Journal: The Journal of Cell Biology

Article Title: Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway

doi: 10.1083/jcb.202002026

Figure Lengend Snippet: Hedgehog pathway activation accumulates CYP7A1 near the ciliary base. (A–D) Quantitative immunofluorescence confocal microscopy for CYP7A1 in relation to the ciliary protein acetylated tubulin (AcTub) and the centriole protein CEP170 demonstrates that CYP7A1 localizes near the ciliary base in NIH 3T3 cells and MEFs after pharmacologic stimulation or genetic de-repression of the Hedgehog pathway. DNA is marked with Hoechst 33342. Main scale bar, 10 µm. Inset scale bar, 1 µm. Student’s t test in C; ANOVA in D. Dotted lines denote average background intensity. (E and F) Superresolution microscopy validates CYP7A1 localization near the ciliary base at the level of CEP170 in NIH 3T3 cells and MEFs. Scale bars, 1 µm. (G) qRT-PCR assessment of Cyp7a1 expression in NIH 3T3 dCas9-KRAB cells after transduction of sg Cyp7a1 demonstrates Cyp7a1 suppression compared with transduction control (ANOVA). (H and I) Quantitative immunofluorescence confocal microscopy for CYP7A1 after transduction of sg Cyp7a1 in NIH 3T3 dCas9-KRAB cells and pharmacologic activation of the Hedgehog pathway confirms that CYP7A1 localizes near the ciliary base (ANOVA). Scale bar, 1 µm. **, P ≤ 0.01; and ***, P ≤ 0.001. Error bars represent SEM. The sample size of each experiment is represented by the number of independent data points on each graph. Each experiment is representative of at least three independent biological replicates.

Article Snippet: HA-tagged Dhcr7 and Cyp7a1 coding DNA sequences were synthesized by Integrated DNA Technologies, cloned into pcDNA3 (Addgene), and transfected using FuGENE HD reagent (E2311; Promega) according to the manufacturer’s instructions.

Techniques: Activation Assay, Immunofluorescence, Confocal Microscopy, Microscopy, Quantitative RT-PCR, Expressing, Transduction, Control

Journal: The Journal of Cell Biology

Article Title: Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway

doi: 10.1083/jcb.202002026

Figure Lengend Snippet: CYP7A1 localizes near the ciliary base. (A) Immunofluorescence confocal microscopy of Ptch1 −/− MEFs transfected with Cyp7a1 Myc in pCMV6-Entry using Lipofectamine demonstrates that exogenous CYP7A1 can localize near the ciliary base, as marked by the centriole protein CEP164, after genetic Hedgehog pathway activation. Scale bar, 1 µm. (B) Immunofluorescence confocal microscopy of MEFs treated with vehicle control or SAG, and transfected with Cyp7a1 HA in pCDNA3 using FuGENE, demonstrates that exogenous CYP7a1 can localize away from the ciliary microenvironment. DNA is marked with Hoechst 33342. Scale bar, 10 µm. (C) qRT-PCR assessment of NIH 3T3 cells and MEFs reveals that pharmacologic activation of the Hedgehog pathway fails to alter expression of Cyp7a1 . (D) Mass spectrometry–based sterolomics of NIH 3T3 dCas9-KRAB cells show that transduction of sg Cyp7a1 does not alter cellular levels of Smoothened-activating oxysterols that are produced by other enzymes. (E) qRT-PCR assessment of NIH 3T3 cells demonstrates that overexpression of Dhcr7 HA or Cyp7a1 HA activates the Hedgehog transcriptional program (ANOVA). *, P ≤ 0.05; ***, P ≤ 0.001. Error bars represent SEM. The sample size of each experiment is represented by the number of independent data points on each graph. Each experiment is representative of at least three independent biological replicates. 24,25-EC, 24,25-epoxycholesterol; 24k-C, 24-keto-cholesterol.

Article Snippet: HA-tagged Dhcr7 and Cyp7a1 coding DNA sequences were synthesized by Integrated DNA Technologies, cloned into pcDNA3 (Addgene), and transfected using FuGENE HD reagent (E2311; Promega) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Confocal Microscopy, Transfection, Activation Assay, Control, Quantitative RT-PCR, Expressing, Mass Spectrometry, Transduction, Produced, Over Expression

Journal: The Journal of Cell Biology

Article Title: Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway

doi: 10.1083/jcb.202002026

Figure Lengend Snippet: CYP7A1 activates the Hedgehog pathway. (A) qRT-PCR assessment of Gli1 expression in NIH 3T3 dCas9-KRAB cells after transduction of sg Cyp7a1 compared with control demonstrates inhibition of the Hedgehog transcriptional program after Cyp7a1 suppression (ANOVA). (B) Mass spectrometry–based sterolomics reveal reduced expression of 7k-C and no change in 7-DHC or 7-DHC oxidation by-products in NIH 3T3 dCas9-KRAB cells transduced with sg Cyp7a1 compared with control (Student’s t test). (C and D) Quantitative immunofluorescence confocal microscopy for Smoothened in relation to the ciliary protein acetylated tubulin (AcTub) in NIH 3T3 dCas9-KRAB cells after transduction of sg Cyp7a1 demonstrates that Cyp7a1 suppression fails to block Smoothened accumulation in cilia in response to SHH compared with either control cells treated with vehicle or cells expressing sg Cyp7a1 treated with vehicle (ANOVA, Student’s t test). Scale bar, 1 µm. Dotted lines denote average background intensity. (E) qRT-PCR assessment of Gli1 expression in NIH 3T3 dCas9-KRAB cells after transduction of sg Cyp7a1 reveals that Cyp7a1 repression attenuates the Hedgehog transcriptional program in response to SHH (ANOVA). *, P ≤ 0.05; ***, P ≤ 0.001. Error bars represent SEM. The sample size of each experiment is represented by the number of independent data points on each graph. Each experiment is representative of at least three independent biological replicates.

Article Snippet: HA-tagged Dhcr7 and Cyp7a1 coding DNA sequences were synthesized by Integrated DNA Technologies, cloned into pcDNA3 (Addgene), and transfected using FuGENE HD reagent (E2311; Promega) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Transduction, Control, Inhibition, Mass Spectrometry, Immunofluorescence, Confocal Microscopy, Blocking Assay

Journal: The Journal of Cell Biology

Article Title: Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway

doi: 10.1083/jcb.202002026

Figure Lengend Snippet: DHCR7 and CYP7A1 cooperate to activate the Hedgehog pathway near the ciliary base. (A) Quantitative immunofluorescence confocal microscopy for CYP7A1 in NIH 3T3 dCas9-KRAB cells shows that Dhcr7 suppression leading to Hedgehog pathway activation accumulates CYP7A1 near the ciliary base (ANOVA). (B) qRT-PCR assessment of Gli1 expression in NIH 3T3 dCas9-KRAB cells transduced with sgCyp7a1 and shRNAs targeting Dhcr7 reveals that concurrent suppression of Cyp7a1 and Dhcr7 attenuates the Hedgehog transcriptional program greater than suppression of either enzyme alone in response to SHH (ANOVA). (C) Quantitative immunofluorescence confocal microscopy for DHCR7 and CYP7A1 in NIH 3T3 cells treated the translation inhibitor CHX cells shows that protein synthesis is required to remove DHCR7 from the ciliary base, but is not required to accumulate CYP7A1 at the ciliary base, in response to Hedgehog pathway activation with SAG (Student’s t test). (D) Quantitative immunofluorescence confocal microscopy for DHCR7 in WT and Ofd1 Gt mouse embryonic stem cells (MESCs) reveals disruption of centriole structure, as marked by centriolin (Cent), reduces DHCR7 localization near the ciliary base. Scale bar, 1 µm (Student’s t test). (E) Superresolution microscopy and the IN/OUT assay using NIH 3T3 cells stably expressing ARL13B with extracellular HA and intracellular mCherry tags validates DHCR7 localization near the ciliary base. Scale bar, 1 µm. (F) Network of Smoothened-activating sterol and oxysterol biosynthesis constructed from the data in this paper and published literature . (G) Model of lipid enrichment in the ciliary microenvironment and regulation of lipids in response to Hedgehog pathway activity. *, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001. Error bars represent SEM. The sample size of each experiment is represented by the number of independent data points on each graph. Each experiment is representative of at least three independent biological replicates.

Article Snippet: HA-tagged Dhcr7 and Cyp7a1 coding DNA sequences were synthesized by Integrated DNA Technologies, cloned into pcDNA3 (Addgene), and transfected using FuGENE HD reagent (E2311; Promega) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Confocal Microscopy, Activation Assay, Quantitative RT-PCR, Expressing, Transduction, Disruption, Microscopy, Stable Transfection, Construct, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Simvastatin promotes NPC 1‐mediated free cholesterol efflux from lysosomes through CYP 7A1/ LXR α signalling pathway in ox LDL ‐loaded macrophages

doi: 10.1111/jcmm.12970

Figure Lengend Snippet: Up‐regulated CYP 7A1 expression underscored the elevation of NPC 1 and LXR α levels by simvastatin in ox LDL ‐loaded macrophages. ( A ) mRNA levels of CYP 7A1 in ox LDL +sim‐treated macrophages were markedly increased compared with Sim only, or ox LDL alone group, or control group. ( B ) Gene interference of CYP 7A1 significantly decreased LXR α and NPC 1 transcriptional levels in ox LDL ‐loaded macrophages on simvastatin. *, # P < 0.05; * versus Ctrl, ox LDL or Sim group; # versus Scram, n = 3. CYP 7A1: Cytochrome P450 7A1.

Article Snippet: All treatment and analysis reagents and biochemical kits utilized in this study were obtained from commercial sources: Cholesterol quantification kit, simvastatin, progesterone, nile red, filipin and 7β‐Hydroxycholesterol (Sigma‐Aldrich, St. Louis, MO, USA); anti‐NPC1 antibody (EMD Millipore, Billerica, MA, USA); Mouse interleukin (IL)‐1 beta/IL‐1F2 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA); GenMute TM siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA); oxLDL (TBARS: 29‐44 nmoles MDA/mg; Alfa Aesar, Ward Hill, MA, USA); rabbit anti‐mouse CD68 antibody (Bioss, Woburn, MA, USA); Alexa fluor 633 goat anti‐rat IgG (Life Technologies, Carlsbad, CA, USA); LXRα siRNA, NPC1 siRNA and LAMP‐1 rat monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); cytochrome P450 7A1 siRNA (Origene, Rockville, MD, USA); solid‐phase extraction (SPE) column (Silicycle, Quebec, QC, Canada); 22(S)‐hydroxycholesterol (D7) (Avanti Polar Lipids, Alabaster, AL, USA); HPLC column, XTerra RP18, 2.1 × 150 mm, 5 μM (Waters Corporation, Milford, MS, USA); and lysosome enrichment kit (Thermo Scientific, Waltham, MA, USA).

Techniques: Expressing, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Simvastatin promotes NPC 1‐mediated free cholesterol efflux from lysosomes through CYP 7A1/ LXR α signalling pathway in ox LDL ‐loaded macrophages

doi: 10.1111/jcmm.12970

Figure Lengend Snippet: Gene interference of CYP 7A1 expression compromised simvastatin‐induced free cholesterol secretion and attenuated the reduction of lysosomal free cholesterol in ox LDL ‐loaded macrophages. ( A ) Confocal microscopy images from nile red, filipin and LAMP 1 staining; ( B ) summarized intensities from free cholesterol staining; ( C ) colocalization coefficient between filipin (free cholesterol) and LAMP 1 (lysosomes), and ( D ) secretion of free cholesterol after CYP 7A1 gene interference. *, # P < 0.05, * versus Scram group; # versus Scram group, n = 8.

Article Snippet: All treatment and analysis reagents and biochemical kits utilized in this study were obtained from commercial sources: Cholesterol quantification kit, simvastatin, progesterone, nile red, filipin and 7β‐Hydroxycholesterol (Sigma‐Aldrich, St. Louis, MO, USA); anti‐NPC1 antibody (EMD Millipore, Billerica, MA, USA); Mouse interleukin (IL)‐1 beta/IL‐1F2 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA); GenMute TM siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA); oxLDL (TBARS: 29‐44 nmoles MDA/mg; Alfa Aesar, Ward Hill, MA, USA); rabbit anti‐mouse CD68 antibody (Bioss, Woburn, MA, USA); Alexa fluor 633 goat anti‐rat IgG (Life Technologies, Carlsbad, CA, USA); LXRα siRNA, NPC1 siRNA and LAMP‐1 rat monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); cytochrome P450 7A1 siRNA (Origene, Rockville, MD, USA); solid‐phase extraction (SPE) column (Silicycle, Quebec, QC, Canada); 22(S)‐hydroxycholesterol (D7) (Avanti Polar Lipids, Alabaster, AL, USA); HPLC column, XTerra RP18, 2.1 × 150 mm, 5 μM (Waters Corporation, Milford, MS, USA); and lysosome enrichment kit (Thermo Scientific, Waltham, MA, USA).

Techniques: Expressing, Confocal Microscopy, Staining

Journal: Journal of Translational Medicine

Article Title: Limonin attenuates hyperlipidemia by regulating the gut microbiota-bile acid-farnesoid X receptor axis

doi: 10.1186/s12967-026-07826-7

Figure Lengend Snippet: Impacts of Limonin upon FXR/FGF15 signaling pathway in hyperlipidemia mice liver. ( A ) mRNA expression levels of fgf15 in liver. ( B ) mRNA expression levels of Fgfr4 in liver. ( C ) mRNA expression levels of Cyp7a1 in liver. ( D ) Representative picture of immunofluorescence expression in mice liver. ( E ) F Immunofluorescence expression of CYP7A1 in liver. ( F ) Immunofluorescence expression of CYP7A1 in liver. ( G ) Immunofluorescence expression of CYP7A1 in liver. ( H ) FGF15 protein expression levels. ( I ) FGFR4 protein expression levels. ( J ) CYP7A1 protein expression levels. ( K ) Representative protein bands detected by WB. Values are denoted by mean ± SD ( n = 3). # P < 0.05, ## P < 0.01 vs. MG and * P < 0.05, ** P < 0.01 vs. NG

Article Snippet: Following blocking in 5% skim milk for 2 h at room temperature, we cultured culture membrane with primary antibody including FXR1 (BOSTER, M03308), FGF15(CUSABIO, O35622 ), ASBT (Cohesion, CQA5895), CYP7A1 (Proteintech, 18054-1-AP), and FGFR4 (BOSTER, BM5163), β-actin (AB_2750915, CPA9121) overnight at 4 °C, followed by reaction of enzyme-labeled secondary antibody for 2 h at room temperature.

Techniques: Expressing, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: Limonin attenuates hyperlipidemia by regulating the gut microbiota-bile acid-farnesoid X receptor axis

doi: 10.1186/s12967-026-07826-7

Figure Lengend Snippet: Graphical illustration of mechanism mediating anti-hyperlipidemia effects of Limonin. The possible mechanisms by which limonin alleviates dyslipidemia are as follows: (1) Reduced relative abundance of BSH-producing microbes induced by Limonin. (2) Boosted conjugated BAs, particularly TCA, T-αMCA, and T-βMCA acting in antagonistic manner on intestinal FXR, (3) The increased conjugated BAs boosted elimination of BAs via feces, and also as endogenous signaling molecules that inhibited activation of intestinal FXR causing FGF15’s reduced production in the distal ileum. (4) The FGF15 through the enterohepatic circulation to enter mice liver, and regulates liver CYP7A1 expression via negative feedback of FGF15-FGFR4 pathway to promoting the synthesis of TC to BA, thereby reducing TC

Article Snippet: Following blocking in 5% skim milk for 2 h at room temperature, we cultured culture membrane with primary antibody including FXR1 (BOSTER, M03308), FGF15(CUSABIO, O35622 ), ASBT (Cohesion, CQA5895), CYP7A1 (Proteintech, 18054-1-AP), and FGFR4 (BOSTER, BM5163), β-actin (AB_2750915, CPA9121) overnight at 4 °C, followed by reaction of enzyme-labeled secondary antibody for 2 h at room temperature.

Techniques: Activation Assay, Expressing