Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B, CAOV3, TYKNU, TOV21G, and TOV112D cells were treated with ( A ) 10µM erastin or ( B ) 250nM RSL3 in the presence or absence of 2-8% ascites from three OVCA patients. Liproxtasin-1 (Lipro-1) (2µM) was used as a control ( n =3/cell line, 24 hours). C-D , cells were treated with 5µM erastin for 20 hours or 2µM RSL3 for 2 hours in the presence or absence of 2% ascites. Lipid peroxidation was measured via flow cytometry analysis of BODIPY TM 581/591 C11 staining ( n provided in panel). E-F , cells were either pre-treated with 8% OVCA ascites (Day 0, 16 hours) or left untreated and subsequently treated with 10µM erastin and 8% OVCA ascites. Cell death was visualized ( E ) and measured ( F ) via the CellTox TM Green assay (Days 2-4) in which green fluorescence indicates cell death ( n =3, scale=80µm). G , cells were treated with 10µM erastin in the presence or absence of 2-8% ascites from liver cirrhosis (LC) or OVCA patients for 24 hours ( n provided in panel, 24 hours). H , micro-organospheres (MOS) were treated with 50µM IKE in the presence or absence of 10-40% OVCA ascites for 72 hours ( n =5). I-J , CAOV3 cells transduced with lentivirus expressing luciferase were resuspended in PBS or human OVCA ascites and IP injected into 6-week-old SCID beige mice. Tumor growth ( I ) was visualized and ( J ) measured as total photon flux (p/s) 10 days after tumor injection via IVIS bioluminescent imaging ( n =6). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way or two-way ( F ) ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method ( D-H ). Statistical significance for J was assessed using the two-tailed Student’s t -test method.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Control, Flow Cytometry, Staining, CellTox Assay, Fluorescence, Transduction, Expressing, Luciferase, Injection, Imaging, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , CAOV3, TYKNU, TOV21G, and TOV112D cells were treated with ( A ) 5µM IKE or ( B ) 2.5µM JKE-1674 in the presence or absence of 2-8% ascites from three OVCA patients. Liproxtasin-1 (Lipro-1) (2µM) was used as a control ( n =3/cell line, 24 hours). C , validation of ascites rescue against si GPX4 (100nM) with three different ascites samples in CAOV3 cells ( n =3, 72 hours). D , si GPX4 knockdown confirmation via qRT-PCR ( n =3). E , CAOV3 cells were treated with 10µM erastin in the presence of either 2% ascites or an additional 2% FBS ( n =3, 24 hours). F-H , CAOV3 cells were treated with ( F ) 200nM staurosporine (STS) for 24 hours, ( G ) 400nM actinomycin D (AMD) for 48 hours, or ( H ) 100nM rapamycin for 48 hours in the presence or absence of 2% ascites ( n =3). I-J , qRT-PCR analysis of ( I ) CHAC1 and ( J ) SLC7A11 expression in CAOV3 cells treated with 5µM erastin in the presence or absence of 10% ascites ( n =3, 16 hours). K-L , western blot analysis of lipid peroxidation levels in tumors harvested 10 days after injection of PBS-resuspended and ascites-resuspended CAOV3 cells in SCID beige mice. Signal quantification is depicted in L ( n =6). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance for C , I , and J was assessed using one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. Statistical significance for L was assessed using the two-tailed Student’s t -test method.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Control, Biomarker Discovery, Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Injection, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-C , ( A ) triglyceride, ( B ) cholesterol, and ( C ) protein levels were measured in regular, dialyzed, and delipidated ascites via Triglyceride-Glo TM , Amplex TM Red Cholesterol, and Pierce TM BCA assays respectively. Protein levels in dialyzed and delipidated ascites were adjusted using regular ascites as control ( n =3). D-E , CAOV3, TYKNU, TOV21G, and TOV112D cells were treated with 2% regular, dialyzed, heat-inactivated, or delipidated ascites collected from three metastatic OVCA patients in the presence or absence of ( D ) 10µM erastin or ( E ) 250nM RSL3 ( n =3/cell-line, 24 hours). F-G , DEG Volcano plots of the RNA-seq pairwise comparison analyses of ( F ) CAOV3 and ( G ) TOV21G cells that were treated with 10% ascites for 16 hours ( n =3, adj. P =0.01). H , Venn diagram comparison of lipid species detected in ascites versus lipid species found to be increased in cells treated with the same ascites ( n =5). I , heat map for sum total of unsaturated fatty acid (UFA) present in increased triglycerides in CAOV3 cells treated with 10% ascites and 5µM erastin for 16 hours. The UFA composition of each triglyceride was recorded and the same UFAs were added together. The sum of each UFA is presented as a number on the heat map ( n =5). All Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Control, RNA Sequencing, Comparison, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A , cell viability was assessed in CAOV3 cells treated with 2% regular, dialyzed, heat-inactivated, or delipidated OVCA ascites and 10µM erastin ( n =3, 24 hours). B , Volcano plot of the differentially expressed genes (DEGs) of RNA-seq analysis for the transcriptome changes of CAOV3 and TOV21G cells treated with 10% ascites for 16 hours. DEGs from each cell line were combined for a pairwise comparison of total DEGs under ascites-treatment ( n =3/cell-line, adj. P =0.01). C , Gene Set Enrichment Analysis (GSEA) for the combined DEGs for the indicated gene-sets from CAOV3 and TOV21G. D , structural lipidomic pairwise comparison analysis for CAOV3 cells treated with 5µM erastin with or without 10% ascites for 16 hours ( n =5, adj. P =0.01). E , pie-chart of the lipid composition and relative abundance of all significantly increased intracellular lipids in the ascites-treated cells. F-G , heat map of all ( F ) significant lipid semiquantitative concentration increases and ( G ) top 50 specific lipid species increases (nmol lipid/mg protein) in erastin and ascites treated cells. H-I , CAOV3 cells were treated with 10% ascites and lipid droplet levels were measured via flow cytometry analysis of BODIPY TM 493/503 staining ( n =3, 16 hours). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using the two-tailed Student’s t -test method ( D , I ), and P values were adjusted using the Benjamini-Hochberg correction method ( D ).

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: RNA Sequencing, Comparison, Concentration Assay, Flow Cytometry, Staining, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A , Cell viability was assessed in CAOV3 cells treated with 10µM erastin, 2% ascites, and 1mM bezafibrate (PPARα agonist), 0.8mM C-75 Trans (FASN inhibitor), 100µM Sulfo-N-succinimidyl oleate (SSO) (CD36 inhibitor), 50µM MF-438 (SCD1 inhibitor), and 80µM BMS-309403 (FABP4 inhibitor) ( n provided in panel, 48 hours). B-D , ( B ) TYKNU, ( C ) TOV21G, and ( D ) TOV112D cells were treated with 10µM erastin, 2% ascites, and 200µM ( B , D ) or 400µM ( C ) bezafibrate ( n =3, 48 hours). E-F , CAOV3 cells were treated with ( E ) 5µM IKE or ( F ) 2.5µM JKE-1674, 2% ascites, and 200µM bezafibrate ( n =3, 48 hours). G-H , CAOV3 cells were treated with ( G ) 10µM erastin, 2% ascites, 200µM bezafibrate, 60µM fenofibrate, 500µM ciprofibrate, or ( H ) 250nM GW7647 ( n provided in panel, 48 hours). I , CAOV3 cells were transduced with a PPRE-luciferase reporter constructed and treated with 2% ascites in the presence or absence of 800µM bezafibrate. Luminescence was measured after 1mM D-luciferin addition ( n =3, 16 hours). J , The log-transformed changes in CAOV3 PPARA target DEGs after 10% ascites exposure ( n =3, 16 hours, adj. P =0.01). The target genes were determined via the PPARGene database ( http://www.ppargene.org/index.php ), which provides all verified PPARα target genes. K , ascites from 7-week-old NSG mice were collected after IVIS imaging. All ascites was pooled together and 8% was added to CAOV3 cells with or without 2.5µM JKE-1674 treatment to assess ferroptosis protection ( n =3). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way (ANOVA), and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Transduction, Luciferase, Construct, Transformation Assay, Imaging, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , cell viability of CAOV3 cells was assessed via treatment with ( A ) 10µM erastin or ( B ) 250 nM RSL3, together with 2% ascites, in the presence or absence of 200µM bezafibrate ( n =3, 48 hours). C-D , Volcano plots of the different lipid species from lipidomic comparison analyses for CAOV3 cells treated 10% ascites ( C ) with or ( D ) without 200µM bezafibrate for 16 hours ( n =5, adj. P =0.01). E , Venn diagram comparison of lipid changes between ascites-only versus ascites-and-bezafibrate treated cells. The heat map depicts the class composition of the 243 lipids that remain unchanged with the ascites-and-bezafibrate treatment. F-G , cells were treated with 10% ascites in the presence or absence of 200µM bezafibrate and lipid droplet levels were measured via flow cytometry analysis of BODIPY TM 493/503 staining ( n =3, 16 hours). H , micro-organospheres (MOS) developed from an OVCA patient were treated with 20% ascites and 50µM IKE, and 1mM bezafibrate or 1mM ciprofibrate to assess resensitivity to ferroptosis ( n =5, 72 hours). I-J , CAOV3 transduced with lentivirus expressing luciferase were treated with either 800µM bezafibrate, 10µM JKE-1674, or both, 5 hours before being injected IP into 6-week-old NSG mice. Tumor growth was then ( I ) visualized and ( J ) measured as total photon flux (p/s) 7 days after injection via IVIS bioluminescent imaging ( n =7). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance for C-D was assessed using the two-tailed Student’s t -test method ( D , I ) and P values were adjusted using the Benjamini-Hochberg correction method ( C-D ). All other statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Comparison, Flow Cytometry, Staining, Transduction, Expressing, Luciferase, Injection, Imaging, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A , Volcano plot of the DEGs of bezafibrate treatment (200µM, 16 hours) from RNA-seq analysis of CAOV3 cells ( n =3, adj. P =0.01). B , Venn diagram comparison of a list of oppositely expressed genes from ascites-treated vs. bezafibrate-treated cells. C-D , western blot analysis of HMGCS2 protein expression upon 10% ascites treatment from three patients with or without 200µM bezafibrate for 16 hours ( n =3). E-F , ferroptosis sensitivity was compared between CAOV3 wildtype (WT) and HMGCS2 KO cells with ( E ) erastin or ( F ) RSL3 treatment ( n =3, 24 hours). G , HMGCS2 expression was restored in HMGCS2 KO cells with hmgcs2 OE plasmid and ferroptosis sensitivity was compared with 10µM erastin or 125nM RSL3 treatment for 24 hours ( n =3). H-I , lipid peroxidation was measured via flow cytometry analysis of BODIPY TM 581/591 C11 staining in HMGCS2 KO cells that were treated with 5µM erastin for 20 hours ( n =3). J-K , HMGCS2 OE cells were treated with 2% ascites for 16 hours and lipid droplet levels were measured via flow cytometry analysis of BODIPY TM 493/503 staining ( n =3). L , cell viability was assessed via treatment with 10µM erastin in the presence or absence of 20µM baicalin with 2% ascites ( n =3, 24 hours). M-N , cells were treated with 10% ascites in the presence or absence of 25µM baicalin (24 hours) and lipid droplet levels were measured as described ( N ) ( n =3). O , cell viability was assessed in cells treated with 10µM erastin in the presence or absence of 100µM malonyl CoA lithium for 24 hours ( n =3). P-Q , cells were treated with 100µM malonyl CoA lithium (16 hours) and lipid droplet levels were measured as described ( Q ) ( n =3). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance for D-O was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. Student’s t -test was performed for Q . All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: RNA Sequencing, Comparison, Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Staining, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , Cell viability was measured in CAOV3 cells transfected with ( A ) 50 nM si HMGCS2 or ( B ) 50 nM si TFRC and treated with 10µM erastin for 24 hours ( n =3). C-D , ( C ) HMGCS2 and ( D ) TFRC silencing was validated via qRT-PCR ( n =3). E-F , ( E ) HMGCS2 and ( F ) TFRC downregulation upon 10% ascites exposure for 16 hours was validated via qRT-PCR ( n =3). G-H , knockout of HMGCS2 was validated in HMGCS2 KO cells via ( G ) qRT-PCR and ( H ) western blot analysis ( n =3). I , HMGCS2 overexpression was validated via western blot analysis ( n =3). J-K , cells overexpressing HMGCS2 were treated with 5µM erastin for 20 hours and ( J ) lipid peroxidation was visualized and ( K ) measured with flow cytometry analysis of BODIPY TM 581/591 C11 staining ( n =3). L , patient overall survival data was extracted from the TCGA database and analyzed via the GEPIA 2 survival analysis tool. All cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Excluding L , statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Transfection, Quantitative RT-PCR, Knock-Out, Western Blot, Over Expression, Flow Cytometry, Staining, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , TFRC membrane expression was measured via flow cytometry analysis in cells treated with 2% ascites for 18 hours and 2µM RSL3 and 800µM bezafibrate for 2 hours ( n =3). C , labile iron levels were measured in cells treated with 2% ascites for 18 hours, and 2µM RSL3 and 800µM bezafibrate for 2 hours ( n =3). Labile iron was measured via calculating the change in Calcein-AM mean fluorescence intensity after treatment with 0.05µM Calcein-AM (15 minutes) and 100µM deferoxamine mesylate (DFO) (1 hour). D , heat map of the free fatty acid (FFA) profile in OVCA ascites, measured via FFA lipidomic profiling ( n =8). E , cell viability was assessed in CAOV3 cells that were treated with 10µM erastin in the presence of either 250µM oleic acid, 150µM linoleic acid or both (250µM, 150µM) ( n =3, 24 hours). F , cell viability was assessed in cells treated with 10µM erastin with or without 200µM bezafibrate and 50µM oleic acid for 24 hours ( n =3). G-I , TFRC membrane expression ( G-H ) and labile iron levels ( I ) were measured with using 50µM oleic acid in place of ascites ( n =3). J , mechanism of action model for ascites protection against ferroptosis and its mitigation by bezafibrate. Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Membrane, Expressing, Flow Cytometry, Fluorescence, Glo Assay, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Melatonin Alleviates Acute Sleep Deprivation-Induced Memory Loss in Mice by Suppressing Hippocampal Ferroptosis

doi: 10.3389/fphar.2021.708645

Figure Lengend Snippet: Effect of melatonin on iron accumulation and iron transporter proteins in the hippocampus of sleep-deprived mice. (A) Relative IOD of iron-positive cells in the hippocampal CA1, CA3, and DG areas ( n = 6). (B) Micrographs depict iron labeling in mouse hippocampal sections. The iron-staining results were processed using ImageJ. Iron deposits were observed as red granules. Bar = 50 μm. (C–E) Relative mRNA levels of iron transporter proteins TFR1, DMT1 and FPN in the hippocampus ( n = 6). (F–I) Relative protein levels of iron transporter proteins TFR1, DMT1 and FPN in the hippocampus ( n = 6). (J) Relative cell viability ( n = 5). (K–L) Relative mRNA levels of iron transporter proteins TFR1, DMT1 and FPN in HT-22 cells exposed to Erastin and melatonin or Fer-1 ( n = 6). (N–Q) Relative protein levels of iron transporter proteins TFR1, DMT1 and FPN in HT-22 cells exposed to Erastin and melatonin or Fer-1 ( n = 6). Differences were assessed using one-way ANOVA. The result represents the mean ± standard error of the mean. Values not sharing a common superscript letter differ significantly at p < 0.05; those with the same letter do not differ significantly ( p ≥ 0.05). CON: control group, SD: sleep deprivation group, SD + L-Mel: SD + low melatonin (20 mg/kg) supplement group, SD + H-Mel: SD + high melatonin (40 mg/kg) supplement group.

Article Snippet: Cells were plated before drug treatment in a 6- or 96-well plate at 10 6 or 10 4 cells/well, respectively, and cultured in DMEM with 10% FBS (complete medium) for 6 h. The cells were then cultured in DMEM without FBS (basal medium) for 12 h. The ferroptosis inducer Erastin (E7781; Sigma, United States), the ferroptosis inhibitor Fer-1, the MT2-selective Mel receptor antagonist 4P-PDOT (1034; Tocris Bioscience, Bristol, United Kingdom), the ERK inhibitor PD98059 (1213; Tocris Bioscience, Bristol, United Kingdom) and the Nrf2 inhibitor ML385 (SML1833; Sigma, United States) were each dissolved in DMSO.

Techniques: Labeling, Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Melatonin Alleviates Acute Sleep Deprivation-Induced Memory Loss in Mice by Suppressing Hippocampal Ferroptosis

doi: 10.3389/fphar.2021.708645

Figure Lengend Snippet: Effect of melatonin on antioxidative activation and lipid peroxidation in the hippocampus of sleep-deprived mice. (A) Relative quantification of the ROS assay in the hippocampus tissue ( n = 6). (B) Fluorescence staining of ROS in HT-22 cells exposed to Erastin and melatonin or Fer-1. Green represents ROS, while blue represents DAPI. Bar = 200 μm. (C) Relative quantification of intracellular ROS level in the HT-22 cells exposed to Erastin and melatonin or Fer-1 (n = 5). (D–F) Relative levels of GPX4, SOD and MDA in the hippocampus ( n = 6). (G-I) Relative levels of GPX4, SOD, and MDA in HT-22 cells exposed to Erastin and melatonin or Fer-1 ( n = 6). Differences were assessed by one-way ANOVA. The result represents the mean ± standard error of the mean. Values not sharing a common superscript letter differ significantly at p < 0.05; those with the same letter do not differ significantly ( p ≥ 0.05). CON: control group, SD: sleep deprivation group, SD + L-Mel: SD + low melatonin (20 mg/kg) supplement group, SD + H-Mel: SD + high melatonin (40 mg/kg) supplement group.

Article Snippet: Cells were plated before drug treatment in a 6- or 96-well plate at 10 6 or 10 4 cells/well, respectively, and cultured in DMEM with 10% FBS (complete medium) for 6 h. The cells were then cultured in DMEM without FBS (basal medium) for 12 h. The ferroptosis inducer Erastin (E7781; Sigma, United States), the ferroptosis inhibitor Fer-1, the MT2-selective Mel receptor antagonist 4P-PDOT (1034; Tocris Bioscience, Bristol, United Kingdom), the ERK inhibitor PD98059 (1213; Tocris Bioscience, Bristol, United Kingdom) and the Nrf2 inhibitor ML385 (SML1833; Sigma, United States) were each dissolved in DMSO.

Techniques: Activation Assay, Quantitative Proteomics, ROS Assay, Fluorescence, Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Melatonin Alleviates Acute Sleep Deprivation-Induced Memory Loss in Mice by Suppressing Hippocampal Ferroptosis

doi: 10.3389/fphar.2021.708645

Figure Lengend Snippet: Effect of melatonin on MT2/ERK/Nrf2 signaling in the HT-22 cells exposed to Erastin. The HT-22 cells exposed to Erastin and melatonin or Fer-1 were pretreated with 4P-PDOT, PD98059 and ML385, respectively. (A) Relative cell viability (%) ( n = 5). (B) Fluorescence staining of ROS. Bar = 200 μm. (C) Relative quantification of intracellular ROS level ( n = 5). (D–J) Relative protein levels of p -ERK1/2, Nrf2, GPX4, TFR1, DMT1 and FPN normalized to β-actin ( n = 6). (K–L) SOD and MDA levels ( n = 6). (M–O) Relative mRNA levels of iron transporter proteins (TFR1, DMT1 and FPN) ( n = 6). Differences were assessed using one-way ANOVA. The result represents the mean ± standard error of the mean. Values not sharing a common superscript letter differ significantly at p < 0.05; those with the same letter do not differ significantly ( p ≥ 0.05).

Article Snippet: Cells were plated before drug treatment in a 6- or 96-well plate at 10 6 or 10 4 cells/well, respectively, and cultured in DMEM with 10% FBS (complete medium) for 6 h. The cells were then cultured in DMEM without FBS (basal medium) for 12 h. The ferroptosis inducer Erastin (E7781; Sigma, United States), the ferroptosis inhibitor Fer-1, the MT2-selective Mel receptor antagonist 4P-PDOT (1034; Tocris Bioscience, Bristol, United Kingdom), the ERK inhibitor PD98059 (1213; Tocris Bioscience, Bristol, United Kingdom) and the Nrf2 inhibitor ML385 (SML1833; Sigma, United States) were each dissolved in DMSO.

Techniques: Fluorescence, Staining, Quantitative Proteomics

Journal: Frontiers in Pharmacology

Article Title: Melatonin Alleviates Acute Sleep Deprivation-Induced Memory Loss in Mice by Suppressing Hippocampal Ferroptosis

doi: 10.3389/fphar.2021.708645

Figure Lengend Snippet: Hypothetical diagram of how melatonin improves SD-induced hippocampal ferroptosis. Exogenous melatonin likely alleviates hippocampal ferroptosis caused by acute SD through by binding to the MT2 receptor and activating ERK/Nrf2 signaling, thereby improving lipid peroxidation and iron transporter disorder. ARE, antioxidant response element; CP, ceruloplasmin; DMT1, divalent metal transporter 1; ERK1/2, extracellular regulated protein kinases; FPN, ferroportin; GPX4, glutathione peroxidase 4; Keap1, Kelch-like ECH-associated protein 1; Mel, melatonin; ML385, Nrf2 inhibitor; MDA, malondialdehyde; MT2, melatonin receptor 2; Nrf2, nuclear factor erythroid 2-related factor 2; PD98059, ERK inhibitor; ROS, reactive oxygen species; SD, sleep deprivation; SOD, superoxide dismutase; STEAP3, 6-transmembrane epithelial antigen of the prostate 3; TFR1, transferrin receptor 1; 4P-PDOT, 4-phenyl-2-propionamidotetralin.

Article Snippet: Cells were plated before drug treatment in a 6- or 96-well plate at 10 6 or 10 4 cells/well, respectively, and cultured in DMEM with 10% FBS (complete medium) for 6 h. The cells were then cultured in DMEM without FBS (basal medium) for 12 h. The ferroptosis inducer Erastin (E7781; Sigma, United States), the ferroptosis inhibitor Fer-1, the MT2-selective Mel receptor antagonist 4P-PDOT (1034; Tocris Bioscience, Bristol, United Kingdom), the ERK inhibitor PD98059 (1213; Tocris Bioscience, Bristol, United Kingdom) and the Nrf2 inhibitor ML385 (SML1833; Sigma, United States) were each dissolved in DMSO.

Techniques: Binding Assay

Journal: Molecular Pain

Article Title: N-Acetylcysteine causes analgesia in a mouse model of painful diabetic neuropathy

doi: 10.1177/1744806920904292

Figure Lengend Snippet: NAC-induced analgesia in the STZ model of painful diabetic neuropathy. Blood glucose levels in mice receiving a single injection of saline or STZ (200 mg/kg, i.p.) are shown in (a), where values are means ± S.E.M. of 7–10 mice. *p < 0.05 (Student’s t-test; t = −8.279). Reductions of mechanical pain thresholds in the same mice at 14 and 21 days following STZ injection are shown in (b), where values are also means ± S.E.M. *p < 0.05 (one-way ANOVA for repeated measures + Duncan method; F (1,13) = 77.224). The effect of a single injection of NAC or pregabalin on mechanical pain thresholds in diabetic mice and non-diabetic control mice are shown in (c), where values are means ± S.E.M. of 7–10 mice. *p < 0.05 (one-way ANOVA + Duncan method applied only to the groups of diabetic mice; F (2,18) = 4.562). The effect of repeated administrations of saline or NAC (100 mg/kg, i.p., once a day for 7 days starting from 21 days after STZ injection) on mechanical pain thresholds are shown in (d and e). In (d), all groups of mice were injected once with saline, LY341495, or sulfasalazine at the end of the chronic treatment with saline or NAC (see Methods). Values are means ± S.E.M. of 6–7 mice per group. p < 0.05 versus the control group receiving repeated injections of saline followed by a single injection of saline (*); or versus the group treated with NAC and a single injection of saline (#) (one-way ANOVA + Duncan method; F (5,35) = 3.364). In (e), all groups of mice were injected once with vehicle, erastin, sorafenib, PD0325901, capsazepine, and mementine+MTEP at the end of the chronic treatment with saline or NAC. Values are means ± S.E.M. of 4–10 mice per group. p < 0.05 versus the control group receiving repeated injections of saline followed by a single injection of vehicle (*); or versus the group treated with NAC and a single injection of vehicle (#); or versus the control group receiving repeated injections of saline followed by a single injection of vehicle ($) (one-way ANOVA + Duncan method; F (11,73) = 7.947). Blood glucose levels in mice receiving a single injection of saline or NAC (100 mg/kg, i.p.) are shown in (f), where values are means ± S.E.M. of 9 mice. In (g), four groups of mice were injected once with vehicle or JNJ47965567 (30 mg/kg) at the end of the chronic treatment with saline or NAC. Values are means ± S.E.M. of 7–10 mice per group. p < 0.05 versus all other groups (one-way ANOVA + Duncan method; F (3,28) = 18.643). NAC: N-acetylcysteine; STZ: streptozotocin.

Article Snippet: NAC, sulfasalazine, and STZ were purchased from Sigma Aldrich (St. Louis, MO); (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY341495), pregabalin, erastin, sorafenib, PD0325901, JNJ479655567, capsazepine, memantine, and 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP) were purchased from Tocris Cookson (Avonmouth, Bristol, UK).

Techniques: Injection, Saline, Control

Journal: Cancer research

Article Title: Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer

doi: 10.1158/0008-5472.CAN-20-3477

Figure Lengend Snippet: (A-D) 3D Matrigel drop invasion assay for DU145, PC3 and C4-2 cells upon erastin (A, B) or RSL3 (C, D) treatment. DU145 and PC3 matrigel drops were treated with erastin (1.25 μM) or RSL3 (125 nM). C4-2 matrigel drops were treated with higher doses of erastin (5 μM) or RSL3 (500 nM) because of their sensitivity to the compounds at higher doses on viability assay (Figure 2A and ​andB).B). Media and treatments were exchanged every three days for six days incubation. The distances of migrated cells away from edge of the matrigel drop were measured as migration (μm) on Day 6. Experiments were performed in duplicate with triplicate wells. Representative experiments and images are shown. Scale bars represent 200 μm. (E-F) Migration assay for DU145 and PC3 cells. Cells were pretreated with erastin (1.25 μM) or RSL3 (125 nM) for 48 hours. Then, 5×104 viable cells were plated into transwell chambers for 20 hours upon erastin (1.25 μM) or RSL3 (125 nM) treatment, then fixed and stained with methanol and 0.01% crystal violet solution. Scale bar=1 mm. Experiments were performed in duplicate with two wells for each condition. Representative experiments and images are shown. For all experiments, DMSO was used as a vehicle control. For treated cells, the relative percentage of migrated cells was calculated relative to the DMSO treated control. Statistical analysis was performed with Student’s t-test (* P<0.05, **P<0.01, *** P<0.001, **** P<0.0001, ns-no significance) and error bars represent mean ± SEM.

Article Snippet: The compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM concentration and stored at −20° C. For in vivo experiments, erastin was obtained from BOC Sciences and (1S,3R)-RSL3 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques: Invasion Assay, Viability Assay, Incubation, Migration, Staining, Control

Journal: Cancer research

Article Title: Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer

doi: 10.1158/0008-5472.CAN-20-3477

Figure Lengend Snippet: (A, B). Viability (%) of prostate cancer cell lines (DU145, PC3, ARCaP, 22RV1, C4-2, LNCaP and H660) following erastin (0, 1.25, 2.5, 5, 10, and 20 μM) (A) or RSL3 (0, 0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 μM) (B) treatment for 72 hours. Experiments were repeated twice independently to verify the reproducibility of the data. Representative experiments are shown. (C, D) ROS measurement by flow cytometry. Cells were treated with erastin (5 μM) (C) or RSL3 (1 μM) (D) for 6 hours followed by incubation with H2DCF for 20 min at 37°C. Relative fluorescence was normalized to untreated control cells and represented as relative percentage of ROS production. Experiments were performed in triplicate and shown. (E, F) Colony formation assay. Prostate cancer cells were grown for nine days in presence of erastin (5 μM) (E) or RSL3 (500 nM) (F). Representative experiments and images are shown. Media containing the indicated compounds was changed every three days. Three independent experiments were performed with triplicate wells. Representative experiments and images are shown. Scale bars represent 4 mm. * P<0.05, **P<0.01, *** P<0.001, **** P<0.0001, ns-no significance, Student’s t-test. Error bars represent mean ± SEM.

Article Snippet: The compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM concentration and stored at −20° C. For in vivo experiments, erastin was obtained from BOC Sciences and (1S,3R)-RSL3 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques: Flow Cytometry, Incubation, Fluorescence, Control, Colony Assay

Journal: Cancer research

Article Title: Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer

doi: 10.1158/0008-5472.CAN-20-3477

Figure Lengend Snippet: (A-D) Colony formation assay. (A, B) C4-2 cells were grown for nine days in presence of erastin (2 μM), enzalutamide (2 μM), abiraterone acetate (2 μM), erastin plus enzalutamide, and erastin plus abiraterone acetate. Media containing the compounds was exchanged every three days. Colonies were then fixed in methanol and stained with crystal violet. After washing and drying plates, plates were scanned on Celigo Imaging Cytometer (Nexcelom Bioscience) and the percentage of the well covered by colonies was quantified in ImageJ. (C, D) Colony formation assay for C4-2 cells upon treatment with RSL3 (50 nM), enzalutamide (2 μM), abiraterone acetate (2 μM), RSL3 plus enzalutamide, and RSL3 plus abiraterone acetate for nine days. Three independent experiments were performed with triplicate wells. Representative experiments and images are shown. Scale bars represent 4 mm. (E-H) 3D Matrigel drop invasion assay. (E, F) C4-2 cells were plated in matrigel drop invasion assay and treated with erastin (5 μM), enzalutamide (5 μM), abiraterone acetate (5 μM), erastin plus enzalutamide, and erastin plus abiraterone acetate (G, H) C4-2 prostate cancer cells were plated in matrigel drop invasion assay and treated with RSL3 (500 nM), enzalutamide (5 μM), abiraterone acetate (5 μM), RSL3 plus enzalutamide, and RSL3 plus abiraterone acetate. Media and treatment were exchanged every three days for six days. Radial migration distance (μm) was measured on Day 6. Experiments were performed in duplicate with triplicate wells. Statistical analysis was performed with Student’s t-test (* P< 0.05, **P<0.01, *** P < 0.001, **** P<0.0001, ns-no significance) and error bars represent mean ± SEM.

Article Snippet: The compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM concentration and stored at −20° C. For in vivo experiments, erastin was obtained from BOC Sciences and (1S,3R)-RSL3 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques: Colony Assay, Staining, Imaging, Cytometry, Invasion Assay, Migration

Journal: Cancer research

Article Title: Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer

doi: 10.1158/0008-5472.CAN-20-3477

Figure Lengend Snippet: (A) Schematic diagram of experimental design using erastin treatment. 5×105 DU145, ARCaP or PC3 (5×105) and NCI-H660 (1×106) cells were mixed in 50 μl of 80% matrigel and implanted subcutaneously (sc) into the flanks of male NSG mice. When tumors averaged 50 to 80 mm3, mice were randomized into vehicle or erastin treatment (20 mg/kg, IP, daily) groups. (B) DU145, ARCaP, PC3, and H660 tumors volumes were measured by caliper (1/2 × (Length × Width × Height)) every three days and presented as fold change over the tumor volume at Day 1. (C) Tumor weights (g) were measured following tumor excision at the experimental end point and graphed as violin plots. (D) Schematic diagram of experimental design upon RSL3 treatment. (E) DU145 and PC3 (5×105) cells were mixed in 80% Matrigel and implanted subcutaneously into flank of NSG male mice. Once the average of tumor volume reached to 50 to 80 mm3, mice were randomized into vehicle or RSL3 treatment (100 mg/kg, IP, biweekly) groups. Tumor volumes (1/2 (Length × Width × Height)) were measured every third day and shown as fold change over Day 1 tumor volume. (F) Tumor weights (g) were measured at the experimental end point. Statistical analysis was performed with Student’s t-test at each time point (* P<0.05, **P<0.01, *** P<0.001, and **** P<0.0001). Error bars signify mean ± SEM.

Article Snippet: The compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM concentration and stored at −20° C. For in vivo experiments, erastin was obtained from BOC Sciences and (1S,3R)-RSL3 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques:

Journal: Cancer research

Article Title: Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer

doi: 10.1158/0008-5472.CAN-20-3477

Figure Lengend Snippet: (A) Schematic diagram of prostate cancer xenograft models upon treatment with erastin (20 mg/kg, IP, daily) and enzalutamide (10 mg/kg, oral gavage, daily). (B) 5×105 C4-2 cells mixed with 80% Matrigel, were implanted bilaterally subcutaneously into the flanks of male NSG mice. Once the average of tumors volume reached to 50 to 80 mm3, mice were randomized into vehicle (n=6), erastin (n=6), enzalutamide (n=6), and erastin plus enzalutamide (n=6) treatment groups. Tumor volumes (1/2 (Length × Width × Height) were measured every third day and represented as fold change over tumor volume at Day 1. (C) Tumor weights (g) were measured after tissue resection at experimental end point. (D) Animal weights were measured every three days upon erastin and enzalutamide combination treatment and plotted. (E) Schematic diagram of prostate cancer xenograft models upon RSL3 (100 mg/kg, IP, twice per week) and enzalutamide (10 mg/kg, oral gavage, daily) treatments. (F) 5×105 C4-2 cells mixed with 80% matrigel, were implanted bilaterally subcutaneously into the flanks of NSG male mice. Mice with tumors with average volume of 50 to 80 mm3, were randomized into vehicle (n=7), RSL3 (n=5), enzalutamide (n=5), and RSL3 plus enzalutamide (n=5) treatment groups. Tumor volumes (1/2 × Length × Width × Height) were measured every 3 days and are shown as fold change when compared to Day 1. (G) Tumor weights (g) at experimental end point are shown. (H) Animal weights were measured every 3 days over the treatment course and plotted. Statistical analysis was performed with Student’s t-test (* P<0.05, **P<0.01, *** P<0.001, and **** P<0.0001) and error bars represent mean ± SEM.

Article Snippet: The compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM concentration and stored at −20° C. For in vivo experiments, erastin was obtained from BOC Sciences and (1S,3R)-RSL3 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Techniques: