Journal: bioRxiv

Article Title: Hydroxylation site-specific and production-dependent roles of endogenous oxysterols in cellular cholesterol homeostasis

doi: 10.1101/2022.05.14.491815

Figure Lengend Snippet: (A) Biosynthesis of oxysterols by cholesterol hydroxylases, CH25H, CYP27A1, CYP46A1, and CYP7A1. (B) CHO-K1 cells were treated without or with 25-HC, 27-HC, 24S-HC, or 7a-HC at different concentration (0, 20, 100, 500, and 2500 nM) for 24 hr. mRNA levels of SREBP-2 and LXR target genes (Hmgcs1 and Abca1, respectively) were analyzed by qPCR. Data represent means ± SD (n = 3). Statistical analyses were performed by one-way ANOVA with Dunnett post hoc test by comparing to the vehicle treatment group (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Sterols were obtained from commercial sources as follows; cholesterol (C8667), 24(S)-hydroxycholesterol (SML1648), 25-hydroxycholesterol (H1015), 27-hydroxycholesterol (SML2042), 7α,25-Dihydroxycholesterol (SML0541), 7-dehydrocholesterol (30800), and 7β-hydroxycholesterol (H6891) from Sigma; desmosterol (NS460402), lanosterol (NS460102), lathosterol (NS460502), and 24,25-dihydrolanosterol (NS460201) from Nagara Science; 7a,27-dihydroxycholesterol (700024P), 7a-hydroxycholesterol (700034P), cholesterol-d7 (700172), 25-hydroxycholesterol-d6 (LM4113-1EA) from AVANTI; 24,25-epoxycholesterol (Ab141633) from Abcam.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Hydroxylation site-specific and production-dependent roles of endogenous oxysterols in cellular cholesterol homeostasis

doi: 10.1101/2022.05.14.491815

Figure Lengend Snippet: (A) Cellular localization of cholesterol hydroxylases. A2058 cells were transfected with the plasmids (pFLAG-CH25H, pCYP27A1-FLAG, pCYP46A1-FLAG, or pCYP7A1-FLAG) and fixed 2 days after transfection. Cholesterol hydroxylases were labeled with anti-FLAG antibody followed by Alexa 488-conjugated anti-mouse IgG (green). The ER and mitochondria were labeled with anti-calnexin and anti-Tom20 antibodies, respectively, followed by Alexa 568-conjugated anti-rabbit IgG (red). Images were taken under a confocal microscopy as described in Materials and Methods. Scale bar, 20 μm except FLAG-CH25H (10 μm). (B and C) Effect of hydroxylase expression on cholesterol homeostatic responses. CHO-K1 cells were transfected with plasmids as above and incubated for 48 hr in medium containing 0.1% FBS. Cells transfected with pCMV10 (mock) were also treated without or with 2.5 μM oxysterols (25-HC, 27-HC, 24S-HC, or 7a-HC) for 16 hr in medium containing 0.1% FBS before harvesting cells. mRNA levels of SREBP-2 target genes (B) and of LXR target genes (C) were analyzed by qPCR. Data represent means ± SD (n = 3). Statistical analyses were performed by one-way ANOVA with Dunnett post hoc test by comparing to mock (*p<0.05, **p<0.01, ***p<0.001). (D) Effect of hydroxylase expression on SREBP-2 processing. CHO-K1 cells were transfected with the plasmids and treated as above. Before harvesting, cells were incubated with MG132 (20 μM) for 2 hr. Expression of SREBP-2 (P, precursor; M, mature form), HMGCR, ABCA1, and FLAG-tagged hydroxylases was analyzed by immunoblot. β-actin serves as a loading control.

Article Snippet: Sterols were obtained from commercial sources as follows; cholesterol (C8667), 24(S)-hydroxycholesterol (SML1648), 25-hydroxycholesterol (H1015), 27-hydroxycholesterol (SML2042), 7α,25-Dihydroxycholesterol (SML0541), 7-dehydrocholesterol (30800), and 7β-hydroxycholesterol (H6891) from Sigma; desmosterol (NS460402), lanosterol (NS460102), lathosterol (NS460502), and 24,25-dihydrolanosterol (NS460201) from Nagara Science; 7a,27-dihydroxycholesterol (700024P), 7a-hydroxycholesterol (700034P), cholesterol-d7 (700172), 25-hydroxycholesterol-d6 (LM4113-1EA) from AVANTI; 24,25-epoxycholesterol (Ab141633) from Abcam.

Techniques: Transfection, Labeling, Confocal Microscopy, Expressing, Incubation, Western Blot

Journal: bioRxiv

Article Title: Hydroxylation site-specific and production-dependent roles of endogenous oxysterols in cellular cholesterol homeostasis

doi: 10.1101/2022.05.14.491815

Figure Lengend Snippet: (A) Diagram of constitutive activation of SREBP-2 in 25RA and SRD15 cells. In these two mutants, SREBP-2 is constitutively activated by different mechanisms. See text for more detail. (B, C) Expression of SREBP-2 target genes in 25RA (B) and SRD-15 (C) cells. On day 0, 25RA, SRD-15, and their parental cells (CHO-K1 and CHO-7, respectively) were seeded into 6-well plates. On day 1, these cells were transfected with different amounts (0.5 and 2.0 μg/well) of plasmid to express FLAG-CH25H or an empty plasmid as indicated. On day 2, mock-transfected cells were either treated or untreated with 25-HC cells (2.5 μM) for 16 hr. On day 3, cells were harvested for RNA isolation. mRNA levels of SREBP-2 target genes were analyzed by qPCR. Data represent means ± S.D. from three biological replicates. Statistical analyses were performed by one-way ANOVA with Dunnett post hoc test (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Sterols were obtained from commercial sources as follows; cholesterol (C8667), 24(S)-hydroxycholesterol (SML1648), 25-hydroxycholesterol (H1015), 27-hydroxycholesterol (SML2042), 7α,25-Dihydroxycholesterol (SML0541), 7-dehydrocholesterol (30800), and 7β-hydroxycholesterol (H6891) from Sigma; desmosterol (NS460402), lanosterol (NS460102), lathosterol (NS460502), and 24,25-dihydrolanosterol (NS460201) from Nagara Science; 7a,27-dihydroxycholesterol (700024P), 7a-hydroxycholesterol (700034P), cholesterol-d7 (700172), 25-hydroxycholesterol-d6 (LM4113-1EA) from AVANTI; 24,25-epoxycholesterol (Ab141633) from Abcam.

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Isolation

Journal: bioRxiv

Article Title: Hydroxylation site-specific and production-dependent roles of endogenous oxysterols in cellular cholesterol homeostasis

doi: 10.1101/2022.05.14.491815

Figure Lengend Snippet: (A) Stabilization of Insig-1 by CH25H expression in CHO cells. On day 1, CHO-K1 and 25RA cells were transfected with plasmid encoding FLAG-CH25H (2 μg/well) as above. On day 2, cells were treated without or with 25-HC (2.5 μM) in the presence or absence of cycloheximide (CHX) (50 μM) for 2 hr as indicated. Immunoblot was performed with antibodies to Insig-1, FLAG, and β-actin. (B) Stabilization of Insig-1 by CH25H expression in HEK293T cells. HEK293T cells were transfected with pFLAG-CH25H (1.0 μg/well) as above. Cells were then treated without or with CHX (50 μM) and 25HC (2.5 μM) for 30 or 90 min as indicated. Stabilization of Insig-1 was examined by immunoblot. Calnexin was used as a loading control. (C) Expression level-dependent effect of CH25H on Insig-1 stabilization. CHO-CH25H tet-on cells were incubated with or without Dox (400 ng/mL or 1000 ng/mL) for 24 hr, followed by the treatment with or without CHX (50 μM) and 25-HC (2.5 μM) for 30 or 90 min as indicated. Insig-1 stabilization was assessed by immunoblot as above. The asterisk denotes a non-specific band.

Article Snippet: Sterols were obtained from commercial sources as follows; cholesterol (C8667), 24(S)-hydroxycholesterol (SML1648), 25-hydroxycholesterol (H1015), 27-hydroxycholesterol (SML2042), 7α,25-Dihydroxycholesterol (SML0541), 7-dehydrocholesterol (30800), and 7β-hydroxycholesterol (H6891) from Sigma; desmosterol (NS460402), lanosterol (NS460102), lathosterol (NS460502), and 24,25-dihydrolanosterol (NS460201) from Nagara Science; 7a,27-dihydroxycholesterol (700024P), 7a-hydroxycholesterol (700034P), cholesterol-d7 (700172), 25-hydroxycholesterol-d6 (LM4113-1EA) from AVANTI; 24,25-epoxycholesterol (Ab141633) from Abcam.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Incubation