Journal: Cell

Article Title: Citrate clearance is a major function of aconitase 2 in the canonical TCA cycle

doi: 10.1016/j.cell.2026.01.028

Figure Lengend Snippet: (A and B) Citrate levels (A) and population doublings (B) in cells expressing indicated sgRNA. (C and D) Population doublings (C) and citrate levels (D) of control (sgAAVS1, –) or ACO2 -edited cells (+) that additionally express either control sgRNA (sgAAVS1) or sgRNA targeting CS , treated as indicated for 72 h (C) or 24 h (D). (E and F) Schematic depicting competition experiment (left). A549 cells with sgAAVS1 expressing mCherry were mixed 1:1 with cells expressing BFP and edited with control (sgAAVS1), sgACO2, sgCS, or both sgACO2 and sgCS and assessed over time (E) or assessed after 21 days in culture with vehicle or indicated concentrations of pyruvate (F). (G) Population doublings of ACO2 -edited cells expressing empty vector (–) or SLC25A1 cDNA (+). Data n = 1 (E) or are mean ± SD with n = 3 (A–D, F, and G) independent replicates. Significance was assessed using two-way ANOVA with Sidak’s multiple-comparisons post-test relative to sgAAVS1 control cells (A), relative to vehicle (C, D, and F) or relative to sgACO2/empty vector cells (G). See also and .

Article Snippet: SLC25A1 Polyclonal antibody , ProteinTech , Cat#15235-1-AP; RRID: AB_2254794.

Techniques: Expressing, Control, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

doi: 10.1038/s41418-018-0101-z

Figure Lengend Snippet: SLC25A1 expression correlates with metastatic disease and promotes stemness in NSCLC. a Representative SLC25A1 immunohistochemical staining of tissue microarrays of lung adenocarcinomas. b Representative images of an immunoreactive tumor, normal adjacent tissue (NAT) and metastatic lymph node. c Quantitative analysis of SLC25A1 staining based on percentage of reactive cells. d The cell cycle profile of H1299 cells grown as monolayers or spheres is shown. e Quantitative real-time PCR (normalized values) on the indicated gene products performed in either monolayer (red bars) or spheres of H1299 cells and analyzed in triplicate samples. f Real-time PCR of the indicated genes performed in naive H1299 or SLC25A1-expressing cells. g – i FACS analysis of CD166, CD133 expression and of the CD44 high, CD24 low population in naive H1299 and SLC25A1-expressing cells. j Quantification of the indicated markers from three independent experiments each with duplicate values. k To study self-renewal, cells from SLC25A1-spheres were sorted for CD44/CD24 high or low populations and re-plated independently as single cells.

Article Snippet: The vector-expressing human SLC25A1 untagged or Flag-Myc epitope tagged were from Origene (#SC120727 and RC200657, respectively).

Techniques: Expressing, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction

Journal: Cell Death and Differentiation

Article Title: The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

doi: 10.1038/s41418-018-0101-z

Figure Lengend Snippet: a Growth curves of H1299 or SLC25A1-expressing cells cultured as spheres, dissociated and counted at each of the indicated time points. The arrows indicate the loss of viability that occurs in the first days of sphere formation, likely reflective of differentiated cells dying by anoikis. b Control or SLC25A1-expressing cells were dissociated and plated as single spheres in semisolid media, methylcellulose. After 1 week, spheres were isolated, dissociated again and re-plated in the same conditions, producing second-generation spheres. c Quantification of the experiments shown in b. d , e FACS analysis of CD133 and CD166 in the indicated cell lines either mock transfected (gray bars) or transfected with the vector expressing SLC25A1 (black bars). f Growth curves of H1299 cells infected with lentivirus control shRNA, or with either of two SLC25A1 shRNAs (825 and 350). g , h Sphere-forming ability of H1299 cells infected with lentivirus control or with the indicated SLC25A1-shRNAs, dissociated and plated as single cells in semisolid media. i CD166 and CD44 expression in cells infected with PLKO control lentivirus or with lentivirus harboring the specific SLC25A1 shRNA. j , k Sphere-forming ability of cells expressing SLC25A1 wild-type or mutant SLC25A1R282G/R285C j , or treated with the SLC25A1 inhibitors BTA (2 mM) and CTPI (1 mM) k . Bars represent the standard deviation, asterisks refer to * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001 by unpaired T -test

Article Snippet: The vector-expressing human SLC25A1 untagged or Flag-Myc epitope tagged were from Origene (#SC120727 and RC200657, respectively).

Techniques: Expressing, Cell Culture, Control, Isolation, Transfection, Plasmid Preparation, Infection, shRNA, Mutagenesis, Standard Deviation

Journal: Cell Death and Differentiation

Article Title: The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

doi: 10.1038/s41418-018-0101-z

Figure Lengend Snippet: Identification and activity of a new SLC25A1 inhibitor. a Comparison of the structure and properties of citrate, BTA, CTPI-1 and CTPI-2 and general structure SLC25A1 inhibitors (top panel): a : Calculated octanol–water partition coefficient; b : Topical polar surface area (TPSA); c : Experimental dissociation constant. d : Docking score calculated by the UCSF DOCK6.7 software. b The structure of the leading compound (CTPI-1) and of the newly identified SLC25A1 inhibitor (CTPI-2) are shown. c A binding model for citrate, CTPI-1 or CTPI-2 in complex with a homology model of human SLC25A1. The Dock6.7 software provided a score for binding and a potential pose for the structure. Refinement of the structure using the AMBER MD module in the UCSF Dock6.7 suite of software was performed to give the optimized models. The model shows the relevant amino acids previously known to be involved in citrate binding, including Lys147, Lys245, Arg285, Lys50 and Arg282. d Proliferation rates assessed with crystal violet of H1299 transduced with control or with the SLC25A1-shRNA lentivirus or fold proliferation changes of cells transfected with the SLC25A1R282G/R285C and treated with CTPI-2, respectively. e Gene expression arrays performed on Illumina Human HT-12 v4 Bead Chip of cells expressing the SLC25A1-shRNA or treated with CTPI-2. A cutoff of fold change >2-fold over control, was used to evaluate the similarity of gene expression profile changes between the shRNA and CTPI-2 treatment. The overlap p- value (hypergeometric test) of <10 −716 is shown. f Comparison of the activity of CTPI-1 and CTPI-2 in sphere-forming assays at the indicated concentrations. Asterisks indicate p -values calculated from cells plated in five or six independent wells and comparisons refer to untreated control cells

Article Snippet: The vector-expressing human SLC25A1 untagged or Flag-Myc epitope tagged were from Origene (#SC120727 and RC200657, respectively).

Techniques: Activity Assay, Comparison, Software, Binding Assay, Transduction, Control, shRNA, Transfection, Gene Expression, Expressing

Journal: Cell Death and Differentiation

Article Title: The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

doi: 10.1038/s41418-018-0101-z

Figure Lengend Snippet: SLC25A1 affects mitochondrial activity and self-renewal in CSCs. a Oxygen consumption rate (OCR) were assessed using the Seahorse Extracellular Flux Analyzer after injection of oligomycin (O), FCCP (F) and Antimycin/Rotenone (A + R), in H1299 cells expressing SLC25A1 grown as monolayer or spheres. OCR was normalized to cell number. To eliminate the influence of the media composition and of attachment, for all these experiments monolayer and sphere cultures were first grown for several days, and subsequently they were dissociated, plated on 96-wells coated with the geltrex ECM and pre-equilibrated in the same media containing serum and 5% glucose for 6–12 h, prior to the assessment of the OCR. BR, MR, SRC refer to basal, maximal respiration and spare respiratory capacity. b OCR rates in control or in cells expressing SLC25A1 shRNAs. c OCR rates of the indicated tumor cells in vehicle (black bars) or CTPI-2 treated (gray bars) cells. In all cases, treatment with CTPI-2 was for 3 h and OCR levels were normalized to cell number. There were no changes in viability during this period of time. Bars represent the standard deviation, asterisks refer to **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 by unpaired T-test

Article Snippet: The vector-expressing human SLC25A1 untagged or Flag-Myc epitope tagged were from Origene (#SC120727 and RC200657, respectively).

Techniques: Activity Assay, Injection, Expressing, Control, Standard Deviation

Journal: Cell Death and Differentiation

Article Title: The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

doi: 10.1038/s41418-018-0101-z

Figure Lengend Snippet: SLC25A1 promotes matrix invasion. a – c Representative results showing that CTPI-2 disrupts self-renewal and sphere-forming capacity of the indicated tumor cells relative to monolayer cultures. d , e Spheres embedded in a collagen-based matrix were imaged immediately after plating (time 0) and for 7–10 consecutive days. Fields were identified by numbers on the bottom of the plate. The white rectangles indicate the enhanced areas of each field shown at the top. Invadopodia formation was calculated by first measuring the areas of the spheres (inner area) and the area of the spheres along with its invadopodia (outer area). The percent difference between the outer area and the inner area was graphed and spheres of similar areas were selected for comparison, so to exclude the influence of different cell number. f , g Similar experiments were performed in SLC25A1-expressing cells.Bars represent the standard deviation, asterisks refer to *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001 by unpaired T-test

Article Snippet: The vector-expressing human SLC25A1 untagged or Flag-Myc epitope tagged were from Origene (#SC120727 and RC200657, respectively).

Techniques: Comparison, Expressing, Standard Deviation

Journal: Cell Death and Differentiation

Article Title: The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

doi: 10.1038/s41418-018-0101-z

Figure Lengend Snippet: SLC25A1 is a key regulator of the citrate pool and redox balance. a Mass spectrometry analysis of citrate in monolayer of sphere cells labeled with [U- 13 C]glucose. b Assessment of citrate levels in mitochondrial or cytoplasmic fractions and in the presence or absence of CTPI-2, added to the media for four hours before the assay. c Levels of the indicated metabolites assessed with LC-MS in the indicated cell lines grown as spheres. d OCR was assessed in untreated or CTPI-2-treated spheres in the presence or absence of membrane permeable methyl-malate, succinate, or citrate over the course of a 3-h period. e Self-renewal in H1299 cells grown as single cells in semisolid media after 1 week of treatment with the indicated metabolites in mock treated or CTPI-2 treated cells. All metabolites were re-added to the culture media every 48 h. f – h qRT-PCR of the IDH1 mRNA f , assessment of NADP/NADPH ratio g and of superoxide levels detected with mitosox red h , in the indicated conditions. i A Heatmap comparison of the changes in metabolite levels between cells expressing SLC25A1 untreated or treated with CTPI-2 is shown. Asterisks indicate metabolites most relevant to the pathways analyzed in this study

Article Snippet: The vector-expressing human SLC25A1 untagged or Flag-Myc epitope tagged were from Origene (#SC120727 and RC200657, respectively).

Techniques: Mass Spectrometry, Labeling, Liquid Chromatography with Mass Spectroscopy, Membrane, Quantitative RT-PCR, Comparison, Expressing

Journal: Cell Death and Differentiation

Article Title: The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

doi: 10.1038/s41418-018-0101-z

Figure Lengend Snippet: Metabolic pathways affected by SLC25A1. a , b Metabolomic analysis with LC-MS of SLC25A1-expressing spheres versus control spheres a and of SLC25A1-expressing spheres untreated versus treated with CTPI-2 b . In a , 1412 metabolites were quantified, 181 of which showed statistically significant changes ( p < 0.05). In b , 413 metabolites were quantified and 91 metabolites showed statistically significant changes ( p < 0.05). Analysis was performed with Morpheus, Metaboanalyst and Ingenuity Pathway analysis (IPA). One minus Pearson correlation’ was used for hierarchical clustering of the metabolites. Panel a shows metabolites induced by SLC25A1 relative to control cells, and panel b shows metabolites that were downregulated by CTPI-2 in SLC25A1-expressing spheres. Pathways that are common in the two comparisons are highlighted in green. c , d Extracellular acidification rates (ECAR) and lactate levels in the indicated growth conditions and in the presence or absence of CTPI-2. e Proposed model for the effects of SLC25A1 and CTPI-2 in monolayer and spheres (see also text for explanation). f Phosphofructokinase (PFK) activity was assessed in cells first grown in monolayer or spheres for 36 h and then treated with citrate (10 mM) overnight. The following day, 5 mM citrate was re-added to the media and cells were either mock treated (lanes 1, 4) or treated with CTPI-2 (lanes 2, 3, 5, 6) for 3 h, before quenching and assessment of PFK activity. Bars represent the standard deviation, asterisks refer to ***P ≤ 0.001 by unpaired T-test

Article Snippet: The vector-expressing human SLC25A1 untagged or Flag-Myc epitope tagged were from Origene (#SC120727 and RC200657, respectively).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Control, Activity Assay, Standard Deviation

Journal: Cell Death and Differentiation

Article Title: The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

doi: 10.1038/s41418-018-0101-z

Figure Lengend Snippet: SLC25A1-dependent mitochondrial respiration drives therapy resistance. a , b T1 and T2 cells were treated with cisplatin (0.5–1 μM) or AZD9291 (1 μM), as indicated in each panel, passaged in the presence of the drugs several times, then subjected to OCR analysis with the Seahorse analyzer. c SLC25A1 protein levels in T1 and T2 cells cultured in the presence or absence of cisplatin. d FACS analysis of CD166 and CD133 markers in the cisplatin or AZD9291-resistant T1 or T2 cells. e – g T1 e , T2 f and T4 g cells were untreated or treated with the indicated drugs. The concentration of drugs employed is indicated at the bottom of each panel. Viability was assessed with Crystal Violet after 5 days of treatment. Relative ( R ) index calculations were used to assess the type of drug interactions and are indicated in each panel. The R index is calculated as the expected cell survival ( S exp ; the product of relative survival in cisplatin and relative survival in CTPI-2) divided by the observed relative survival in the presence of both drugs ( S obs ). S exp / S obs = 1.0 denotes an additive interaction, whereas >1.0 denotes a synergistic interaction, R index values approaching 2.0 are indicative of strong synergy. h Kaplan–Meier survival curves relative to SLC25A1 expression in lung AdenoCa patients that received chemotherapy. Red and black lines indicate high and low SLC25A1 expression, respectively. Analysis was performed by using the Km plotter database ( http://kmplot.com/analysis/ ). Bars represent the standard deviation, asterisks refer to **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 by unpaired T-test

Article Snippet: The vector-expressing human SLC25A1 untagged or Flag-Myc epitope tagged were from Origene (#SC120727 and RC200657, respectively).

Techniques: Cell Culture, Concentration Assay, Expressing, Standard Deviation

Journal: Cell

Article Title: Regulation of HIV-1 Gag-Pol Expression by Shiftless, an Inhibitor of Programmed −1 Ribosomal Frameshifting

doi: 10.1016/j.cell.2018.12.030

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 32 p-ctp , Perkin Elmer , BLU508H250UC.

Techniques: Virus, Recombinant, Transfection, Luciferase, Lysis, Reporter Assay, SYBR Green Assay, Control, Sequencing, shRNA, Software

Journal: Bone

Article Title: HIF-1α mediates osteoclast-induced disuse osteoporosis via cytoophidia in the femur of mice.

doi: 10.1016/j.bone.2022.116648

Figure Lengend Snippet: Fig. 4. HIF-1α regulated the IMPDH and CTPS cytoophidia in osteoclasts. (A) The RNA-seq analysis of the osteoclasts induced by BMMs from the control and CKO mice indicated that the mRNA expression level of IMPDH1 and CTPS increased slightly. (B) Western blotting quantified the protein expressions of CTPS1 and IMPDH2, which showed that their expressions increased with the HIF-1α accumulation (n = 3 per group). (C) The IMPDH2 and CTPS1 immunofluorescence co-staining on the 2nd, 3rd, 4th and 6th day of induction with RANKL showed that these nucleic acid metabolic enzymes aggregated in the cytoplasm with special shapes (white arrowheads). The positive signal is in the shape of a dot, rod, short line, and ring. (D) In vivo, IMPDH2 and CTPS1 co-staining was performed. In osteoclasts, the dot and linear microstructure are visible. Moreover, the expression of these enzymes in the osteoclasts was significantly higher in the unloaded control group than the expression in the unloaded CKO group. (E) The IMPDH2 immunofluo rescence staining indicated that IMPDH2 cytoophidia (white arrowheads) increased in the CoCl2 group whereas the IMPDH2 cytoophidia decreased in the HIF-1α KO groups treated with or without CoCl2. (F) The CTPS1 immunofluorescence staining indicated that the CTPS1 cytoophidia increased in the CoCl2 group whereas the CTPS1 cytoophidia decreased in the HIF-1α KO groups treated with or without CoCl2. (G) The quantitative analysis of the IMPDH2 expression in the four groups. (H) The quantitative analysis of the CTPS1 expression in the four groups. Bars = 100 μm. The data were expressed as the median and interquartile range. Significance is presented in graphs and was determined by Student's t-test. Ab breviations: CTPS, cytidine-5-triphosphate synthase; DMSO, dimethyl sulfoxide; IMPDH, inosine 5-monophosphate dehydrogenase; MPA, mycophenolic acid.

Article Snippet: The primary antibodies used were mouse Hypoxyprobe antibody (HypoxyprobeTM-1Kit, USA), mouse anti-HIF-1α (BF8002, 1:200; Affinity, China), rabbit anti-HIF-1α (ab179483, 1:200; Abcam, USA), mouse anti-CTSK (sc-48353, 1:200; Santa Cruz, USA), rabbit antiCTP synthase (15914-1-AP, 1:200; Proteintech, China), rabbit antiIMPDH2 (12948-1-AP, 1:200; Proteintech, China) and rabbit anti-cMyc (ab32072; 1:200; Abcam, USA).

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Immunofluorescence, Staining, In Vivo