function as the substrate for the generation of formate by MTHFD1-like (MTHFD1L). Mitochondrial formate is transported to the cytoplasm and used to regenerate 10-formyl-THF and 5,10-methylene-THF by MTHFD1. Cytoplasmic 10-formyl-THF will be used for de novo purine synthesis whereas 5,10-methylene-THF will be used as a methyl donor in the methionine cycle or for thymidylate synthesis. Excess formate is excreted from the cell in a process termed formate overflow. b , ALDH1L2 expression determined by western blot in mouse tissues from Aldh1l2 WT or KO mice and representative of both male and female mice and three independent experiments. c , ALDH1L2 and ALDH1L1 mRNA normalized expression (normalized transcripts per million (nTPM)) from human tissue samples extracted from the consensus dataset of the Human Protein Atlas . d , ALDH1L2 staining on formalin-fixed paraffin-embedded (FFPE) sections from Aldh1l2 WT mouse pancreas, representative of six individual mice. e , ALDH1L2 staining on FFPE sections from Aldh1l2 WT and Aldh1l2 KO mouse pancreas, representative of six individual mice. f , g , Aldh1l2 ( f ) and Aldh1l1 ( g ) mRNA expression measured by RT–qPCR in a subpopulation of healthy mouse pancreas isolated by flow cytometry. Acinar cells were gated as CD45 − Epcam + CD133 + SSC hi gating, ductal cells as CD45 − EpCAM + CD133 + SSC lo gating and stromal cells as CD45 − EpCAM − Pdpn + . The remaining CD45 − EpCAM − Pdpn − cells contain mostly endothelial cells. Data represent the mean ± s.e.m. of individual female mice pooled from two independent experiments. CD45 − EpCAM − Pdpn − ( n = 10), acinar ( n = 6), ductal ( n = 5) and stromal ( n = 8). h , ALDH1L2, amylase and CK19 protein expression in ex vivo cultured acinar cells. The same samples were run on two separate blots and probed for ALDH1L2/CK19 and AMYLASE/VINCULIN. Representative of 2-4 independent experiments.

Article Snippet: A ) SHMT2, MTHFD2 and MTHFD1L RNA normalized expression (nTPM) from human tissue samples extracted from the consensus dataset of the human protein atlas .

Techniques: Expressing, Western Blot, Staining, Formalin-fixed Paraffin-Embedded, Quantitative RT-PCR, Isolation, Flow Cytometry, Ex Vivo, Cell Culture

Journal: Nature Metabolism

Article Title: ALDH1L2 regulates reactive oxygen species and acinar-to-ductal metaplasia in the pancreas

doi: 10.1038/s42255-026-01456-5

Figure Lengend Snippet: A ) SHMT2 and MTHFD1L mRNA expression in pancreatic cancer patients from the PAAD TCGA data set (n = 179) compared to normal tissue expression from GTEx data (n = 171). Data represented as mean ± SD and obtained from GEPIA B ) ALDH1L2 protein expression in 6 human pancreatic cancer cell lines and one ALDH1L2 -positive breast cancer cell line (MDA-MB-468). C ) ALDH1L2 immunohistochemistry staining of pancreas tissue of 25-week-old KC mice. D ) Aldh1l2 mRNA staining by RNAscope of pancreas tissue from 15-week-old KC mice. E ) Percentage of CD3 + (left panel) and FOXP3 + (right panel) cells in the pancreatic tumour tissue of 9 week old KPC mice as quantified by immunochemistry staining. Data represented as mean ± SD (range [min-max] for left panel: WT group [0.19-2.75] Aldh1l2 KO group [0.26-4.39], right panel: WT group [0.11-1.28] KO group [0.05-1.66]) of n = 19 WT and n = 24 KO field of view taken on 7 mice from each background. Unpaired t test with welch correction for left panel and unpaired t test for right panel. F ) Amylase (brown) and CK19 (pink) immunohistochemistry double staining in pancreatic tissue of 15-week-old WT and Aldh1l2 KO mice. Representative of n = 10 per genotype. G ) Ectopic over-expression of ALDH1L2 in clones following transfection of the pancreatic cancer cell line, KPC-7, as measured by western blot. H ) Growth curve of pancreatic cancer cell line overexpressing ALDH1L2 (ALDH1L2-OE KPC-7, clone #23) or not after subcutaneous injection in syngeneic C57bl/6 mice. Data represented as ± SEM of n = 6 WT and n = 6 ALDH1L2 overexpressing tumours. Representative of 2 independent experiments. Two-way anova. I ) ADM quantification in pancreatic tissue of 25-week-old WT ( Pdx1 Cre ;Kras LSL-G12D/+ ; Trp53 LSL-R172H/+ ; Tigar +/+ , n = 6 ) and Tigar KO (n = 8) KC mice. Data is represented as mean ± SD. Unpaired t-test. *p-value = 0.0392. J ) ADM quantification in pancreatic tissue of 6-week-old WT ( Pdx1 Cre ;Kras G12D/+ ; Trp53 fl/fl ;Tigar +/+ , n = 7 ) and Tigar KO (n = 4) KFC mice. Data is represented as mean ± SD. Unpaired t-test, *p-value = 0.0275. K ) Tigar mRNA expression measured by RT-qPCR in subpopulation of healthy female mouse pancreas isolated by flow cytometry. Data represented as the mean ± SEM of individual mice pooled from 2 independent experiments. CD45 − EpCAM − Pdpn − (n = 10), acinar (n = 6), ductal (n = 5), stromal (n = 8).

Article Snippet: A ) SHMT2, MTHFD2 and MTHFD1L RNA normalized expression (nTPM) from human tissue samples extracted from the consensus dataset of the human protein atlas .

Techniques: Expressing, Immunohistochemistry, Staining, RNAscope, Double Staining, Over Expression, Clone Assay, Transfection, Western Blot, Injection, Quantitative RT-PCR, Isolation, Flow Cytometry