27ohc  (Santa Cruz Biotechnology)

Journal: Biochemical Journal

Article Title: Proteomic analysis reveals inhibition of mevalonate and glycolysis pathways in hepatocytes by 27-hydroxycholesterol

doi: 10.1042/BCJ20253035

Figure Lengend Snippet: ( A ) FACS plot showing frequency of apoptosis in AML12 cell populations following 5-FU (500 μM, positive control) and 27OHC (1, 2.5 μg/ml) treatment for 24 h. ( B ) TUNEL staining following 27OHC (2.5 μg/ml) treatment. DNase was used as a positive control. n =3 independent biological replicates for ( A, B ). ( C ) AML12 cells were incubated with different concentrations of 27OHC for 24 h. The cell viability was measured using the WST-1 assay ( n =4). Statistical significance is from the pooled data of the multiple independent experiments. Values are presented as the mean ± S.E.M. * P <0.05 and *** P <0.001. N.S indicates not significant. 5-FU, 5-fluorouracil.

Article Snippet: 27OHC was purchased from Tocris Bioscience (cat. no. 3907).

Techniques: Positive Control, TUNEL Assay, Staining, Incubation, WST-1 Assay

Journal: Biochemical Journal

Article Title: Proteomic analysis reveals inhibition of mevalonate and glycolysis pathways in hepatocytes by 27-hydroxycholesterol

doi: 10.1042/BCJ20253035

Figure Lengend Snippet: ( A ) Venn diagram of the 4028 identified proteins. ( B ) Volcano plot (left) displays the overall proteins identified with a P -value of <0.05. Overall, 38 and 26 proteins were significantly up-regulated and down-regulated, respectively, following 27OHC (2.5 μg/ml) treatment ( n =3). The right panel represents proteins with a P -value of <0.05 and a 2 log 2 FC cutoff. ( C ) Comparative canonical pathway analyses using Ingenuity Pathway Analysis (IPA). Orange and blue indicate canonical pathways with a positive or negative Z‐score value of ≥2.0, respectively, for pathway activation. ( D ) IPA of glycolysis signaling in the proteome. ( E ) IPA of cholesterol biosynthesis signaling in the proteome. ( F ) IPA of heme degradation signaling in the proteome. Red and green indicate the increased and decreased protein levels, respectively. Orange and blue indicate the predicted activation and inhibition, respectively.

Article Snippet: 27OHC was purchased from Tocris Bioscience (cat. no. 3907).

Techniques: Activation Assay, Inhibition

Journal: Biochemical Journal

Article Title: Proteomic analysis reveals inhibition of mevalonate and glycolysis pathways in hepatocytes by 27-hydroxycholesterol

doi: 10.1042/BCJ20253035

Figure Lengend Snippet: AML12 cells were incubated with the indicated concentrations of cholesterol ( A ) or 27OHC ( B ) for 24 h. The expression of Hmgs, Hmgr, Mvk, Mvd, Idi1, Fpps, and Srebp2 genes was measured using qPCR. The expression of each gene was normalized to that of Gapdh . ( C ) Mouse primary hepatocytes were incubated with 27OHC or the vehicle control for 24 h. The expression of indicated genes was measured using qPCR. ( D ) Following 12 h or 24 h incubation with 2.5 μg/ml 27OHC, total lysates were prepared from AML12 cells to detect precursor and mature SREBP2 proteins using immunoblot assay. ( E ) Total cholesterol levels in AML12 cells were quantified using filipin staining after incubation with 5 mM methyl-β-cyclodextrin (MCD) or 2.5 μg/ml 27OHC (scale bars, 50 μm). n =3 independent biological replicates. Values are presented as the mean ± S.E.M. * P <0.05, ** P <0.01, and *** P <0.001.

Article Snippet: 27OHC was purchased from Tocris Bioscience (cat. no. 3907).

Techniques: Incubation, Expressing, Control, Western Blot, Staining

Journal: Biochemical Journal

Article Title: Proteomic analysis reveals inhibition of mevalonate and glycolysis pathways in hepatocytes by 27-hydroxycholesterol

doi: 10.1042/BCJ20253035

Figure Lengend Snippet: AML12 cells were incubated with different doses of cholesterol ( A ) or 27OHC ( B ) for 24 h. The expression of Pfkl, Aldob, Tpi1, Pgk1, Pkm, and Pklr genes was measured using qPCR. ( C ) Mouse primary hepatocytes were incubated with 27OHC or vehicle control for 24 h. The expression of the Pfkl, Tpi1, and Pgk1 genes was measured using qPCR. ( D ) Inhibition of l -lactate production following treatment with 27OHC (2.5 μg/ml) for 48 h was assessed using the glycolysis assay kit. 2-DG was used as the positive control. ( E ) Serial measurements of the extracellular acidification rate (ECAR, left). Glycolytic capacity and glycolytic reserve of cell (right) were quantified following 27OHC treatment (2.5 μg/ml). n =3 independent biological replicates. Values are presented as the mean ± S.E.M. * P <0.05, ** P <0.01, and *** P <0.001.

Article Snippet: 27OHC was purchased from Tocris Bioscience (cat. no. 3907).

Techniques: Incubation, Expressing, Control, Inhibition, Positive Control

Journal: Biochemical Journal

Article Title: Proteomic analysis reveals inhibition of mevalonate and glycolysis pathways in hepatocytes by 27-hydroxycholesterol

doi: 10.1042/BCJ20253035

Figure Lengend Snippet: ( A ) Relative mRNA levels of Ho1 in AML12 cells treated with either vehicle or 2.5 µg/ml 27OHC for 24 h. ( B ) NRF2 and HO-1 protein levels following 27OHC (2.5 μg/ml) treatment of Nrf2 siRNA-transfected AML12 cells were assessed using immunoblotting. Actin was used as the loading control. ( C ) Flow cytometry analysis of reactive oxygen species (ROS) levels in AML12 cells treated with vehicle or 2.5 µg/ml 27OHC for 24 h. ROS levels were detected using DCFDA/FITC-A staining. Mean fluorescence intensity (MFI) indicates the proportion of cells with elevated ROS levels. ( D ) Total glutathione (top left), reduced glutathione (GSH, top right), oxidized glutathione (GSSG, bottom left), and GSH:GSSG ratio (bottom right) were determined in AML12 cells treated with either vehicle or 2.5 µg/ml 27ohc for 24 h. n =3 independent biological replicates. Values are presented as the mean ± S.E.M. ** P <0.01.

Article Snippet: 27OHC was purchased from Tocris Bioscience (cat. no. 3907).

Techniques: Transfection, Western Blot, Control, Flow Cytometry, Staining, Fluorescence

Journal: Biochemical Journal

Article Title: Proteomic analysis reveals inhibition of mevalonate and glycolysis pathways in hepatocytes by 27-hydroxycholesterol

doi: 10.1042/BCJ20253035

Figure Lengend Snippet: ( A ) Expression levels of endogenous (left) and transfected HIF-1α (right) were determined in whole-cell lysates following 27OHC (2.5 μg/ml) treatment. Quantitative analysis of HIF-1α expression was presented in a bar graph. β-Actin was used as the loading control. ( B and C ) AML12 cells were grown under normoxic or hypoxic (2% O 2 ) conditions for 24 h with 27OHC. ( B ) Intracellular ROS levels were detected using DCFDA/FITC-A staining. ( C ) Glycolytic gene expression under hypoxic conditions was analyzed. n =3 independent biological replicates. Values are presented as the mean ± S.E.M. * P <0.05, ** P <0.01, and *** P <0.001.

Article Snippet: 27OHC was purchased from Tocris Bioscience (cat. no. 3907).

Techniques: Expressing, Transfection, Control, Staining, Gene Expression

Journal: Biochemical Journal

Article Title: Proteomic analysis reveals inhibition of mevalonate and glycolysis pathways in hepatocytes by 27-hydroxycholesterol

doi: 10.1042/BCJ20253035

Figure Lengend Snippet: ( A ) Oil Red O staining was used to evaluate intracellular lipid accumulation in cells treated with vehicle, 27OHC, palmitic/oleic acid (PA/OA), or PA/OA in combination with 27OHC (2.5 μg/ml). Scale bars: 50 µm. Lower panel indicated the quantification of Oil Red O absorbance at 500 nm. Data are presented as fold change relative to vehicle control. n =3 independent biological replicates. Values are presented as the mean ± S.E.M. *** P <0.001. ( B ) Working model.

Article Snippet: 27OHC was purchased from Tocris Bioscience (cat. no. 3907).

Techniques: Staining, Control

Journal: Cells

Article Title: A Role for ER-Beta in the Effects of Low-Density Lipoprotein Cholesterol and 27-Hydroxycholesterol on Breast Cancer Progression: Involvement of the IGF Signalling Pathway?

doi: 10.3390/cells11010094

Figure Lengend Snippet: Effects of exogenous 27OHC and LDL on cell growth, migration/invasion, and EMT markers. Crystal violet staining proliferation assay results for ( A ) MCF-7 and ( B ) MDA-MB-231 after being dosed with LDL (80 and 100 μg/mL) for 48 h and with 27OHC (0.1 and 1 μM) for 48 h for ( C ) MCF-7 and ( D ) MDA-MB-231. A trans-well assay was used to detect cell migration and invasion after being dosed with LDL or 27OHC, the migrated cells were stained with crystal violet, and images were taken (×20 magnification). Quantification of ( E ) MCF-7 cell migration after a 24 h incubation period, ( F ) MDA-MB-231 cell migration after 6 h incubation period. Quantification of ( G ) MCF-7 cell invasion after 24 h incubation period and ( H ) MDA-MB-231 cell invasion after a 6 h incubation period. Western immunoblot analysis was performed to show the protein abundance of EMT markers, fibronectin, and vimentin, and their densitometric analyses were corrected in ( I ) MCF-7 and ( J ) MDA-MB-231 after being dosed with 27OHC (0.1 μM) and LDL (80 μg/mL). p -values were determined by using one-way statistical analysis in GraphPad Prism: ANOVA test plus least significant difference (LSD) post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001). Scale bar represents 100 μM.

Article Snippet: 27OHC (purity ≥ 99.9%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in absolute ethanol to a stock solution of 1000 μM, and stored at −80 °C.

Techniques: Migration, Staining, Proliferation Assay, Incubation, Western Blot, Quantitative Proteomics

Journal: Cells

Article Title: A Role for ER-Beta in the Effects of Low-Density Lipoprotein Cholesterol and 27-Hydroxycholesterol on Breast Cancer Progression: Involvement of the IGF Signalling Pathway?

doi: 10.3390/cells11010094

Figure Lengend Snippet: Cholesterol increases proliferation and migration through 27OHC production in breast cancer cells. Using the Muse cell analyser, we assessed the cell number for ( A ) MCF-7 and ( D ) MDA-MB-231 after being transfected with CYP27A1 siRNA and non-silencing RNA and being dosed with (LDL 80 μg/mL) for 48 h. A trans-well assay was used to detect cell migration and invasion after being transfected with CYP27A1 siRNA and dosed with (LDL 80 μg/mL). The migrated cells were stained with crystal violet, and images were taken (×20 magnification). The migrated cells were quantified for ( B ) MCF-7 after 24 h and ( E ) MDA-MB-231 after 6 h of incubation time. Western immunoblot analysis was performed to show the protein abundance of CYP27A1 MCF ( C ) for MCF-7 and ( F ) MDA-MB-231 with or without LDL and CYP27A1 silencing. Additionally, the relative fold changes of CYP27A1 against loading control β-actin were measured ( C , F ). p -values were determined by using the one-way statistical analysis of GraphPad Prism: ANOVA test plus least significant difference (LSD) post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001). Scale bar represents 100 μM.

Article Snippet: 27OHC (purity ≥ 99.9%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in absolute ethanol to a stock solution of 1000 μM, and stored at −80 °C.

Techniques: Migration, Transfection, Staining, Incubation, Western Blot, Quantitative Proteomics, Control

Journal: Cells

Article Title: A Role for ER-Beta in the Effects of Low-Density Lipoprotein Cholesterol and 27-Hydroxycholesterol on Breast Cancer Progression: Involvement of the IGF Signalling Pathway?

doi: 10.3390/cells11010094

Figure Lengend Snippet: Effects of 27-hydroxycholesterol on cell growth and migration/invasion in the presence or absence of ER-α. Using the Muse cell analyser, we assessed the cell number for ( A ) MCF-7 after being transfected with ER-α siRNA (20 nm) and non-silencing RNA (20 nm) and dosed with 27OHC (0.1 μM) for 48 h. ( B ) Western immunoblot analysis was performed to show the protein abundance of ER-α in MCF-7 with or without ER-α silencing and 27OHC. β-Actin was used as a loading control. A trans-well migration/invasion assay was used for the migrated cells after 24 h, the cells were stained with crystal violet, and images were taken (×20 magnification). We quantified ( C ) cell migration and ( D ) invasion in MCF-7 cells after being transfected with ER-α siRNA and dosed with 27OHC. ( E ) Densitometry of the Western blot showing the protein abundance of ER-α, LXR-β, and ER-β after normalization to the reference protein GAPDH and B-actin. p -values were determined by using the one-way statistical analysis of GraphPad Prism: ANOVA test plus least significant difference (LSD) post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001). Scale bar represents 100 μM.

Article Snippet: 27OHC (purity ≥ 99.9%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in absolute ethanol to a stock solution of 1000 μM, and stored at −80 °C.

Techniques: Migration, Transfection, Western Blot, Quantitative Proteomics, Control, Invasion Assay, Staining

Journal: Cells

Article Title: A Role for ER-Beta in the Effects of Low-Density Lipoprotein Cholesterol and 27-Hydroxycholesterol on Breast Cancer Progression: Involvement of the IGF Signalling Pathway?

doi: 10.3390/cells11010094

Figure Lengend Snippet: 27OHC increases cell migration/invasion in ER-α-negative breast cancer cells via ER-β. A trans-well assay was used to detect ( A ) migration and ( B ) invasion after 6 h for MDA-MB-231 after being transfected with ER-β siRNA (60 nm) and non-silencing RNA (60 nm) and dosed with 27OHC (0.1 μM). ( C ) Western blotting was used to detect the abundance of ER-β with or without with ER-β silencing. The densitometry quantification of ER-β was assessed after normalization to β-actin. ( D ) We quantified cell migration after being dosed with 27OHC (0.1 μM) and PHTPP (5 μM). The migrated and invaded cells were stained with crystal violet, and images were taken (×20 magnification). ( E ) Scatterplot analysis of the TCGA data of 504 invasive breast cancer carcinomas for the mRNA expression of ER-β represented as the median expression of CYP27A1. ( F ) Scatterplot analysis of the METABRIC data of 1193 invasive breast cancer carcinomas for the mRNA expression of IGF-I represented as median expression of CYP27A1. ( G ) Scatterplot analysis of the TCGA data of 504 invasive breast cancer carcinomas for the mRNA expression of CYP27A1 represented as the median expression of ER-β. Results are presented as mean +/− SEM in ( A – D ); in ( E ), the horizontal line presents the mean. p -values were determined by using the one-way statistical analysis of GraphPad Prism: one-way ANOVA followed by least significant difference (LSD) post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001) ( A – D ); ( E ) Mann–Whitney test. Scale bar represents 100 μM.

Article Snippet: 27OHC (purity ≥ 99.9%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in absolute ethanol to a stock solution of 1000 μM, and stored at −80 °C.

Techniques: Migration, Transfection, Western Blot, Staining, Expressing, MANN-WHITNEY

Journal: Cells

Article Title: A Role for ER-Beta in the Effects of Low-Density Lipoprotein Cholesterol and 27-Hydroxycholesterol on Breast Cancer Progression: Involvement of the IGF Signalling Pathway?

doi: 10.3390/cells11010094

Figure Lengend Snippet: Interactions between the IGF system and cholesterol metabolism in TNBC. Cell proliferation for ( A ) MDA-MB-231 treated with (LDL 80 μg/mL) in the presence or absence of a tyrosine kinase inhibitor (AG1024; 2 µM) for 48 h, assessed using a crystal violet proliferation assay. Cell migration for ( B ) MDA-MB-231 cells after being dosed with LDL with or without AG1024 (2 µM). The migrated cells were stained with crystal violet, and images were taken (×20 magnification). Western blotting results for ( C ) MDA-MB-231 after being dosed with 27OHC (0.1 µM) and LDL (80 µg/mL) for 48 h; their densitometry analyses of fold changes of IGF-IRβ against loading control β-actin. A radioimmunoassay was used to measure IGF-I concentrations in ( D ) MDA-MB-231 cells after being dosed with 27OHC (0.1 µM) and LDL (80 µg/mL) for 48 h, as well as ( E ) levels of IGF1 in the presence or absence of CYP27A1 in MDA-MB-231 after being dosed with LDL in the presence or absence of CYP27A1 for 48 h. ( F ) Scatterplot analysis of the TCGA data of 504 invasive breast cancer carcinomas for the mRNA expression of IGF-I represented as the median expression of CYP27A1. ( G ) Scatterplot analysis of the METABRIC data of 1193 invasive breast cancer carcinomas for the mRNA expression of IGF-I represented as the median expression of CYP27A1. Data representative of mean ± SEM ( n = 3). p -values were determined by using the one-way statistical analysis of GraphPad Prism: ANOVA test plus least significant difference (LSD) post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001) ( A – F ); ( G ) Mann–Whitney test. Scale bar represents 100 μM.

Article Snippet: 27OHC (purity ≥ 99.9%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in absolute ethanol to a stock solution of 1000 μM, and stored at −80 °C.

Techniques: Proliferation Assay, Migration, Staining, Western Blot, Control, RIA Assay, Expressing, MANN-WHITNEY

Journal: International Journal of Lipids

Article Title: Lipid Components in the Dynamin Fraction Prepared from Rat Brain

doi: 10.14302/issn.2835-513x.ijl-18-2122

Figure Lengend Snippet: Figure 3. Mass-spectrum of three OHCs (A; 7OHC, B; 24OHC, C; 27OHC) and peaks #1~#5

Article Snippet: 27-hydroxycholesterol (27OHC) was from Avanti Polar Lipids, Inc. 24(S)-hydroxycholesterol (24OHC) was obtained from Focus Biomolecules.

Techniques: