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: 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