Journal: Experimental and Therapeutic Medicine

Article Title: Dendrobium nobile Lindl. alkaloids improve lipid metabolism by increasing LDL uptake through regulation of the LXRα/IDOL/LDLR pathway and inhibition of PCSK9 expression in HepG2 cells

doi: 10.3892/etm.2025.12796

Figure Lengend Snippet: DNLA suppresses LXRα and IDOL protein expression in HepG2 cells. (A) Protein expression of LXRα and IDOL in HepG2 cells was assessed by western blotting. Normalized intensity of (B) LXRα and (C) IDOL vs. GAPDH, presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01 and **** P<0.0001. DNLA, Dendrobium nobile Lindl. alkaloids; LPS, lipopolysaccharides; LXRα, liver X receptor α; IDOL, inducible degrader of the low-density lipoprotein receptor.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed dry milk in Tris-buffer solution with 0.1% Tween-20 (TBST) at room temperature for 2 h before being incubated overnight at 4˚C with the following primary antibodies (1:1,000): Rabbit anti-LDLR (cat. no. ab30532; Abcam), rabbit anti-PCSK9 (cat. no. ab185194; Abcam), rabbit anti-IDOL (cat. no. bs-9674R; BIOSS), rabbit anti-SREBP2 (cat. no. ab30682; Abcam), rabbit anti-LXRα (cat. no. bs-10311R; BIOSS), rabbit anti-HNF1α (cat. no. 89670S; Cell Signaling Technology, Inc.), rabbit anti-3-hydroxy-3-methyl glutaryl-coenzyme A reductase (HMGCR; cat. no. ab242315; Abcam), rabbit anti-cytochrome P450 (CYP) 7A1 (cat. no. bs-21430R; BIOSS) and mouse anti-GAPDH (cat. no. 97166S; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Dendrobium nobile Lindl. alkaloids improve lipid metabolism by increasing LDL uptake through regulation of the LXRα/IDOL/LDLR pathway and inhibition of PCSK9 expression in HepG2 cells

doi: 10.3892/etm.2025.12796

Figure Lengend Snippet: T0901317, an LXRα agonist, reverses the decrease in IDOL and LXRα expression induced by DNLA and reduces the LDLR content in HepG2 cells. (A) Protein expression of LXRα, IDOL and LDLR in HepG2 cells assessed by western blotting. Normalized intensity of (B) LXRα, (C) IDOL and (D) LDLR vs. GAPDH, presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001. DNLA, Dendrobium nobile Lindl. alkaloids; LPS, lipopolysaccharides; LXRα, liver X receptor α; IDOL, inducible degrader of the low-density lipoprotein receptor; LDLR, low-density lipoprotein receptor.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed dry milk in Tris-buffer solution with 0.1% Tween-20 (TBST) at room temperature for 2 h before being incubated overnight at 4˚C with the following primary antibodies (1:1,000): Rabbit anti-LDLR (cat. no. ab30532; Abcam), rabbit anti-PCSK9 (cat. no. ab185194; Abcam), rabbit anti-IDOL (cat. no. bs-9674R; BIOSS), rabbit anti-SREBP2 (cat. no. ab30682; Abcam), rabbit anti-LXRα (cat. no. bs-10311R; BIOSS), rabbit anti-HNF1α (cat. no. 89670S; Cell Signaling Technology, Inc.), rabbit anti-3-hydroxy-3-methyl glutaryl-coenzyme A reductase (HMGCR; cat. no. ab242315; Abcam), rabbit anti-cytochrome P450 (CYP) 7A1 (cat. no. bs-21430R; BIOSS) and mouse anti-GAPDH (cat. no. 97166S; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Schematic representation of the synthetic luciferase reporters constructs. ChoRE, carbohydrate response element; LXRE, LXR response element. B. Huh7 cells cultured in 25 mM glucose were transfected with synthetic luciferase reporters containing ChoRE+LXRE, ChoRE-only, or LXRE-only, and plasmids expressing ChREBPα, the activated quadruple mutant ChREBP-Q, or ChREBPβ, together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. C. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ or Lpk -driven luciferase reporter luciferase reporter, and plasmids expressing ChREBPα, ChREBP-Q, ChREBPβ together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. Data are presented as mean ± SEM (n=3-7). Significant differences are shown as *p < 0.05, **p < 0.01, ***p < 0.001 compared to control within the same ChREBP isoform transfection. ns, not significant.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Luciferase, Construct, Cell Culture, Transfection, Expressing, Mutagenesis, Control

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Co-immunoprecipitation (CoIP) of LXRα and ChREBPα expressed in COS-1 cells cultured in 25 mM glucose. Lysates were immunoprecipitated with ChREBP and LXRα antibodies, and input and immunoprecipitated proteins immunoblotted the same antibodies (n=3). One representative western blot is shown. B. Distribution of ChREBP-LXR peak pairs. ChREBP ChIP-seq data from fasted and high-carbohydrate refed mouse liver ( Poungvarin et al , 2015 ) and LXR ChIP-seq data from T0901317-treated mouse liver ( Boergesen et al , 2012 ) were reanalyzed to generate a genome-wide map of ChREBP and LXR binding sites. The top 10,000 peaks from each dataset were used to calculate the Peak(ChREBP)-to-Peak(LXR) distance, and all peak pairs with a peak-to-peak distance < 1,000 bp were plotted against the number of peak pairs. C. Localization of ChREBP-LXR peak pairs. The Peak(ChREBP)-to-Peak(LXR) distance were plotted against the position relative to transcription start site (TSS) of the closest gene. Blue dots, verified LXR/ChREBP target genes, Acaca, Mlxipl (Chrebpα and β), Pklr, Scd1, Srebf1 and Txnip . Orange curve, moving average (window: 100 bp) of peak-to-peak distance as a function of distance to TSS. D. Global landscape of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks on Chromatin 3. Square brackets indicate the scale maxima of log2(ChIP/input) ratios. E. Local pattern of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks on the promoter regions of the ChREBP target genes Mlxipl ( Chrebpβ ) and Pklr ( Lpk ). Square brackets indicate the scale maxima of ChIP/input ratios. F. Genomic positions of the ChREBP-LXR peak pairs. The peak pairs were mapped to genomic elements using Rgmatch. The elements shown are grouped as follows: 5’ far, 25 kb to 5 kb upstream of TSS; Promoter, < 5 kb upstream of TSS; TSS, -200 bp to + 200 bp; E1, first exon; I1, first intron; 3’ near, < 5 kb downstream of TSS; 3’ far, 5 kb to 25 kb downstream of TSS. G. Genome-wide co-occurrence of mouse hepatic transcription factors (TFs). Comparison of LXR and ChREBP with published binding profiles of PPARα ( Boergesen et al , 2012 ) and FXR ( Ijssennagger et al , 2016 ). Forbes coefficients (FC) of genomic co-occurrence between ChREBP, LXR, FXR and PPARα were calculated using the Genomic HyperBrowser.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Immunoprecipitation, Cell Culture, Western Blot, ChIP-sequencing, Genome Wide, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ (n=3) or Lpk -driven luciferase reporter (n=6), and plasmids expressing ChREBPα/Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Six hours post transfection, cells were treated with 2.5 mM or 25 mM glucose for 18 hours. Dual luciferase reporter assays were performed 24 hours post transfection. B. LXRαβ wild type (WT), LXRα -/- , LXRβ -/- and LXRα -/- β -/- mice were fasted for 24 hours (white bars) or fasted for 24 hours and refed for 12 hours (black bars) (n=5-8 mice per group). Hepatic gene expression of Lpk ( Pklr ), Chrebpβ and Chrebpα was analyzed by quantitative RT-PCR and normalized to Tbp . Data are presented as mean ± standard error of the mean (SEM). Significant differences are shown as *p < 0.05, **p < 0.01, ***p < 0.001 compared to control within the same treatment, and # p < 0.05, ## p < 0.01, ### p < 0.001 between indicated groups.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Cell Culture, Transfection, Luciferase, Expressing, Control, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Left panels: Local pattern of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks in the promoter region of Lpk ( Pklr ), Txnip, Fasn and Scd1 . Square brackets indicate the scale maxima of ChIP/input ratios. Arrows indicate the genomic locations of quantitative RT-PCR primers. Right panel: AML12 cells transfected with ChREBPα/Mlxγ and LXRα/RXRα were treated with DMSO (0.1%) or GW3965 (10 µM) for 18 hours. ChREBP or LXR binding to genomic location indicated in the right panels were detected by ChIP using antibodies against ChREBP, LXR or IgG as negative control. Data are presented as mean ± SEM (n=3-5). Significant differences are shown as **p < 0.01, ***p < 0.001 compared to ChIP-IgG, and # p < 0.05, ## p < 0.01, ### p < 0.001 between DMSO and GW3965 groups. ns, not significant. B. CoIP of LXRα and ChREBPα, expressed in COS-1 cells cultured in 25 mM glucose, treated with DMSO (0.1%) or GW3965 (1 µM) for 18 hours. Lysates were immunoprecipitated with ChREBP antibody (n=3). Input and immunoprecipitated proteins were immunoblotted with ChREBP or LXRα antibodies. One representative western blot is shown.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Quantitative RT-PCR, Transfection, Binding Assay, Negative Control, Cell Culture, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Cholesterol Efflux on Endothelial Dysfunction Caused by Oxidative Stress

doi: 10.3390/ijms24065939

Figure Lengend Snippet: H 2 O 2 can promote cholesterol accumulation by oxidative stress. ( A , B ) The protein levels of LXRα, LXRβ, HMGCR, ABCA1, and ABCG1 were measured by a Western blot analysis. ( C ) The accumulation of ER cholesterol after filipin staining was visualized under a fluorescence microscope (magnification 5×). n = 3, * p < 0.05, and ** p < 0.001 vs. the C group (treated by PBS). C, control group; H 2 O 2 , cells treated with H 2 O 2 (500 μmol/L) for 24 h. The scale of the pictures was 100 μm.

Article Snippet: The antibodies are as follows: MCP-1 (WL02966, Wanlei, Shenyang, China), 1:500; CD106/VCAM-1 (WL02474, Wanlei, Shenyang, China), 1:500; GRP78 (66574-1-Ig, Proteintech, Hubei, China), 1:10000; GAPDH (HRP-60004, Proteintech, Hubei, China), 1:40000; β-catenin (17565-1-AP, Proteintech, Hubei, China), 1:4000; ICAM-1 (10831-1-AP, Proteintech, Hubei, China), 1:1000; ABCA1 (D155299, BBI, Shanghai, China), 1:500; ABCG1 (AP6529A, Abgent, San Diego, USA), 1:500; LXRα (D198471, BBI, Shanghai, China), 1:2000; LXRβ (A04523-2, BOSTER, Hubei, China), 1:1000; LRP6 (AP1191, ABclonal, Hubei, China), 1:1000; HMGCR (AP11955B, Abgent, San Diego, CA, USA), 1:1000 and p-β-catenin (DF2989, Affinity, Jiangsu, China), 1:2000.

Techniques: Western Blot, Staining, Fluorescence, Microscopy, Control