hydroxycholesterol d6 d6 27ohc  (Croda International Plc)

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: 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: