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: Aco2 fl/fl animals were generated by Taconic/Cyagen.

Techniques: Expressing, Control, Plasmid Preparation

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) Principal component (PC) analysis of the top 500 genes with the highest row variance from RNA sequencing data of A549 cells with control (sgAAVS1) or ACO2 editing with additional control (sgAAVS1) or CS editing. (B) Dot plot depicting Reactome gene sets enriched among genes increased in ACO2 -edited cells treated with pyruvate compared with vehicle. (C) Violin plot depicting Z scores of ISR-related genes in A549 cells with control (sgAAVS1; –) or ACO2 editing (+) with additional control (sgAAVS1; –) or CS editing (+) cultured in vehicle (–) or pyruvate (+) for 24 h. (D) Immunoblot of A549 cells with control (sgAAVS1; –) or ACO2 editing. (E) Median fluorescent intensity (MFI) depicting L-HPG incorporation of A549 cells with control (sgAAVS1) or ACO2 editing cultured as indicated for 24 h. Dotted line depicts L-HPG incorporation in cells treated with cycloheximide (CHX). (F) MFI depicting OPP incorporation in A549 cells with control (sgAAVS1) or ACO2 editing cultured as indicated for 48 h. The dotted line depicts OPP incorporation in cells treated with CHX. Values are multiplied by 10E− 4. (G) Immunoblot of A549 cells with control (sgAAVS1; –) or ACO2 editing (+) with additional control (sgAAVS1; –) or CS editing cultured as indicated for 24 h. (H and I) Percent EdU-positive cells (H) and cell cycle distribution (I) in A549 cells with control (sgAAVS1; –) or ACO2 editing cultured as indicated for 48 h. **** p < 3E− 6 (I). RNA sequencing data reflect n = 2 independent replicates, shown as the average frequency distribution in (C). Data are mean ± SD with n = 2 (H for just sgAAVS1 + vehicle condition) or n = 3 (E, F, H, and I) independent replicates. Significance was assessed using two-way ANOVA with Sidak’s multiple-comparisons post-test (C), relative to vehicle treatment (E and H) or relative to sgAAVS1+pyruvate (I). See also .

Article Snippet: Aco2 fl/fl animals were generated by Taconic/Cyagen.

Techniques: RNA Sequencing, Control, Cell Culture, Western Blot

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 concentration in serum (A) and citrate levels in tissues (B) from indicated mice 10 days post tamoxifen administration (serum: n = 11 mice/genotype; other tissues: n = 5–11 mice/genotype). (C and D) Representative hematoxylin & eosin (top) and immunohistochemistry for NGAL (bottom) in kidneys from indicated mice 20–25 days post tamoxifen administration (C). Black arrows indicate areas of renal tubular degeneration. Quantification of tubular pathology and NGAL immunolabeling (D) ( n = 6 kidneys). (E) Relative metabolite levels in kidneys of indicated mice harvested 21 days post tamoxifen administration ( n = 9 Aco2 +/+ and n = 8 Aco2 fl/fl ). (F) Quantification of p-eIF2α relative to total eIF2α from immunoblot of kidneys harvested 21 days post tamoxifen administration, shown in ( n = 6 Aco2 +/+ and n = 7 Aco2 fl/fl ). (G) Gene expression of selected ATF4 target and proximal tubule cell marker genes in kidneys harvested 21 days post tamoxifen administration ( n = 9 Aco2 +/+ and n = 8 Aco2 fl/fl ). Data are mean ± SD with independent replicates as indicated for each graph. Significance was assessed using an unpaired two-tailed t test (A and F), a two-way ANOVA with Sidak’s multiple-comparisons post-test relative to Aco2 +/+ control (B, E, and G), or Fisher’s exact test (D). See also .

Article Snippet: Aco2 fl/fl animals were generated by Taconic/Cyagen.

Techniques: Concentration Assay, Immunohistochemistry, Immunolabeling, Western Blot, Gene Expression, Marker, Two Tailed Test, Control

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) Serum citrate concentration in mice provided drinking water supplemented with NaCl (control) or 3% citrate 10 days post tamoxifen administration (control water: n = 12 Aco2 +/+ , 11 Aco2 fl/fl ; citrate water: n = 14 Aco2 +/+ , 10 Aco2 fl/fl ). (B) Survival curves of indicated mice post tamoxifen (TAM) administration (control water: n = 4 Aco2 +/+ , 9 Aco2 fl/fl ; citrate water: n = 9 Aco2 +/+ , 12 Aco2 fl/fl ). (C) Violin plot depicting Z scores of 122 ISR-related genes in kidneys harvested from mice provided regular water or water supplemented with 3% citrate 10 days post tamoxifen administration ( n = 6 for all conditions except n = 7 for Aco2 fl/fl mice on 3% citrate). (D) Normalized enrichment scores of gene sets related to kidney injury or proximal tubule (PT) identity from RNA sequencing of kidneys described in (C), comparing Aco2 fl/fl vs. Aco2 +/+ animals on regular water (left column) or 3% citrate (right column). Selected gene sets represent genes increased or decreased following acute kidney injury (AKI), associated with AKI in PT cells specifically, or clusters associated with normal PT identity (segments 1–3) or injury. (E) SLC13A2 and SLC13A5 expression in cancer cell lines of kidney ( n = 51) or liver ( n = 27) lineage (TPM, transcripts per million). (F) Citrate levels in HepG2 cells cultured as indicated for 24 h. (G and H) Percent DAPI-positivity (G) or population doublings (H) in HepG2 cells cultured as indicated for 3 days. (I) Percent DAPI-positive cells of indicated genotype cultured in 5 mM (A549) or 1 mM (Calu1) citrate for 3 days. (J) Cellular competition experiment mixing ACO2 -edited cells expressing GFP (“sgACO2-GFP”) 1:1 with cells expressing SLC13A2 cDNA and either sgAAVS1 or sgACO2 and passaged with vehicle or indicated concentrations of citrate for 21 days. Shown are the percent changes in green fluorescent protein (GFP)-negative cells cultured with citrate relative to vehicle at each time point. (K) Immunoblot of cells with control (sgAAVS1; –) or ACO2 editing (+) expressing EGFP (–) or SLC13A2 cDNA (+), cultured as indicated for 24 h. Data are n = 1 (J) or mean ± SD with n = 3 (F–I) independent replicates. Significance was assessed using two-way ANOVA with Sidak’s multiple-comparisons post-test as indicated (C), relative to control water (A), relative to vehicle (F–H), or relative to sgAAVS1 + SLC13A2 -expressing cells (I), using log-rank (Mantel-Cox) test (B), or using the fgsea package in R (D). See also .

Article Snippet: Aco2 fl/fl animals were generated by Taconic/Cyagen.

Techniques: Concentration Assay, Control, RNA Sequencing, Expressing, Cell Culture, Western Blot