Journal: Cell metabolism

Article Title: Oncogene amplification in growth factor signaling pathways renders cancers dependent on membrane lipid remodeling

doi: 10.1016/j.cmet.2019.06.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse Monoclonal anti-LPCAT1, Clone 8B6E9 (WB 1:10000) , Proteintech , Cat#66044–1-lg; RRID:AB_11045658.

Techniques: Recombinant, Membrane, Transfection, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Negative Control, Empire Assay, Plasmid Preparation, shRNA, Control, Sequencing, Software

Journal: Nature Communications

Article Title: Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils

doi: 10.1038/s41467-025-59050-4

Figure Lengend Snippet: A The UMAP plot shows five epithelial subtypes in distinct colors, with bar graphs indicating their quantities. B The UMAP plot (left) shows the distributions of control, resistant, and sensitive groups, with cell proportions (center) and subtype compositions (right). C Heatmap of top 10 signature genes for three groups. D CellChat circle plot shows differences in intercellular communication number and strength between two groups. E The UMAP plot shows 11 immune cell subtypes, with bar graphs indicating their numbers. F The UMAP plot shows 11 immune subtypes across two groups. G CellChat analysis compares epithelial-immune information flow in Resi and Sens groups, with relative (left) and absolute (right) flow shown. H Scatter plot of ANNEXIN pathway signaling in cell clusters from CellChat analysis. Bubble plots from CellChat ( I ) and CellPhoneDB (J) show epithelial subtype communication with Neu1 receptors and ligands. P values were calculated using a permutation test, assessing the significance of cell-cell communication by comparing observed mean expression with a null distribution generated by random permutations ( I ). P values were calculated using a permutation test, which generates a null distribution by randomly shuffling cell labels to determine the specificity of interactions ( J ). P values are one-sided and exact. K Violin plot (left) and FeaturePlot (right) show Anxa1 expression in epithelial cells across groups. The box plot elements are defined as described in the Statistics and Reproducibility section. Each group includes cells from 3 mice. P values were calculated one-way ANOVA. L Violin plot showing Anxa1 expression across epithelial cell subtypes. The box plot elements are defined as described in the Statistics and Reproducibility section. Each group includes cells from 9 mice. P values were calculated one-way ANOVA. M Representative IHC staining and score of Anxa1 in HNSCC. n = 6 control, n = 6 resistant, n = 8 sensitive mice. Scale bar = 65 μm. Data shown as as mean ± SD. P values were calculated one-way ANOVA with Tukey’s multiple comparisons test. Source data and exact p values are provided as a file.

Article Snippet: Following a previously published method, HL60 cells were incubated with 500 nM ANXA1 (MCE, HY-P7512) for 6 h, or sorted Fpr1+ neutrophils were incubated with 500 nM Anxa1 (MCE, HY-P72078) for 6 h . Following the manufacturer’s instructions, Fpr1+ neutrophils treated with or without Anxa1 were stained with MitoTracker Red (Thermo Fisher, A66443) and Hoechst 33342 (Beyotime, R0305S-6) under light-protected conditions at 37 °C for 20 min. After staining, images were captured using a super-resolution microscope (Nikon, Tokyo, Japan) in SIM mode.

Techniques: Control, Expressing, Generated, Immunohistochemistry

Journal: Nature Communications

Article Title: Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils

doi: 10.1038/s41467-025-59050-4

Figure Lengend Snippet: A The experimental design of the HNSCC tumorigenesis model and treatment strategy in the Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. B Representative image of tongue visible lesions in Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. n = 6 mice in each group. Scale bar, 2 mm. C Quantification of HNSCC lesion number and lesion area (mm 3 ) in Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. D Representative H&E staining of HNSCC and Quantification of HNSCC invasion grades in Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. Scale bar, 65 μm. n = 6 mice in each group. Statistical significance was assessed using the Pearson chi-square test. P value is exact and two-sided. Representative IHC staining and IHC Score of Ki67 ( E ), Anxa1 ( F ), and Fpr1 ( G ) in HNSCC of Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. The scale bar is 65 μm. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. Representative immunofluorescence staining of Gzma+γδT ( H ) or Gzma+Cd8 T ( I ) cells in mouse HNSCC tissues from Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. Epithelial tissues were stained with anti-Gzma antibody (green) and anti-Tcr g/d ( H ) or anti-Cd8a ( I ) antibody (red). Cell nuclei were stained with DAPI (blue). Scale bar, 30 μm. On the right, the corresponding percentages of Gzma+γδT ( H ) or Gzma+Cd8 T ( I ) cells in terms of area and number are provided. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. Source data and exact p values are provided as a file.

Article Snippet: Following a previously published method, HL60 cells were incubated with 500 nM ANXA1 (MCE, HY-P7512) for 6 h, or sorted Fpr1+ neutrophils were incubated with 500 nM Anxa1 (MCE, HY-P72078) for 6 h . Following the manufacturer’s instructions, Fpr1+ neutrophils treated with or without Anxa1 were stained with MitoTracker Red (Thermo Fisher, A66443) and Hoechst 33342 (Beyotime, R0305S-6) under light-protected conditions at 37 °C for 20 min. After staining, images were captured using a super-resolution microscope (Nikon, Tokyo, Japan) in SIM mode.

Techniques: Two Tailed Test, Staining, Immunohistochemistry, Immunofluorescence

Journal: Nature Communications

Article Title: Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils

doi: 10.1038/s41467-025-59050-4

Figure Lengend Snippet: A Venn diagram of genes identified by FindMarkers and SCENIC analyses. B Violin plot (left) shows Sox9 expression across groups, and FeaturePlot (right) depicts Sox9 expression in epithelial cells. The box plot elements are defined as described in the Statistics and Reproducibility section. Each group includes cells from 3 mice. P values were calculated one-way ANOVA. C Violin plot showing Sox9 expression across epithelial cell subtypes. The box plot elements are defined as described in the Statistics and Reproducibility section. Each group includes cells from 9 mice. P values were calculated one-way ANOVA. D Representative IHC staining and score of Sox9 in HNSCC. n = 6 control, n = 6 resistant, n = 8 sensitive mice. Scale bar = 65 μm. Data shown as mean ± SD. P values were calculated one-way ANOVA with Tukey’s multiple comparisons test. E ChIP of genomic DNA from resistant mouse HNSCC tissues was performed with a Sox9-specific antibody. Left: gel image, right: qPCR analysis of Anxa1 promoter ChIP signal relative to IgG group. Two primer pairs were used. The experiment was independently repeated three times. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. F Luciferase reporter assay assessed Anxa1 promoter activity in Moc1 cells. A schematic diagram shows cloning the Anxa1 promoter into the pZX001 vector to generate pGL3 luciferase plasmid. Binding motif information was retrieved from footprintDB database. G The relative activity of the Anxa1 promoter was detected by luciferase assay across the three groups. The experiment was independently repeated three times. Data shown as mean ± SD. P values were calculated one-way ANOVA with Tukey’s multiple comparisons test. H Representative immunofluorescence colocalization of Anxa1 (red) and Sox9 (green) in Resi mouse HNSCC tissues. Nuclei counterstained with DAPI (blue). Scale bar = 20 μm. Source data and exact p values are provided as a file.

Article Snippet: Following a previously published method, HL60 cells were incubated with 500 nM ANXA1 (MCE, HY-P7512) for 6 h, or sorted Fpr1+ neutrophils were incubated with 500 nM Anxa1 (MCE, HY-P72078) for 6 h . Following the manufacturer’s instructions, Fpr1+ neutrophils treated with or without Anxa1 were stained with MitoTracker Red (Thermo Fisher, A66443) and Hoechst 33342 (Beyotime, R0305S-6) under light-protected conditions at 37 °C for 20 min. After staining, images were captured using a super-resolution microscope (Nikon, Tokyo, Japan) in SIM mode.

Techniques: Expressing, Immunohistochemistry, Control, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay, Cloning, Plasmid Preparation, Binding Assay, Immunofluorescence

Journal: Nature Communications

Article Title: Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils

doi: 10.1038/s41467-025-59050-4

Figure Lengend Snippet: A The experimental design of the HNSCC tumorigenesis model and treatment strategy in the Resi; DTR+ and Resi; DTR− groups. B Representative image of tongue visible lesions in Resi; DTR+ and Resi; DTR− groups. n = 6 mice in each group. Scale bar, 2 mm. C Quantification of HNSCC lesion number and lesion area (mm 3 ) in Resi; DTR+ and Resi; DTR− groups. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. D Representative H&E staining of HNSCC and Quantification of HNSCC invasion grades in Resi; DTR+ and Resi; DTR− groups. Scale bar, 65 μm. n = 6 mice in each group. Statistical significance was assessed using the Pearson chi-square test. P value is exact and two-sided. Representative IHC staining and IHC Score of Ki67 ( E ), Sox9 ( F ), Anxa1 ( G ), and Fpr1 ( H ) in HNSCC of Resi; DTR+ and Resi; DTR− groups. The scale bar is 65 μm. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. Representative immunofluorescence staining of Gzma+γδT ( I ) or Gzma+Cd8 T ( J ) cells in mouse HNSCC tissues from Resi; DTR+ and Resi; DTR− groups. Epithelial tissues were stained with anti-Gzma antibody (green) and anti-Tcr g/d ( I ) or anti-Cd8a ( J ) antibody (red). Cell nuclei were stained with DAPI (blue). Scale bar, 30 μm. On the right, the corresponding percentages of Gzma+γδT ( I ) or Gzma+Cd8 T ( J ) cells in terms of area and number are provided. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. Source data and exact p values are provided as a file.

Article Snippet: Following a previously published method, HL60 cells were incubated with 500 nM ANXA1 (MCE, HY-P7512) for 6 h, or sorted Fpr1+ neutrophils were incubated with 500 nM Anxa1 (MCE, HY-P72078) for 6 h . Following the manufacturer’s instructions, Fpr1+ neutrophils treated with or without Anxa1 were stained with MitoTracker Red (Thermo Fisher, A66443) and Hoechst 33342 (Beyotime, R0305S-6) under light-protected conditions at 37 °C for 20 min. After staining, images were captured using a super-resolution microscope (Nikon, Tokyo, Japan) in SIM mode.

Techniques: Two Tailed Test, Staining, Immunohistochemistry, Immunofluorescence

Journal: Antioxidants (Basel, Switzerland)

Article Title: Mitochondrial Reactive Oxygen Species Formation Determines ACSL4/LPCAT2-Mediated Ferroptosis.

doi: 10.3390/antiox12081590

Figure Lengend Snippet: Figure 1. Quantification of protein and mRNA levels and sensitivity of empty vector control and ACSL4/LPCAT2 overexpressing HEK293T cells against RSL3. (A–D) Overexpression of ACSL4 and LPCAT2 by the sleeping beauty system was confirmed by (A) Western blot analysis, which was quantified for protein levels (B) ACSL4 (n = 10) (C) LPCAT2 (n = 8) and (D) GPx4 (n = 4) and depicted as fold protein level normalized to controls. (E,F) Quantitative PCR analysis of untreated LV and ACSL4/LPCAT2 OE cells. Relative gene expression was calculated for (E) ACSL4 and (F) LPCAT2 by normalization to GAPDH RNA level and LV control conditions (data are given as individual data points ± SD; n = 4 replicates per group). Sensitivity against RSL3 was analyzed by (G) MTT assay after 16 h of RSL3 treatment (percentage of control condition) and (H) xCELLigence real-time impedance measurement evaluated 19 h after treatment onset. Data are given as mean ± SD (n = 8 replicates). (I) Cell death was quantified by FACS analysis of PI staining after 16 h of RSL3 treatment (5000 cells per replicate of n = 3 replicates, percentage of gated cells). (J) Representative dot plots of the FACS measurements. *** p < 0.001; ** p < 0.01 compared to (untreated) control (ANOVA, Scheffé’s test).

Article Snippet: ACSL4 (1:500 in 5% blocking buffer; Santa Cruz Biotechnology, Heidelberg, Germany), LPCAT2 (1:6000 in 5% blocking buffer; Proteintech, Planegg-Martinsried, Germany), GPx4 (1:500 in 5% blocking buffer, Abcam, Berlin, Germany), or xCT (1:50,000 in 5% blocking buffer, Proteintech, Planegg-Martinsried, Germany) were incubated overnight at 4 ◦C.

Techniques: Plasmid Preparation, Control, Over Expression, Western Blot, Real-time Polymerase Chain Reaction, Gene Expression, MTT Assay, Staining

Journal: Antioxidants (Basel, Switzerland)

Article Title: Mitochondrial Reactive Oxygen Species Formation Determines ACSL4/LPCAT2-Mediated Ferroptosis.

doi: 10.3390/antiox12081590

Figure Lengend Snippet: Figure 2. DFO and Fer-1 prevent ACSL4/LPCAT2 OE cells from increased mitochondrial ROS production and loss of mitochondrial membrane potential and metabolic activity. Metabolic activity of HEK293T cells was evaluated by MTT assays after 16 h of treatment with (A) 0.8 µM RSL3 and co-treatment with 2.5–10 µM deferoxamine (DFO); (B) 0.18 µM RSL3 and co-treatment with 1–15 µM ferrostatin-1 (Fer-1); and (C) 0.2 µM RSL3 and co-treatment with 10 µM Fer-1, 10 µM DFO, 10 µM zileuton (Zil), 0.5 µM ST1853 (ST) and 5 µM PD146176 (PD). Data are shown as percentage of control condition of n = 8 replicates. (D) Mitochondrial ROS formation and (F) mitochondrial membrane potential were quantified by FACS analysis of MitoSOX or TMRE stained cells after 0.2 µM RSL3 and co-treatment with 10 µM DFO and Fer-1 for 16 h (5000 cells per replicate of n = 3 replicates, percentage of gated cells). (E,G) Histograms of the respective FACS measurements with gating. *** p < 0.001; ** p < 0.01 compared to control condition, ### p < 0.001; ## p < 0.01; and # p < 0.05 compared to RSL3-treated control condition (ANOVA, Scheffé’s test).

Article Snippet: ACSL4 (1:500 in 5% blocking buffer; Santa Cruz Biotechnology, Heidelberg, Germany), LPCAT2 (1:6000 in 5% blocking buffer; Proteintech, Planegg-Martinsried, Germany), GPx4 (1:500 in 5% blocking buffer, Abcam, Berlin, Germany), or xCT (1:50,000 in 5% blocking buffer, Proteintech, Planegg-Martinsried, Germany) were incubated overnight at 4 ◦C.

Techniques: Membrane, Activity Assay, Control, Staining

Journal: Antioxidants (Basel, Switzerland)

Article Title: Mitochondrial Reactive Oxygen Species Formation Determines ACSL4/LPCAT2-Mediated Ferroptosis.

doi: 10.3390/antiox12081590

Figure Lengend Snippet: Figure 3. Troglitazone and Rosiglitazone inhibit ferroptosis in ACSL4/LPCAT2 overexpressing cells.

Article Snippet: ACSL4 (1:500 in 5% blocking buffer; Santa Cruz Biotechnology, Heidelberg, Germany), LPCAT2 (1:6000 in 5% blocking buffer; Proteintech, Planegg-Martinsried, Germany), GPx4 (1:500 in 5% blocking buffer, Abcam, Berlin, Germany), or xCT (1:50,000 in 5% blocking buffer, Proteintech, Planegg-Martinsried, Germany) were incubated overnight at 4 ◦C.

Techniques:

Journal: Antioxidants (Basel, Switzerland)

Article Title: Mitochondrial Reactive Oxygen Species Formation Determines ACSL4/LPCAT2-Mediated Ferroptosis.

doi: 10.3390/antiox12081590

Figure Lengend Snippet: Figure 4. FACS analysis and Seahorse measurement demonstrate mitochondrial involvement in ACSL4 and LPCAT2 OE cells. (A) Mitochondrial ROS formation and (C) mitochondrial membrane potential were quantified by FACS analysis of MitoSOX or TMRE stained cells after 0.3, 0.6, and 0.9 µM RSL3 treatment for 16 h (5000 cells per replicate of n = 3 replicates, calculated as percentage of gated cells). (B,D) Representative histograms of the respective FACS measurement with gating. (E) Mitochondrial respiration and (F) glycolysis were detected by the seahorse system after 16 h of 0.8 µM RSL3 treatment (n = 6–8 replicates per condition). *** p < 0.001; ** p < 0.01; and * p < 0.05 compared to untreated control conditions (ANOVA, Scheffé’s test).

Article Snippet: ACSL4 (1:500 in 5% blocking buffer; Santa Cruz Biotechnology, Heidelberg, Germany), LPCAT2 (1:6000 in 5% blocking buffer; Proteintech, Planegg-Martinsried, Germany), GPx4 (1:500 in 5% blocking buffer, Abcam, Berlin, Germany), or xCT (1:50,000 in 5% blocking buffer, Proteintech, Planegg-Martinsried, Germany) were incubated overnight at 4 ◦C.

Techniques: Membrane, Staining, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Mitochondrial Reactive Oxygen Species Formation Determines ACSL4/LPCAT2-Mediated Ferroptosis.

doi: 10.3390/antiox12081590

Figure Lengend Snippet: Figure 5. Mitochondrial ROS scavenging by mitoquinone prevents RSL3-mediated ferroptosis in ACSL4/LPCAT2 OE cells. (A) Metabolic activity and (B) ATP levels were determined by MTT or ATP assay after 16 h treatment with 0.1 µM RSL3. Data are shown as percentage of control conditions, n = 8 replicates. (C) Mitochondrial ROS formation, (E) mitochondrial membrane potential, and (G) cell death were quantified by FACS analysis of MitoSOX, TMRE, or PI-stained cells after co-treatment with 0.125 µM or 0.25 µM mitoquinone (MitoQ) and 0.4 µM (MitoSOX) or 0.8 µM RSL3 (TMRE, PI) for 16 h (5000 cells per replicate of n = 3 replicates, percentage of gated cells). (D,F,H) Representative histograms, or dot plots of the respective FACS measurements with gating. (I,K) Mitochondrial respiration and (J,L) glycolysis measurements by the seahorse XF analyzer of LV (I,J) and OE (K,L) cells treated with 0.4 µM RSL3 and co-treated with 0.25 µM MitoQ for 16 h (n = 5–8 replicates per condition). *** p < 0.001 compared to control condition, ### p < 0.001; # p < 0.05 compared to RSL3-treated control condition (ANOVA, Scheffé’s test).

Article Snippet: ACSL4 (1:500 in 5% blocking buffer; Santa Cruz Biotechnology, Heidelberg, Germany), LPCAT2 (1:6000 in 5% blocking buffer; Proteintech, Planegg-Martinsried, Germany), GPx4 (1:500 in 5% blocking buffer, Abcam, Berlin, Germany), or xCT (1:50,000 in 5% blocking buffer, Proteintech, Planegg-Martinsried, Germany) were incubated overnight at 4 ◦C.

Techniques: Activity Assay, ATP Assay, Control, Membrane, Staining

Journal: Antioxidants (Basel, Switzerland)

Article Title: Mitochondrial Reactive Oxygen Species Formation Determines ACSL4/LPCAT2-Mediated Ferroptosis.

doi: 10.3390/antiox12081590

Figure Lengend Snippet: Figure 6. Metabolic intervention fails to prevent ferroptosis in ACSL4/LPCAT2 OE cells. (A) Metabolic activity was determined by MTT assay after 16 h treatment with 1 µM RSL3 and co-treatment with 5 mM metformin. Data are shown as percentage of control conditions, n = 8 replicates. (B) Cell death was measured by PI staining after treating HEK293T cells with 0.1 µM RSL3 and 5 mM metformin for 16 h (5000 cells per replicate of n = 3 replicates, percentage of gated cells). (C,E) Mitochondrial respiration and (D,F) glycolysis measurements by the Seahorse XF analyzer of LV (B,C) and OE (D,E) cells treated with 1 µM RSL3 and co-treated with 5 mM metformin for 16 h (n = 5–8 replicates per condition). (G) MTT assay after 16 h treatment with 1 µM RSL3 and co-treatment with 0.5 or 2 µM 4-octyl-itaconate (4OI), 5 or 10 µM phenformin, and 5 or 20 mM itaconate (Data are shown as percentage of control condition, n = 8 replicates). (H) Metabolic activity was determined by MTT assay after 16 h of exposure to 0.15 µM RSL3 in DMEM medium containing 4, 2, or 0 mM glutamine. Data are shown as percentage of control conditions of n = 8 replicates. (I) To confirm the MTT results, cells were measured by PI staining 18 h after treatment with 0.6 µM RSL3 under conditions of 4, 2, 1, or 0 mM glutamine in DMEM medium (5000 cells per replicate of n = 3 replicates, percentage of gated cells). *** p < 0.001; ** p < 0.01; and * p < 0.05 compared to control conditions and ### p < 0.001; and ## p < 0.01 compared to RSL3-treated control cells (ANOVA, Scheffé’s test).

Article Snippet: ACSL4 (1:500 in 5% blocking buffer; Santa Cruz Biotechnology, Heidelberg, Germany), LPCAT2 (1:6000 in 5% blocking buffer; Proteintech, Planegg-Martinsried, Germany), GPx4 (1:500 in 5% blocking buffer, Abcam, Berlin, Germany), or xCT (1:50,000 in 5% blocking buffer, Proteintech, Planegg-Martinsried, Germany) were incubated overnight at 4 ◦C.

Techniques: Activity Assay, MTT Assay, Control, Staining