hmgcs2 orf clone  (GenScript corporation)

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: For transient overexpression of HMGCS2 , 100ng of mouse hmgcs2 ORF clone with a pCMV6-Entry backbone (Origene, MG208162) was diluted in Opti-MEM™ I (9µL).

Techniques: RNA Sequencing Assay, 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: For transient overexpression of HMGCS2 , 100ng of mouse hmgcs2 ORF clone with a pCMV6-Entry backbone (Origene, MG208162) was diluted in Opti-MEM™ I (9µL).

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 , 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: For generating a stable HMGCS2 overexpressing cell line, the human HMGCS2 ORF clone with a pCMV6-Entry backbone (Origene, RC208128) was cut and ligated into lentiviral vector (Origene, PS100069) according to the manufacturer’s guidelines.

Techniques: RNA Sequencing Assay, 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: For generating a stable HMGCS2 overexpressing cell line, the human HMGCS2 ORF clone with a pCMV6-Entry backbone (Origene, RC208128) was cut and ligated into lentiviral vector (Origene, PS100069) according to the manufacturer’s guidelines.

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 , 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: For generating a stable HMGCS2 overexpressing cell line, the human HMGCS2 ORF clone with a pCMV6-Entry backbone (Origene, RC208128) was cut and ligated into lentiviral vector (Origene, PS100069) according to the manufacturer’s guidelines.

Techniques: RNA Sequencing Assay, 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: For generating a stable HMGCS2 overexpressing cell line, the human HMGCS2 ORF clone with a pCMV6-Entry backbone (Origene, RC208128) was cut and ligated into lentiviral vector (Origene, PS100069) according to the manufacturer’s guidelines.

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