Journal: International Journal of Molecular Sciences

Article Title: Idi1 and Hmgcs2 Are Affected by Stretch in HL-1 Atrial Myocytes

doi: 10.3390/ijms19124094

Figure Lengend Snippet: Selected gene expressions revealed by PCR assay for fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism: Comparison of stretched and non-stretched HL-1 atrial myocytes.

Article Snippet: The pCMV6- Hmgcs2 (Myc-DDK-tagged) was cloned by OriGene (Rockville, MD, USA) with Sgf I-Rsr II as restriction sites.

Techniques: Comparison

Journal: International Journal of Molecular Sciences

Article Title: Idi1 and Hmgcs2 Are Affected by Stretch in HL-1 Atrial Myocytes

doi: 10.3390/ijms19124094

Figure Lengend Snippet: “Superpathway of cholesterol biosynthesis” in the Ingenuity Pathway Analysis. The only genes in the “superpathway of cholesterol biosynthesis” were Idi1 and Hmgcs2 .

Article Snippet: The pCMV6- Hmgcs2 (Myc-DDK-tagged) was cloned by OriGene (Rockville, MD, USA) with Sgf I-Rsr II as restriction sites.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Idi1 and Hmgcs2 Are Affected by Stretch in HL-1 Atrial Myocytes

doi: 10.3390/ijms19124094

Figure Lengend Snippet: Quantitative determination of mRNAs of ( A ) isopentenyl-diphosphate delta isomerase ( Idi1 ) and ( B ) 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 ( Hmgcs2 ), by real-time RT-PCR in non-stretched and stretched HL-1 atrial myocytes. * p < 0.05.

Article Snippet: The pCMV6- Hmgcs2 (Myc-DDK-tagged) was cloned by OriGene (Rockville, MD, USA) with Sgf I-Rsr II as restriction sites.

Techniques: Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Idi1 and Hmgcs2 Are Affected by Stretch in HL-1 Atrial Myocytes

doi: 10.3390/ijms19124094

Figure Lengend Snippet: Quantitative determination of mRNAs by real-time RT-PCR in the HL-1 atrial myocytes. ( A ) Genetic silencing of Idi1 mRNA; ( B ) genetic modification (plasmid transfection, pCMV6- Hmgcs2 ) of Hmgcs2 mRNA. * p < 0.05.

Article Snippet: The pCMV6- Hmgcs2 (Myc-DDK-tagged) was cloned by OriGene (Rockville, MD, USA) with Sgf I-Rsr II as restriction sites.

Techniques: Quantitative RT-PCR, Modification, Plasmid Preparation, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Idi1 and Hmgcs2 Are Affected by Stretch in HL-1 Atrial Myocytes

doi: 10.3390/ijms19124094

Figure Lengend Snippet: Immunoblot results for DDK ( A ), Hmgcs2 ( B ), and Idi1 ( C ) proteins in the non-stretched group, stretched group, and stretched group with plasmid transfection of pCMV6- Hmgcs2 (Myc-DDK-tagged) ( A , B ), and the stretched group with RNA interference of Idi1 ( C ). Plasmid transfection of pCMV6- Hmgcs2 (Myc-DDK-tagged) in stretched HL-1 atrial myocytes was confirmed by significantly increased expressions of DDK ( A ) and Hmgcs2 ( B ) in stretched HL-1 atrial myocytes that received plasmid transfection. Silencing of Idi1 in stretched HL-1 atrial myocytes was confirmed by significantly decreased expression of Idi1 ( C ) in stretched HL-1 atrial myocytes that had received RNA interference. * p < 0.05.

Article Snippet: The pCMV6- Hmgcs2 (Myc-DDK-tagged) was cloned by OriGene (Rockville, MD, USA) with Sgf I-Rsr II as restriction sites.

Techniques: Western Blot, Plasmid Preparation, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Idi1 and Hmgcs2 Are Affected by Stretch in HL-1 Atrial Myocytes

doi: 10.3390/ijms19124094

Figure Lengend Snippet: Left panel: Comparison of fraction of HL-1 atrial myocytes expressing Nile red in non-stretched HL-1 atrial myocytes, stretched HL-1 atrial myocytes, and stretched HL-1 atrial myocytes with genetic modification (plasmid transfection, pCMV6- Hmgcs2 ) of Hmgcs2 mRNA. Right panel: Fluorescence intensity measurements for Nile red expression in non-stretched HL-1 atrial myocytes, stretched HL-1 atrial myocytes, and stretched HL-1 atrial myocytes with genetic modification of Hmgcs2 mRNA. * p < 0.05.

Article Snippet: The pCMV6- Hmgcs2 (Myc-DDK-tagged) was cloned by OriGene (Rockville, MD, USA) with Sgf I-Rsr II as restriction sites.

Techniques: Comparison, Expressing, Modification, Plasmid Preparation, Transfection, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Idi1 and Hmgcs2 Are Affected by Stretch in HL-1 Atrial Myocytes

doi: 10.3390/ijms19124094

Figure Lengend Snippet: Primer sequences for real-time PCR.

Article Snippet: The pCMV6- Hmgcs2 (Myc-DDK-tagged) was cloned by OriGene (Rockville, MD, USA) with Sgf I-Rsr II as restriction sites.

Techniques:

Journal: Cell Death and Differentiation

Article Title: Ketogenesis contributes to intestinal cell differentiation

doi: 10.1038/cdd.2016.142

Figure Lengend Snippet: Knockdown of HMGCS2 attenuates spontaneous differentiation of Caco-2 cells. Caco-2 cells, stably transfected with control or HMGCS2 shRNA, were incubated 3, 6 and 12 days after confluency to differentiation. (a) Total RNA was extracted, and SI, KRT20 and p21Waf1 mRNA expression was assessed by real-time RT-PCR. (n=3, data represent mean±S.D.; *P<0.01 versus pre-confluent; #P<0.01 versus control shRNA). Data are from one of three independent experiments with similar results. (b) Cells were lysed and western blot analysis was performed using antibodies against p21Waf1, CDX2, villin, HMGCS2 and β-actin. The images are representative of three independent experiments

Article Snippet: Flag-tagged human HMGCS2 was from Origene (Rockville, MD, USA).

Techniques: Knockdown, Stable Transfection, Transfection, Control, shRNA, Incubation, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death and Differentiation

Article Title: Ketogenesis contributes to intestinal cell differentiation

doi: 10.1038/cdd.2016.142

Figure Lengend Snippet: HMGCS2 contributes to intestinal differentiation. (a) Caco-2 cells were transfected with empty vector (control) or transfected with Flag-HMGCS2 constructs. After 48 h, cells were lysed and extracted for protein. p21Waf1, CDX2, H3K9ac, HMGCS2, Flag-tagged HMGCS2 and β-actin were determined by western blotting. The images are representative of three independent experiments. p21Waf1, CDX2 and H3K9ac signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (n=3, data represent mean±S.D.; *P<0.01 versus control vector). (b and c) LS174T cells were infected with a recombinant adenovirus encoding the human HMGCS2 or vector control encoding GFP. After 48 h, cells were lysed and extracted for RNA and protein. (b) p21Waf1, CDX2, H3K9ac, HMGCS2 and β-actin were determined by western blotting. The images are representative of three independent experiments. p21Waf1, CDX2 and H3K9ac signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (n=3, data represent mean±S.D.; *P<0.01 versus GFP control). (c) CDX2 mRNA expression was assessed by real-time RT-PCR. (n=3, data represent mean±S.D.; *P<0.01 versus GFP control). Data are from one of three independent experiments with similar results. Overexpression of HMGCS2 inhibits HDAC and increases p21Waf1 and CDX2 expression in Caco-2 and LS174T cells. (d) Immunohistochemical analysis of HMGCS2 protein expression in normal human small intestine. Human normal small intestine sections were fixed and stained with primary anti-human HMGCS2 antibody. HMGCS2 is specifically expressed in the more differentiated region (i.e., villus; arrows). Scale bars=50 μm. The images are representative of five cases

Article Snippet: Flag-tagged human HMGCS2 was from Origene (Rockville, MD, USA).

Techniques: Transfection, Plasmid Preparation, Control, Construct, Western Blot, Infection, Recombinant, Expressing, Quantitative RT-PCR, Over Expression, Immunohistochemical staining, Staining

Journal: Cell Death and Differentiation

Article Title: Ketogenesis contributes to intestinal cell differentiation

doi: 10.1038/cdd.2016.142

Figure Lengend Snippet: Enhanced intestinal cell differentiation by ketone diet. Mice were fed with normal chow (n=5) or a ketogenic diet (n=5) for 14 days. (a) Small intestinal mucosal protein lysates were extracted for western blot detection of H3K9ac, MUC2, p21Waf1, p-S6, S6, HMGCS2 and β-actin protein expression. Each well represents a different mouse from the relevant group. (b) Representative Fast Red staining of the small intestine revealed an increase in IAP expression. (c) Representative AB staining of the small intestine revealed an increase in mucinous goblet cells in ketogenic diet-fed mice compared with control mice (arrows). (d) Quantification of AB-positive cells in control and ketogenic diet-fed mice. (n=15 (3 crypts per mouse)), data represent mean±S.D.; *P<0.01 versus control diet). (e) Representative IHC staining of the small intestine for LYZ showed the increase in Paneth cells (arrows) in ketogenic diet-fed mice compared with control mice. (f) Quantification of LYZ-positive cells in control and ketogenic diet-fed mice. (n=15 (3 crypts per mouse), data represent mean±S.D.; *P<0.01 versus control diet). (g) Representative IHC staining (arrows) for CDX2 demonstrated increased expression in the intestinal epithelium of ketogenic diet-fed mice compared with control mice. Scale bars=50 μm

Article Snippet: Flag-tagged human HMGCS2 was from Origene (Rockville, MD, USA).

Techniques: Cell Differentiation, Western Blot, Expressing, Staining, Control, Immunohistochemistry

Journal: Cell Death and Differentiation

Article Title: Ketogenesis contributes to intestinal cell differentiation

doi: 10.1038/cdd.2016.142

Figure Lengend Snippet: Cross-talk between mTORC1 and HMGCS2/βHB signaling. (a) HT29 cells were transfected with NTC or mTOR siRNA. After incubation for 48 h, cells were lysed and western blot analysis was performed using antibodies against HMGCS2, mTOR and β-actin. Knockdown of mTOR increased HMGCS2 expression in HT29 cells. The images are representative of three independent experiments. HMGCS2 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (n=3, data represent mean±S.D.; *P<0.01 versus NTC siRNA). (b) Mouse small intestinal mucosal protein extracted from mice treated without (control, n=3) or with rapamycin (n=3) for 6 days. Western blotting was performed for the expression of the indicated proteins. Each well represents a different mouse from the relevant group. Treatment with rapamycin significantly increased HMGCS2 expression in intestinal epithelium. (c) HT29 cells were treated with βHB at various dosages for 48 h. Cells were lysed and western blot analysis was performed using antibodies against p-S6 and S6. Treatment with βHB inhibited mTOR signaling as shown by the decreased phosphorylation of S6. The images are representative of three independent experiments. (d) LS174T cells were infected with a recombinant adenovirus encoding the human HMGCS2 or vector control encoding GFP. After 48 h, cells were lysed and western blot performed for the detection of the indicated proteins. Overexpression of HMGCS2 inhibited mTOR signaling as shown by the decreased expression of p-S6. p-S6 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to total S6. (n=3, data represent mean±S.D.; *P<0.01 versus GFP control). The images are representative of three independent experiments. (e) mTOR/HMGCS2/βHB pathway model. Inhibition of mTORC1 increases ketogenesis and contributes to intestinal cell differentiation. In contrast, increase in ketogenesis inhibits mTOR signaling and induces differentiation. mTORC1 acts cooperatively with HMGCS2/βHB to maintain intestinal homeostasis

Article Snippet: Flag-tagged human HMGCS2 was from Origene (Rockville, MD, USA).

Techniques: Transfection, Incubation, Western Blot, Knockdown, Expressing, Control, Phospho-proteomics, Infection, Recombinant, Plasmid Preparation, Over Expression, Inhibition, Cell Differentiation

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, 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