Journal: Acta Pharmaceutica Sinica. B

Article Title: Vitamin A and its analogues modulate MUFAs metabolism to improve ferroptosis and aging by direct targeting of ACSL3

doi: 10.1016/j.apsb.2025.11.004

Figure Lengend Snippet: Vitamin A and its metabolites inhibit ferroptosis independent of antioxidant capacity or RAR/RXR activation. (A) Illustration of the structures of VA and ATRA. (B) Measurement of the EC 50 values of VA and ATRA towards RSL3 (0.5 μmol/L) or Erastin (10 μmol/L) induced ferroptosis in HT-1080 cells. Cells were treated with drugs for 24 h. (C) RAL suppressed RSL3 and Erastin-induced ferroptosis in HT-1080 cells. (D) Viability of HT-1080 GPX4 -KO cells treated with the indicated compounds for 24 h after withdrawal of Fer-1, which is essential for the survival of GPX4 -KO cells. (E) The antioxidative capacity of VA (50 μmol/L), ATRA (50 μmol/L) and RAL (50 μmol/L) was analyzed by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Trolox (50 μmol/L) was used as a positive control. (F) The effect of VA, ATRA and RAL on the autoxidation of C11 BODIPY 581/591 initiated by AAPH in EggPC liposomes was measured by FENIX assay. Fer-1 was used as a positive control. (G) Protein expression levels of GPX4, SLC7A11, ACSL4, SCD1, FSP1, HO-1 and NRF2 in HT-1080 cells were analyzed by Western blot. Cells were treated with VA (1 μmol/L) or ATRA (10 μmol/L) in the presence or absence of RSL3 (0.25 μmol/L) for 2.5 h or Erastin (10 μmol/L) for 8 h. (H) Protein expression levels of IRP1, TFRC and FTH1 in HT-1080 cells were analyzed by Western blot. Cells were treated with VA (1 μmol/L) or ATRA (10 μmol/L) in the presence or absence of RSL3 (0.25 μmol/L) for 2.5 h or Erastin (10 μmol/L) for 8 h. (I, J) Cell viability of HT-1080 cells after co-treatment with RSL3 and VA or ATRA in the presence of pan-RAR antagonist AGN193109 (20 μmol/L) (I) or pan-RXR antagonist HX531 (20 μmol/L) (J). (K) The protective effects of VA or ATRA on RSL3-induced ferroptosis in RXRA and RXRB knockout HT-1080 cells. (L) Viability of HT-1080 cells treated with different doses of RAR agonists Ch55 and Adapalene (ADA) under RSL3 (0.5 μmol/L) and Erastin (10 μmol/L)-induced ferroptosis for 24 h. Data shown represent the mean ± SD ( n = 3). ∗∗ P < 0.01, and ∗∗∗ P < 0.001, indicating significant differences between groups. ns means no significance.

Article Snippet: Primary antibodies used were as follows: ACSL3 (Santa Cruz, sc-166374), ACSL3 (Proteintech, 20710-1-AP), ACSL4 (Proteintech, 66617-1-Ig), SLC7A11 (CST, 12691S), GPX4 (HuaBio, ER1803-15), IRP1 (CST, 20272S), TFRC (CST, 13113S), FTH1 (CST, 4393S), RXRA/RXRB (CST, 8589), SCD1 (Proteintech, 28678-1-AP), FSP1 (Proteintech, 20886-1-AP), HSP90 (Proteintech, 13171-1-AP), β -actin (Proteintech, 81115-1-RR) and Vinculin (Sigma, V9131).

Techniques: Activation Assay, Positive Control, Liposomes, Expressing, Western Blot, Knock-Out

Journal: Acta Pharmaceutica Sinica. B

Article Title: Vitamin A and its analogues modulate MUFAs metabolism to improve ferroptosis and aging by direct targeting of ACSL3

doi: 10.1016/j.apsb.2025.11.004

Figure Lengend Snippet: VA analogues inhibit ferroptosis through the regulation of MUFA metabolism. (A, B) Lipidomic analysis of MUFAs and PUFAs in membrane lipids including PE (A) and PC (B) following VA (5 μmol/L) and D3 (5 μmol/L) treatment for 12 h in HT-1080 cells. (C) Viability HT-1080 cells treated with RSL3 (0.5 μmol/L) and VA (1 μmol/L) or ATRA (10 μmol/L) in the presence or absence of SCD1 inhibitor A939572 (20 μmol/L) for 24 h. (D) Validation of SCD1 KO in HT-1080 cells. (E) The levels of OA-CoA were decreased in SCD1 knockout HT-1080 cells. (F) Viability of Vector and SCD1 KO cells treated with RSL3 for 24 h. (G) SCD1 KO compromised the anti-ferroptotic effect of VA analogues in HT-1080 cells. Cells were treated with RSL3 (0.5 μmol/L) and VA (1 μmol/L), ATRA (10 μmol/L) or D3 (5 μmol/L) for 24 h. (H, I) Supplement of OA restored the protective effect of VA analogues against ferroptosis. Vector and SCD1 KO HT-1080 cells were pretreated with OA in (10 and 20 μmol/L in panel H, and 10 μmol/L in panel I, followed by treatment with RSL3 (0.5 μmol/L) and VA (1 μmol/L), ATRA (10 μmol/L) or D3 (5 μmol/L) for 24 h. All the data are presented as the mean ± SD ( n = 3). ∗ P < 0.05, ∗∗∗ P < 0.001, indicating significant differences between groups.

Article Snippet: Primary antibodies used were as follows: ACSL3 (Santa Cruz, sc-166374), ACSL3 (Proteintech, 20710-1-AP), ACSL4 (Proteintech, 66617-1-Ig), SLC7A11 (CST, 12691S), GPX4 (HuaBio, ER1803-15), IRP1 (CST, 20272S), TFRC (CST, 13113S), FTH1 (CST, 4393S), RXRA/RXRB (CST, 8589), SCD1 (Proteintech, 28678-1-AP), FSP1 (Proteintech, 20886-1-AP), HSP90 (Proteintech, 13171-1-AP), β -actin (Proteintech, 81115-1-RR) and Vinculin (Sigma, V9131).

Techniques: Analogues, Membrane, Biomarker Discovery, Knock-Out, Plasmid Preparation

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: An approach for quantifying mitochondrial and cytosolic metabolite levels. a Rapid cell fractionation effectively removes cytosolic components (green) without damaging mitochondrial membrane. Mitochondria retain MitoTracker (red) after digitonin-based cell fractionation. While nucleus (blue) remains intact within the mitochondrial fraction, it should not bias the mitochondrial metabolite pool size measurements due to the free diffusion of small molecules through nuclear pore complexes into the cytosolic fraction. b The purity of the mitochondria and cytosol-enriched fractions in terms of the expression of mitochondrial (citrate synthase; CS, red) and cytosolic marker proteins (glyceraldehyde-phosphate dehydrogenase; GAPDH, green) via quantitative western blot (three biological replicates are shown). c – d The extent of cross-contamination between the mitochondrial and cytosolic fractions based on mitochondrial and cytosolic protein (western blot, green) and small-molecule markers (LC-MS, orange). Tetramethylrhodamine methyl ester (TMRM) was introduced as a mitochondria-specific small-molecule marker. e A comparison of the sum of measured metabolite pools in the two subcellular fractions to that measured in whole-cell extracts; a cumulative distribution shows the number of metabolites ( y -axis) for which the sum of pool sizes in the two fractions deviates by different extent from the whole-cell measurements ( x -axis). f Delay in the quenching of metabolism in the mitochondrial fraction (by 1, 5, 10, and 30 min) leads to reduced pool sizes due to the diffusion of small molecules out of mitochondria; a cumulative distribution shows the number of metabolites ( y -axis) for which the fractional pool size in the mitochondrial fraction drops by different extent ( x -axis). g The deconvolution function used to infer metabolite pool size ratio in mitochondria versus cytosol ( y -axis) given the measured pool size ratio ( x -axis), considering the cross-contamination between the two fractions. Confidence intervals of the deconvoluted pool size rations are shown considering 5% (orange) and 10% (green) coefficient of variance (CV) in the measured pool sizes in the mitochondrial and cytosolic fractions. h The ratio of metabolite pool sizes in mitochondria versus cytosol for 42 metabolites in HeLa cells under standard normoxic condition; glycolytic and pentose phosphate pathway metabolites (orange) and TCA cycle metabolites (green). Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Cell Fractionation, Membrane, Diffusion-based Assay, Expressing, Marker, Western Blot, Liquid Chromatography with Mass Spectroscopy, Comparison

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: Compartment-specific isotope tracing in mitochondria and cytosol. a A schematic description of the spatial-fluxomics approach. b TCA cycle metabolism and associated reactions involving acetyl-CoA and fatty acid metabolism. c Isotopic labeling kinetics of citrate m + 2 in mitochondria versus cytosol when feeding HeLa cells with [U- 13 C]-glucose; showing the ratio between the relative abundance of the m + 2 form of citrate in the mitochondria (out of the entire mitochondrial pool) divided by the relative abundance of citrate m + 2 in cytosol. d – f The isotopic labeling kinetics of citrate m + 4 ( d ), glutamine m + 5 ( e ), and glutamate m + 5 ( f ) when feeding [U- 13 C]-glutamine. Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Isotopic Labeling

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: A quantitative view of mitochondrial and cytosolic fluxes in the TCA cycle and citrate metabolism under normoxia. a – d Mass-isotopomer labeling kinetics of citrate in mitochondria ( a ) and cytosol ( b ), and malate in mitochondria ( c ) and cytosol ( d ) when feeding HeLa cells with [U- 13 C]-glutamine under standard normoxic conditions. e Measured isotopic labeling ratio in HeLa cells for citrate m + 5 /m + 4 and malate m + 3 /m + 4 in media (red) in comparison to the expected labeling via computational simulation, considering the measured labeling kinetics of citrate and malate in mitochondria (green) and cytosol (orange). f Gibbs free energy of mitochondrial (IDH2/3) and cytosolic (IDH1) IDH isozymes (in the oxidative direction) in HeLa cells under normoxia (green) and hypoxia (red). g Mitochondrial and cytosolic fluxes, showing percentage from citrate synthase flux (which is 0.48 mM h −1 ). Arrow represents the direction of net flux; number represents net flux in the direction of the arrow and number in parenthesis correspond to the backward flux. Confidence intervals for estimated fluxes are shown in Supplementary Data . h The measured mass-isotopomer distribution of palmitate when feeding cells with [U- 13 C]-glutamine (green) is consistent with the simulated fit (orange). For the simulation, acetyl-CoA labeling was assumed to follow a binomial distribution with a probability of 7.1% having m + 2 labeling form. i Validation of the method based on knock-down of IDH1 or IDH2 genes and following citrate isotopic labeling after feeding cells with [U- 13 C]-glutamine. Upon IDH1 knockdown, the ratios between citrate m + 5 and m + 4 in mitochondria and cytosol are similar, indicating that all reductive glutamine flux occurred in mitochondria (where citrate m + 4 is produced from malate m + 4). Meanwhile, IDH2 knockdown resulted in a higher citrate m + 5 to m + 4 ratio in cytosol, indicating that reductive IDH1 remains active. n.s. not significant. * P < 0.05 and ** P < 0.01 by two-sample t -test. Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Labeling, Isotopic Labeling, Comparison, Biomarker Discovery, Knockdown, Produced

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: Metabolic rewiring of mitochondrial and cytosolic fluxes under hypoxia. a, b Mass-isotopomer labeling kinetics of citrate in mitochondria ( a ) and cytosol ( b ) when feeding HeLa cells with [U- 13 C]-glutamine under hypoxia. c Mitochondrial to cytosolic metabolite pool size ratios ( y -axis) under normoxia (green) and hypoxia (orange) in HeLa cells; average fold change in whole-cell pool size under normoxia and hypoxia is indicated by color ( x -axis). d Mitochondrial and cytosolic fluxes in HeLa cells under hypoxia, showing percentages from citrate synthase flux in normoxia. Arrow represents the direction of net flux; number represents net flux in the direction of the arrow and number in parenthesis correspond to the backward flux. Confidence intervals for estimated fluxes shown in Supplementary Data . * P < 0.05, ** P < 0.01, and *** P < 0.001 by two-sample t -test. Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Labeling

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: Reversed CS flux supports cell survival and growth in SDH-deficient cells. a , b Isotopic labeling kinetics of cytosolic and mitochondrial citrate m + 5 and mitochondrial malate m + 3 when feeding SDH-WT ( a ) and SDH-KO cells ( b ) with [U- 13 C]-glutamine. c Mitochondrial to cytosolic metabolite pool size ratios ( y -axis) in SDH-WT (green) and SDH-KO cells (orange); average fold change in whole-cell pool size upon SDH deficiency is indicated by color ( x -axis). d Gibbs free energy of mitochondrial (IDH2/3) and cytosolic (IDH1) IDH isozymes (in the oxidative direction) in SDH-proficient (green) and deficient cells (orange). e Mitochondrial and cytosolic fluxes in SDH-KO cells, showing percentages from citrate synthase flux in the SDH-positive cells. Arrow represents the direction of net flux; number represents net flux in the direction of the arrow and number in parenthesis correspond to the backward flux. Confidence intervals for estimated fluxes shown in Supplementary Data 8. f Protein expression of mitochondrial and cytosolic markers in isolated mitochondria from SDH-WT and KO cells. g Fractional labeling of acetyl-CoA (m + 2) and malate (m + 4) in isolated mitochondria cultured with [U- 13 C]-citrate. h Fractional labeling of acetyl-CoA (m + 2) and malate (m + 4) in isolated SDH-KO mitochondria cultured with [U- 13 C]-citrate with or without ATP and ACLY inhibitor (BMS-303141). i Relative cell viability for SDH-WT (green) and SDH-KO cells (orange) following the addition of increasing concentration of dichloroacetate (DCA). XTT cell viability assay was performed 72 h after DCA treatment. j Relative metabolite pool sizes in SDH-WT (green) and SDH-KO cells (orange) with 10 mM DCA (24 h); compared to untreated control. k Metabolite fractional labeling from [U- 13 C]-glutamine in SDH-KO cells without (green) or with (orange) 10 mM DCA (24 h). * P < 0.05, ** P < 0.01, and *** P < 0.001 by multiple t -test analysis with FDR correction ( f ) or by two-sample t -test ( c , e , g , and h ). n.d. not detected. Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Isotopic Labeling, Expressing, Isolation, Labeling, Cell Culture, Concentration Assay, Viability Assay, Control