Journal: International Journal of Molecular Sciences

Article Title: Compound K Attenuates the Development of Atherosclerosis in ApoE −/− Mice via LXRα Activation

doi: 10.3390/ijms17071054

Figure Lengend Snippet: Compound K attenuates atherosclerosis lesion formation and fatty liver in apoE −/− mice. En face ( A ) and aortic sinus section ( B ), (zoomed areas were marked with red square) images of atherosclerosis were analysed by NIS-Elements image analysis software, and the cartograms were shown as ( D , E ). Levels of cholesterol concentration in mice aorta were extracted and measured ( F ). Levels of lipid deposition in apoE −/− mice livers were measured using Oil-Red O staining ( C ). Images of stained liver histological sections were analysed by NIS-Elements image analysis software ( G ). Data were presented as mean ± SEM ( n = 6) and analysed by ANOVA with Dunnett’s post-hoc analysis. * p < 0.05 vs. control; # p < 0.05 vs. model; ## p < 0.01 vs. model.

Article Snippet: The macrophages were incubated with 100 μg/mL oxidized low density lipoprotein (ox-LDL, MDA 30 μM, Institute of Basic Medicine, Peking Union Medical College, Beijing, China) in 6-well plates for 24 h. Then cells were administrated with ox-LDL free medium containing CD36 blocking antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000) and compound K (0 μM, 3.3 μM, 10 μM, 30 μM) or GW3965 (3.3 μM) or compound K (10 μM) plus 10 mM GGPP for another 24 h. Cells of control group were incubated over the same time period in culture media without any treatment.

Techniques: Software, Concentration Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Compound K Attenuates the Development of Atherosclerosis in ApoE −/− Mice via LXRα Activation

doi: 10.3390/ijms17071054

Figure Lengend Snippet: Effects of compound K on serous lipid profile and inflammatory cytokines in apoE −/− mice. Serous total cholesterol (TC), low density lipoprotein cholesterol (LDL), very low density lipoprotein cholesterol (VLDL), high density lipoprotein cholesterol (HDL) concentrations and triglyceride (TG) in apoE −/− mice treated with different concentrations of compound K were measured using Olympus AU-2700 automatic biochemical analyzer ( A ); Levels of serum IL-1β, IL-6 and TNF-α were detected by Mcytomag-70K-3 Mouse Cytokine/Chemokine Magnetic Bead Panel ( B ). Data are presented as mean ± SEM ( n = 6) and analysed by ANOVA with Dunnett’s post-hoc analysis. * p < 0.05 vs. control; # p < 0.05 vs. model; ## p < 0.01 vs. model.

Article Snippet: The macrophages were incubated with 100 μg/mL oxidized low density lipoprotein (ox-LDL, MDA 30 μM, Institute of Basic Medicine, Peking Union Medical College, Beijing, China) in 6-well plates for 24 h. Then cells were administrated with ox-LDL free medium containing CD36 blocking antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000) and compound K (0 μM, 3.3 μM, 10 μM, 30 μM) or GW3965 (3.3 μM) or compound K (10 μM) plus 10 mM GGPP for another 24 h. Cells of control group were incubated over the same time period in culture media without any treatment.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Compound K Attenuates the Development of Atherosclerosis in ApoE −/− Mice via LXRα Activation

doi: 10.3390/ijms17071054

Figure Lengend Snippet: Effects of compound K on inflammasome activity in apoE −/− mice aorta. The level of cleaved-IL-1β in mice aorta was detected by immunoblotting. Treatments of compound K (3, 9 mg/kg) significantly attenuated the increase of cleaved-IL-1β ( A ) expression. To investigate the probably mechanism, the levels of NLRP3, caspase-1 and nuclear NF-κB p65 ( B ) were detected by western blotting, and normalized to β-actin. Data are presented as mean ± SEM ( n = 6) and analysed by ANOVA with Dunnett’s post-hoc analysis. * p < 0.05 vs. control; # p < 0.05 vs. model; ## p < 0.01 vs. model.

Article Snippet: The macrophages were incubated with 100 μg/mL oxidized low density lipoprotein (ox-LDL, MDA 30 μM, Institute of Basic Medicine, Peking Union Medical College, Beijing, China) in 6-well plates for 24 h. Then cells were administrated with ox-LDL free medium containing CD36 blocking antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000) and compound K (0 μM, 3.3 μM, 10 μM, 30 μM) or GW3965 (3.3 μM) or compound K (10 μM) plus 10 mM GGPP for another 24 h. Cells of control group were incubated over the same time period in culture media without any treatment.

Techniques: Activity Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Compound K Attenuates the Development of Atherosclerosis in ApoE −/− Mice via LXRα Activation

doi: 10.3390/ijms17071054

Figure Lengend Snippet: Compound K promotes the expression of reverse cholesterol transport (RCT) related proteins. Levels of LXRα, ABCA1 and ABCG1 in aorta ( A ); ABCG5 and ABCG8 in intestine ( B ); and SREBP-1c ( C ) in liver were detected by immunoblotting and normalized to β-actin. Different from GW3965, treatment of compound K could significantly reduce the expression of SREBP-1c. Data are presented as mean ± SEM ( n = 6) and analysed by ANOVA with Dunnett’s post-hoc analysis. * p < 0.05 vs. control; # p < 0.05 vs. model; ## p < 0.01 vs. model.

Article Snippet: The macrophages were incubated with 100 μg/mL oxidized low density lipoprotein (ox-LDL, MDA 30 μM, Institute of Basic Medicine, Peking Union Medical College, Beijing, China) in 6-well plates for 24 h. Then cells were administrated with ox-LDL free medium containing CD36 blocking antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000) and compound K (0 μM, 3.3 μM, 10 μM, 30 μM) or GW3965 (3.3 μM) or compound K (10 μM) plus 10 mM GGPP for another 24 h. Cells of control group were incubated over the same time period in culture media without any treatment.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Compound K Attenuates the Development of Atherosclerosis in ApoE −/− Mice via LXRα Activation

doi: 10.3390/ijms17071054

Figure Lengend Snippet: Effects of compound K on formation of foam cells and activity of inflammasome. Foam cells derived from macrophages treated by different concentration compound K were stained by Oil-red O. The visible positive stained lipid drops were detected under microscope ( A ); Concentrations of cholesteryl ester in foam cells were detected ( B ); Levels of LXRα, ABCA1 and ABCG1 in foam cells were detected by immunoblotting and normalized to β-actin ( C ); The level of cleaved-IL-1β in cholesterol crystal (1 mg/mL) stimulated macrophages was detected by immunoblotting ( D ); To investigate the probably mechanism, the levels of NLRP3, caspase-1 and nuclear NF-κB p65 were detected by western blot, and normalized to β-actin ( D ). Data are presented as mean ± SEM ( n = 6). * p < 0.05 vs. control; * p < 0.01 vs. control; # p < 0.05 vs. model; ## p < 0.01 vs. model.

Article Snippet: The macrophages were incubated with 100 μg/mL oxidized low density lipoprotein (ox-LDL, MDA 30 μM, Institute of Basic Medicine, Peking Union Medical College, Beijing, China) in 6-well plates for 24 h. Then cells were administrated with ox-LDL free medium containing CD36 blocking antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000) and compound K (0 μM, 3.3 μM, 10 μM, 30 μM) or GW3965 (3.3 μM) or compound K (10 μM) plus 10 mM GGPP for another 24 h. Cells of control group were incubated over the same time period in culture media without any treatment.

Techniques: Activity Assay, Derivative Assay, Concentration Assay, Staining, Microscopy, Western Blot

Journal: Advanced Science

Article Title: Pirin Inhibits FAS‐Mediated Apoptosis to Support Colorectal Cancer Survival

doi: 10.1002/advs.202301476

Figure Lengend Snippet: NFκB2 is required for FAS transactivation. A) Gel shift assay was performed to observe the retardation of GST, GST‐p50 or GST‐p52 to mobility of DNA probe covering 3′‐2a regions of FAS promoter. Probe and protein concentration were 20 n m . B) Gel shift assay was performed with probe 3′‐2a, GST‐p52 protein and increasing doses of GST‐PIR protein. The final concentration of probe was 20 n m . C) Gel shift assay was performed with probe 3′‐2a and indicated proteins. The final concentration of probe was 20 n m . D) Flag‐tagged PIR and HA‐tagged RELB were transiently expressed in HEK293T cells. Co‐IP assay were performed with anti‐Flag agarose (Flag IP), followed by detection of Flag‐PIR and HA‐RELB in the immuno‐precipitates. E) Co‐IP assays were performed with endogenous proteins to determine the association of PIR with RELB in HCT116 cells. F) Flag‐PIR, Flag‐RELB and HA‐p52 were transiently expressed in HEK293T cells. Co‐IP were performed to determine whether PIR disrupts the interaction between RELB and p52. G) Flag‐RELB and Flag‐p52 were transiently expressed in HCT116 cells, followed by knockdown of PIR with sh PIR and further reconstitution of PIR with rHA‐PIR. Chromatin immunoprecipitation (ChIP) was then performed to determine the binding of p52/RELB to FAS promoter using anti‐Flag antibody. H) HCT116 cells were transfected with Flag‐RELB and Flag‐p52, followed by treatment with DMSO or TphA (50 µm, 12 h). ChIP assays were then performed as in (G). I) HCT116 cells were firstly expressed for sh GFP (as control) and sh NFκB2 individually, to create cell lines with NFκB1 knockdown. 24 hours later, each group of cells were further expressed for sh GFP or sh PIR ‐1. After cultured for another 72 hours, cells were evaluated for survival rate by flow cytometry (upper panel) and expression levels of indicated proteins by WB (lower panel). Data represent the mean±SD. (n = 5, unpaired Student's t ‐test, ** p < 0.01, *** p < 0.001). J) HCT116 cells were transfected with indicated plasmids. After 72 hours of transfection, cells were determined for survival rate (upper panel) and expression levels of proteins (lower panel). Date represents the mean±SD. (n = 4, unpaired Student's t ‐test, *** p < 0.001). K) HEK293T cells were co‐transfected with luciferase reporter plasmids carrying FAS full‐length promoter ( FAS ‐Luc) or its 3′‐2a deletion mutants ( FAS ‐3′‐2a‐Luc), along with different doses of Flag‐p52/RELB complex. After 24 hours of transfection, luciferase activities were determined and normalized to the first column (upper panel). Protein levels were determined by WB (lower panel). Data are presented as mean±SD. (n = 3, unpaired Student's t ‐test, ** p < 0.01, *** p < 0.001, n.s.: no significant difference).

Article Snippet: Triphenyl Compound A (TphA) , Santa Cruz , sc‐364144A.

Techniques: Gel Shift, Protein Concentration, Concentration Assay, Co-Immunoprecipitation Assay, Knockdown, Chromatin Immunoprecipitation, Binding Assay, Transfection, Control, Cell Culture, Flow Cytometry, Expressing, Luciferase

Journal: Advanced Science

Article Title: Pirin Inhibits FAS‐Mediated Apoptosis to Support Colorectal Cancer Survival

doi: 10.1002/advs.202301476

Figure Lengend Snippet: PIR suppresses FAS‐dependent apoptosis by switching off NF‐κB2‐FAS axis. A) CT26 cells were treated with cetuximab (CTX, 10 µg mL −1 ) and TphA (50 µg mL −1 ) alone or in combination for 24 hours, followed by detection of survival rate (upper panel, n = 5, unpaired Student's t ‐test, *** p < 0.001) and FAS protein level (lower panel). B and C) Allograft tumors assays based on mouse CT26 cells were conducted according to the procedure indicated in upper panel of (B). Mice were sacrificed at day 24 th . The representative in situ tumors (B) and metastatic intestinal tumors (C) from mice treated with CTX and TphA alone or in combination were shown. s.c.: subcutaneous injections; i.p.: intraperitoneal injections. Scale bars represent 5 mm. D) Colon tumor numbers (left panel) and tumor‐bearing colons (right panel) of the same mice in (B) and (C) were presented (n = 5, unpaired Student's t ‐test, ** p < 0.01). E and F) Quantitative analysis of the percentage of CD8 + T cells (E, n = 5, unpaired Student's t ‐test, * p < 0.05, *** p < 0.001). IHC was performed with CD8 + T cell specific antibody to determine CD8 + T cell infiltration in situ tumors (F). Scale bars represent 200 and 20 µm separately.

Article Snippet: Triphenyl Compound A (TphA) , Santa Cruz , sc‐364144A.

Techniques: In Situ

Journal: Advanced Science

Article Title: Pirin Inhibits FAS‐Mediated Apoptosis to Support Colorectal Cancer Survival

doi: 10.1002/advs.202301476

Figure Lengend Snippet:

Article Snippet: Triphenyl Compound A (TphA) , Santa Cruz , sc‐364144A.

Techniques: Virus, Recombinant, Cell Isolation, Sequencing, Software