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: Drug Metabolism and Disposition

Article Title: Pediatric Cytochrome P450 Activity Alterations in Nonalcoholic Steatohepatitis

doi: 10.1124/dmd.117.077644

Figure Lengend Snippet: Metabolic AUC ratio of losartan in CYP2C9 polymorphic subjects Data are presented as mean (S.D., n ).

Article Snippet: Paraxanthine, paraxanthine-D3, losartan carboxylic acid (E3174), and 5-hydroxyomeprazole were purchased from Toronto Research Chemicals (Toronto, ON, Canada).

Techniques: Clinical Proteomics, Control, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine

doi: 10.3390/ijms22105160

Figure Lengend Snippet: OXA (17-33) A increases [Ca 2+ ] i via choline-Sigma-1R-dependent mechanism. ( A ) Illustration of average Ca 2+ transients (± SD) induced by OXA (17-33) (10 nM) alone (left) and in the presence of VU0155056 (1 μM), PLD inhibitor (middle), and BD1047 (50 µM) or NE-100 (5 µM), Sigma-1R antagonists (right). ( B ) Comparison of the amplitude of the increase in [Ca 2+ ] i (average + SD) in each condition. Inhibition of PLD or antagonism of Sig-1R reduces the Ca 2+ response elicited by OXA (17-33) (10 nM); p < 0.05 as compared to amplitude of [Ca 2+ ] i increase produced by OXA (17-33) (*) or produced in the presence of the inhibitors ( # ) ( n = 6 neurons/condition).

Article Snippet: VU0155056, a PLD inhibitor [ ], was purchased from Avanti Polar Lipids (Alabaster, AL, USA).

Techniques: Comparison, Inhibition, Produced